CAZypedia - User contributions [en-ca]

File:GDPFuc.png

2026-02-05T22:28:28Z

Spencer Williams: Spencer Williams uploaded a new version of File:GDPFuc.png

File:GDPFuc.png

2026-02-05T22:27:25Z

Spencer Williams: Spencer Williams uploaded a new version of File:GDPFuc.png

Glycoside Hydrolase Family 31

2025-08-27T00:50:17Z

Spencer Williams: /* References */

{{CuratorApproved}}
* [[Author]]: [[User:Ran Zhang|Ran Zhang]], [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Steve Withers|Steve Withers]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''
|-
|'''Clan'''
|GH-D
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH31.html
|}
</div>

== Substrate specificities ==
CAZy Family GH31 has been reviewed and classified on the basis of sequence and structures into subfamilies <cite>#Arumapperuma2023</cite>. Family GH31 is one of the two major families of [[glycoside hydrolases]], along with GH13, that contain α-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal α-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to α-glucosidases, GH31 also contains α-xylosidases, isomaltosyltransferases, maltase/glucoamylases and sulfoquinovosidases. Sulfoquinovosidases (SQases) cleave the α-glycosidic linkage of α-sulfoquinovosides (glycosides of 6-sulfoglucose), present within sulfoquinovosyl diacylglyceride, a sulfolipid produced by photosynthetic organisms including plants <cite>Speciale2016</cite>. SQases are also present in family [[GH188]]. Another mechanistically interesting activity is the non-hydrolytic [[Alpha-glucan lyases|α-glucan lyases]], which cleave α-glucans to give 1,5-anhydro-fructose. GH31 enzymes are found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two human digestive GH31 enzymes are both duplicated genes, each with dual specificities. In light of the sequence and functional diversity of GH31 members, this family has been divided into subfamilies <cite>Arumapperuma2023</cite>.

== Kinetics and Mechanism ==
Enzymes of family GH31 are [[retaining]] α-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement <cite>Chiba1979</cite>. GH31 enzymes (except for the [[α-glucan lyases]]) are believed to follow the classical [[Koshland double-displacement mechanism]]. <cite>Frandsen1998</cite> This has been strongly supported by labeling of the [[catalytic nucleophile]] of several GH31 enzymes using conduritol B epoxide <cite>Iwanami1995</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>Quaroni1976</cite> and human lysosomal α-glucosidase <cite>Hermans1991</cite>. Later studies on an α-glucosidase from ''Aspergillus niger'' <cite>Lee2001</cite>, an α-xylosidase from ''Escherichia coli'' <cite>Lovering2005</cite>, YihQ sulfoquinovosidase from ''Escherichia coli'' <cite>Speciale2016</cite>, and an α-xylosidase from ''Cellvibrio japonicus'' <cite>Larsbrink2011</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent [[intermediate]]s. Subsequently, retention of the anomeric configuration was directly observed by H-1 NMR during the hydrolysis of a natural xylogluco-oligosaccharide substrate by ''C. japonicus'' Xyl31A <cite>Larsbrink2012</cite>, and of a synthetic α-sulfoquinovoside by ''E. coli'' YihQ sulfoquinovosidase <cite>Speciale2016</cite>.

The [[α-glucan lyases]] from GH31 cleave α-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose (see [[Alpha-glucan lyases|Lexicon]]). Detailed mechanistic studies have been carried out on ''Gracilariopsis'' α-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>Lee2002 Lee2003</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme [[intermediate]] by 5-fluoro-β-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two α-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-α-D-glucopyranosyl fluoride.

== Catalytic Residues ==
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>Frandsen1998 Lovering2005 Lee2003</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the [[catalytic nucleophile]], corresponding to Asp224 of ''Aspergillus niger'' α-glucosidase within the sequence IDM <cite>Iwanami1995 Hermans1991</cite>. This was confirmed by using the more reliable 5-fluoro-α-D-glucopyranosyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>Lee2001</cite>.

The [[general acid/base]] residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' α-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>Okuyama2001</cite>. This was subsequently confirmed by the crystallographic studies on α-xylosidase (YicI) from ''Escherichia coli'' <cite>Lovering2005</cite> and successfully engineering YicI into the first α-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>Kim2006</cite>.

The [[catalytic nucleophile]] in ''Gracilariopsis'' α-1,4-glucan lyase has been identified as Asp553 in the sequence QDM, through the use of 5-fluoro-β-L-idopyranosyl fluoride <cite>Lee2002</cite>. This corresponds precisely to the position of the [[catalytic nucleophile]] in the GH31 glycosidases. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the [[catalytic nucleophile]] as it departs <cite>Lee2003</cite>.

== Three-dimensional structures ==
The first crystal structure of a GH31 enzyme was that of the α-xylosidase YicI from ''Escherichia coli'', published in 2005 <cite>Lovering2005</cite>. Since that time, a number of structures have continued to emerge. Among these, the crystallographic study of the ''Sulfolobus solfataricus'' α-glucosidase (MalA) is notable as the first experimental report to highlight the 3-D structural relationship of GH31 enzymes to those of [[GH27]] and [[GH36]] <cite>Ernst2006</cite>; these three families now compose clan [http://www.cazy.org/fam/acc_GH.html#table GH-D]. The structure of the N-terminal domain of human intestinal maltase-glucoamylase was the first from a eukaryotic member of GH31 <cite>Sim2008</cite>. These structures reveal a common (β/α)8 barrel catalytic domain. Most GH31 members are multi-domain proteins, while the specific function (if any) of these accessory domains is generally unknown. An exception is the α-xylosidase, ''Cj''Xyl31A of ''C. japonicus'', in which a PA-14 domain that is rare among GH31 members is suggested to confer increased catalytic specificity toward large oligosaccharide substrates <cite>Larsbrink2011 Larsbrink2012</cite>. The X-ray structure of an inactive mutant of ''E. coli'' YihQ sulfoquinovosidase in complex with a substrate revealed that sulfonate recognition was achieved by a triad of W304, R301 and Y508 (the latter through a bridging water molecule) <cite>Speciale2016</cite>.

== Family Firsts ==

;'''First stereochemical outcome'''
:Determined for several α-glucosidases by a combination of polarimetric and reducing end measurements <cite>Chiba1979</cite>

;'''First [[catalytic nucleophile]] identification'''
:Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling <cite>Quaroni1976</cite>

;'''First [[general acid/base]] residue identification'''
:''Schizosaccharomyces pombe'' α-glucosidase by sequence comparison and kinetic studies of the mutants <cite>Okuyama2001</cite>

;'''First three-dimensional structure of GH31 enzymes'''
:''Escherichia coli'' α-xylosidase (YicI) <cite>Lovering2005</cite>

== References ==
<biblio>
#Arumapperuma2023 pmid=36806678

#Chiba1979 pmid=376499
#Frandsen1998 pmid=9620260
#Iwanami1995 pmid=7766184
#Quaroni1976 pmid=776963
#Hermans1991 pmid=1856189
#Lee2001 pmid=11583585
#Lovering2005 pmid=15501829
#Lee2002 pmid=11982345
#Lee2003 pmid=14596624
#Okuyama2001 pmid=11298744
#Kim2006 pmid=16478160
#Ernst2006 pmid=16580018
#Sim2008 pmid=18036614
#Larsbrink2011 pmid=21426303
#Larsbrink2012 pmid=22961810
#Speciale2016 pmid=26878550
#Arumapperuma2023 pmid=36806678
</biblio>


[[Category:Glycoside Hydrolase Families|GH031]]

Glycoside Hydrolase Family 31

2025-08-27T00:49:49Z

Spencer Williams: /* Substrate specificities */ added sentence and link to subfamily classification

{{CuratorApproved}}
* [[Author]]: [[User:Ran Zhang|Ran Zhang]], [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Steve Withers|Steve Withers]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''
|-
|'''Clan'''
|GH-D
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH31.html
|}
</div>

== Substrate specificities ==
CAZy Family GH31 has been reviewed and classified on the basis of sequence and structures into subfamilies <cite>#Arumapperuma2023</cite>. Family GH31 is one of the two major families of [[glycoside hydrolases]], along with GH13, that contain α-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal α-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to α-glucosidases, GH31 also contains α-xylosidases, isomaltosyltransferases, maltase/glucoamylases and sulfoquinovosidases. Sulfoquinovosidases (SQases) cleave the α-glycosidic linkage of α-sulfoquinovosides (glycosides of 6-sulfoglucose), present within sulfoquinovosyl diacylglyceride, a sulfolipid produced by photosynthetic organisms including plants <cite>Speciale2016</cite>. SQases are also present in family [[GH188]]. Another mechanistically interesting activity is the non-hydrolytic [[Alpha-glucan lyases|α-glucan lyases]], which cleave α-glucans to give 1,5-anhydro-fructose. GH31 enzymes are found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two human digestive GH31 enzymes are both duplicated genes, each with dual specificities. In light of the sequence and functional diversity of GH31 members, this family has been divided into subfamilies <cite>Arumapperuma2023</cite>.

== Kinetics and Mechanism ==
Enzymes of family GH31 are [[retaining]] α-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement <cite>Chiba1979</cite>. GH31 enzymes (except for the [[α-glucan lyases]]) are believed to follow the classical [[Koshland double-displacement mechanism]]. <cite>Frandsen1998</cite> This has been strongly supported by labeling of the [[catalytic nucleophile]] of several GH31 enzymes using conduritol B epoxide <cite>Iwanami1995</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>Quaroni1976</cite> and human lysosomal α-glucosidase <cite>Hermans1991</cite>. Later studies on an α-glucosidase from ''Aspergillus niger'' <cite>Lee2001</cite>, an α-xylosidase from ''Escherichia coli'' <cite>Lovering2005</cite>, YihQ sulfoquinovosidase from ''Escherichia coli'' <cite>Speciale2016</cite>, and an α-xylosidase from ''Cellvibrio japonicus'' <cite>Larsbrink2011</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent [[intermediate]]s. Subsequently, retention of the anomeric configuration was directly observed by H-1 NMR during the hydrolysis of a natural xylogluco-oligosaccharide substrate by ''C. japonicus'' Xyl31A <cite>Larsbrink2012</cite>, and of a synthetic α-sulfoquinovoside by ''E. coli'' YihQ sulfoquinovosidase <cite>Speciale2016</cite>.

The [[α-glucan lyases]] from GH31 cleave α-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose (see [[Alpha-glucan lyases|Lexicon]]). Detailed mechanistic studies have been carried out on ''Gracilariopsis'' α-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>Lee2002 Lee2003</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme [[intermediate]] by 5-fluoro-β-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two α-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-α-D-glucopyranosyl fluoride.

== Catalytic Residues ==
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>Frandsen1998 Lovering2005 Lee2003</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the [[catalytic nucleophile]], corresponding to Asp224 of ''Aspergillus niger'' α-glucosidase within the sequence IDM <cite>Iwanami1995 Hermans1991</cite>. This was confirmed by using the more reliable 5-fluoro-α-D-glucopyranosyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>Lee2001</cite>.

The [[general acid/base]] residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' α-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>Okuyama2001</cite>. This was subsequently confirmed by the crystallographic studies on α-xylosidase (YicI) from ''Escherichia coli'' <cite>Lovering2005</cite> and successfully engineering YicI into the first α-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>Kim2006</cite>.

The [[catalytic nucleophile]] in ''Gracilariopsis'' α-1,4-glucan lyase has been identified as Asp553 in the sequence QDM, through the use of 5-fluoro-β-L-idopyranosyl fluoride <cite>Lee2002</cite>. This corresponds precisely to the position of the [[catalytic nucleophile]] in the GH31 glycosidases. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the [[catalytic nucleophile]] as it departs <cite>Lee2003</cite>.

== Three-dimensional structures ==
The first crystal structure of a GH31 enzyme was that of the α-xylosidase YicI from ''Escherichia coli'', published in 2005 <cite>Lovering2005</cite>. Since that time, a number of structures have continued to emerge. Among these, the crystallographic study of the ''Sulfolobus solfataricus'' α-glucosidase (MalA) is notable as the first experimental report to highlight the 3-D structural relationship of GH31 enzymes to those of [[GH27]] and [[GH36]] <cite>Ernst2006</cite>; these three families now compose clan [http://www.cazy.org/fam/acc_GH.html#table GH-D]. The structure of the N-terminal domain of human intestinal maltase-glucoamylase was the first from a eukaryotic member of GH31 <cite>Sim2008</cite>. These structures reveal a common (β/α)8 barrel catalytic domain. Most GH31 members are multi-domain proteins, while the specific function (if any) of these accessory domains is generally unknown. An exception is the α-xylosidase, ''Cj''Xyl31A of ''C. japonicus'', in which a PA-14 domain that is rare among GH31 members is suggested to confer increased catalytic specificity toward large oligosaccharide substrates <cite>Larsbrink2011 Larsbrink2012</cite>. The X-ray structure of an inactive mutant of ''E. coli'' YihQ sulfoquinovosidase in complex with a substrate revealed that sulfonate recognition was achieved by a triad of W304, R301 and Y508 (the latter through a bridging water molecule) <cite>Speciale2016</cite>.

== Family Firsts ==

;'''First stereochemical outcome'''
:Determined for several α-glucosidases by a combination of polarimetric and reducing end measurements <cite>Chiba1979</cite>

;'''First [[catalytic nucleophile]] identification'''
:Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling <cite>Quaroni1976</cite>

;'''First [[general acid/base]] residue identification'''
:''Schizosaccharomyces pombe'' α-glucosidase by sequence comparison and kinetic studies of the mutants <cite>Okuyama2001</cite>

;'''First three-dimensional structure of GH31 enzymes'''
:''Escherichia coli'' α-xylosidase (YicI) <cite>Lovering2005</cite>

== References ==
<biblio>
#Arumapperuma2023 pmid=36806678

#Chiba1979 pmid=376499
#Frandsen1998 pmid=9620260
#Iwanami1995 pmid=7766184
#Quaroni1976 pmid=776963
#Hermans1991 pmid=1856189
#Lee2001 pmid=11583585
#Lovering2005 pmid=15501829
#Lee2002 pmid=11982345
#Lee2003 pmid=14596624
#Okuyama2001 pmid=11298744
#Kim2006 pmid=16478160
#Ernst2006 pmid=16580018
#Sim2008 pmid=18036614
#Larsbrink2011 pmid=21426303
#Larsbrink2012 pmid=22961810
#Speciale2016 pmid=26878550
</biblio>


[[Category:Glycoside Hydrolase Families|GH031]]

Glycoside hydrolases

2025-06-24T06:37:57Z

Spencer Williams: /* NAD-dependent hydrolysis */

{{CuratorApproved}}
* Authors: [[User:Steve Withers|Steve Withers]], [[User:Spencer Williams|Spencer Williams]]
* Responsible Curator: [[User:Spencer Williams|Spencer Williams]]
----
== Overview ==
Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N- and S-linked glycosides.

[[Image:GHs.png|center|400px]]

== Classification ==
Glycoside hydrolases can be classified in many different ways. The following paragraphs list several different ways, the utility of which depends on the context in which the classification is made and used.

=== Endo/exo ===
''exo''- and ''endo''- refers to the ability of a glycoside hydrolase to cleave a substrate at the end (most frequently, but not always the non-reducing end) or within the middle of a chain <cite>DaviesHenrissat1995</cite>. For example, most cellulases are ''endo''-acting, whereas LacZ β-galactosidase from ''E. coli'' is ''exo''-acting. A general [[sub-site nomenclature]] exists to demarcate substrate binding in glycosidase active-sites.

[[Image:exo_endo.png|center|600px]]

=== Enzyme Commission (EC) number ===
EC numbers are codes representing the Enzyme Commission number. This is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. Every EC number is associated with a recommended name for the respective enzyme. EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number. A necessary consequence of the EC classification scheme is that codes can be applied only to enzymes for which a function has been biochemically identified. Additionally, certain enzymes can catalyze reactions that fall in more than one class. These enzymes must bear more than one EC number.

=== Mechanistic classification ===
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below.<cite>Gebler1992</cite> However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years, as discussed below.

[[Image:Retaining&inverting_GH.png|center|500px]]

=== Sequence-based classification ===
[[Sequence-based classification]] uses algorithmic methods to assign sequences to various families. The glycoside hydrolases have been classified into more than 100 families <cite>Henrissat1991</cite>; this is permanently available through the Carbohydrate Active enZyme database <cite>CAZyURL</cite>. Each family (GH family) contains proteins that are related by sequence, and by corollary, fold. This allows a number of useful predictions to be made since it has long been noted that the catalytic machinery and molecular mechanism is conserved for the vast majority of the glycosidase families <cite>Gebler1992</cite> as well as the geometry around the glycosidic bond (irrespective of naming conventions) <cite>Henrissat1995</cite>. Usually, the mechanism used (ie [[retaining]] or [[inverting]]) is conserved within a GH family. One notable exception is the glycoside hydrolases of family [[GH97]], which contains both retaining and inverting enzymes; a glutamate acts as a [[general base]] in inverting members, whereas an aspartate likely acts as a [[catalytic nucleophile]] in retaining members <cite>Gloster2008</cite>. Another mechanistic curiosity are the glycoside hydrolases of familes [[GH4]] and [[GH109]] which operate through an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s <cite>Yip2007</cite>. This allows a single enzyme to hydrolyze both α- and β-glycosides.

Classification of families into larger groups, termed '[[clans]]' has been proposed <cite>Henrissat1996</cite>. A '[[clan]]' is a group of families that possess significant similarity in their tertiary structure, catalytic residues and mechanism. Families within clans are thought to have a common evolutionary ancestry. For an updated table of glycoside hydrolase clans see the CAZy Database <cite>CAZyURL</cite>.

== Mechanism ==
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below <cite>Koshland1953</cite>. However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years.

===Inverting glycoside hydrolases===
Hydrolysis of a glycoside with net inversion of anomeric configuration is generally achieved via a one step, single-displacement mechanism involving [[oxocarbenium ion]]-like [[transition state]]s, as shown below. The reaction typically occurs with [[general acid]] and [[general base]] assistance from two amino acid side chains, normally glutamic or aspartic acids, that are typically located 6-11 A apart<cite>McCarter1994</cite>.

[[Image:Inverting glucosidase mechanism.png|centre|600px]]

====Glycosyl-phosphate cleaving enzymes that lack a general acid====
A subset of family [[GH92]] α-mannosidases catalyze the hydrolysis of mannose-1-phosphate linkages found in the mannose-1-phosphate-6-mannose groups of yeast mannoproteins. In these enzymes the usual [[general acid]] glutamic acid found in other members of this family is replaced by a glutamine. It has been suggested that the phosphate aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst <cite>Tiels2012</cite>. This replacement may also reduce charge repulsion between the glutamic acid residue and the anionic phosphate aglycon. A related example may be found in the case of the family [[GH1]] myrosinases.

===Retaining glycoside hydrolases===
====Classical Koshland retaining mechanism====
Hydrolysis with net retention of configuration is most commonly achieved via a two step, double-displacement mechanism involving a covalent glycosyl-enzyme [[intermediate]], as is shown in the figure below. Each step passes through an [[oxocarbenium ion]]-like [[transition state]]. Reaction occurs with acid/base and nucleophilic assistance provided by two amino acid side chains, typically glutamate or aspartate, located 5.5 A apart. In the first step (often called the glycosylation step), one residue plays the role of a nucleophile, attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme [[intermediate]]. At the same time the other residue functions as an acid catalyst and protonates the glycosidic oxygen as the bond cleaves. In the second step (known as the deglycosylation step), the glycosyl enzyme is hydrolyzed by water, with the other residue now acting as a base catalyst deprotonating the water molecule as it attacks. The pKa value of the acid/base group cycles between high and low values during catalysis to optimize it for its role at each step of catalysis <cite>McIntosh1996</cite>. In the case of sialidases, the catalytic nucleophile is a tyrosine residue (see below). This mechanism was originally proposed by Dan Koshland, although at the time the identities of the residues was unclear <cite>Koshland1953</cite>.

[[Image:Retaining_glycosidase_mechanism.png|centre|600px]]

====Neighboring group participation====
=====By a neighboring 2-acetamido group=====
Enzymes of [[GH18]], [[GH20]], [[GH25]], [[GH56]], [[GH84]], [[GH85]] and [[GH123]] hydrolyse substrates containing an ''N''-acetyl (acetamido) or ''N''-glycolyl group at the 2-position. These enzymes have no catalytic nucleophile: rather they utilize a mechanism in which the 2-acetamido group acts as an intramolecular nucleophile. Neighboring group participation by the 2-acetamido group leads to formation of an oxazoline (or more strictly an [[oxazolinium ion]]) [[intermediate]]. This mechanism was deduced from X-ray structures of complexes of chitinases with natural inhibitors <cite>Terwisscha1995</cite>, from the potent inhibition afforded by a stable thiazoline analogue of the oxazoline <cite>Knapp1996 Mark2001</cite>, and from detailed mechanistic analyses using substrates of modified reactivity <cite>Vocadlo2005</cite>. Typically, a stabilizing residue (a carboxylate) stabilizes the charge development in the [[transition state]]. Not all enzymes that cleave substrates possessing a 2-acetamido group utilize a neighboring groups participation mechanism; enzymes from [[GH3]] and [[GH22]] utilize a classical retaining mechanism with an enzymic nucleophile. Other hexosaminidases such as those of [[Glycoside Hydrolase Family 19]] utilize an inverting mechanism.

[[Image:Hex_neighboring_mechanism.png|centre|700px]]

=====By a neighboring 2-hydroxyl group=====
Enzymes of [[Glycoside Hydrolase Family 99]] hydrolyse α-mannoside substrates and lack a enzymic catalytic nucleophile. These enzymes use a mechanism in which the 2-hydroxyl group acts as an intramolecular nucleophile. Neighboring group participation by the 2-hydroxy group leads to formation of an epoxide (or more strictly a 1,2-anhydro sugar). This mechanism was implicated from 3D X-ray structures of complexes of bacterial endomannosidase/endomannanase with iminosugar inhibitors <cite>Thompson2012</cite>. Detailed mechanistic analyses using carbocyclic analogues of the 1,2-anhydro sugar, quantum mechanic/molecular mechanics computational modelling, and kinetic isotope effects <cite>Sobala2020</cite>.

====Myrosinases: Retaining glycoside hydrolases that lack a general acid and utilize an exogenous base====
Glycoside hydrolases termed myrosinases catalyze the hydrolysis of anionic thioglycosides (glucosinolates) found in plants. They are found in [[Glycoside Hydrolase Family 1]]. In these enzymes the usual acid/base glutamic acid found in other members of this family is replaced by a glutamine. This likely reduces charge repulsion between the anionic aglycon sulfate. The unusual aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst. However, since a base catalyst is required for the second step (hydrolysis or deglycosylation) these enzymes require an alternative basic group. This is provided by the co-enzyme L-ascorbate <cite>Burmeister2000</cite>.

[[Image:myrosinase.png|centre|550px]]

====Alternative nucleophiles====
Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of [[GH33]] and [[GH34]], and 2-keto-3-deoxy-D-lyxo-heptulosaric acid hydrolases of [[GH143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>. A cysteine nucleophile has been demonstrated for Zn2+-dependent arabinofuranosidases of families [[GH127]] and [[GH146]] through X-ray crystallography of the covalently labelled nucleophilic cysteine and MD and QM/MM simulations <cite>#McGregor2021</cite>.

[[Image:sialidase_mechanism.png|center|700px]]

====NAD-dependent hydrolysis====
The members of [[GH4]], [[GH109]], [[GH177]], [[GH179]], and [[GH188]] use a mechanism that requires an NAD cofactor, which remains tightly bound throughout catalysis. The mechanism proceeds via anionic [[transition state]]s with elimination and redox steps rather than the classical mechanisms proceeding through[[oxocarbenium ion]]-like transition states. As shown below for a 6-phospho-β-glucosidase, the mechanism involves an initial oxidation of the 3-hydroxyl of the substrate by the enzyme-bound NAD cofactor. This increases the acidity of the C2 proton such that an E1cb elimination can occur with assistance from an enzymatic base. The α,β-unsaturated [[intermediate]] formed then undergoes addition of water at the anomeric centre and finally the ketone at C3 is reduced to generate the free sugar product. Thus, even though glycosidic bond cleavage occurred via an elimination mechanism, the overall reaction is hydrolysis. This mechanism was elucidated through a combination of stereochemical studies by NMR, kinetic isotope effects, linear free energy relationships, X-ray crystallography and UV/Vis spectrophotometry <cite>Yip2004 Rajan2004</cite>.

[[Image:Family_4_mechanism.png|center|700px]]

== References ==
<biblio>
#McCarter1994 pmid=7712292
#Koshland1953 Koshland, D. (1953) ''Biol. Rev.'' 28, 416.
#McIntosh1996 pmid=8756457
#Gebler1992 pmid=1618761
#Henrissat1995 pmid=7624375
#Terwisscha1995 pmid=7495789
#Mark2001 pmid=11124970
#Knapp1996 Knapp, S., Vocadlo, D., Gao, Z. N., Kirk, B., Lou, J. P., and Withers, S. G. (1996) ''Journal of the American Chemical Society 118'', 6804-6805.
#Vocadlo2005 pmid=16171396
#Burmeister2000 pmid=10978344
#Amaya2004 pmid=15130470
#Watts2003 pmid=12812490
#Watson2003 pmid=14580216
#Yip2004 pmid=15237973
#Rajan2004 pmid=15341727
#CAZyURL Carbohydrate Active Enzymes database; URL http://www.cazy.org/
#Henrissat1991 pmid=1747104
#Henrissat1996 pmid=8687420
#Gloster2008 pmid=18848471
#Yip2007 pmid=17676871
#Tiels2012 pmid=23159880
#DaviesHenrissat1995 pmid=8535779
#Ndeh2017 pmid=28329766
#Sobala2020 pmid=32490192
#McGregor2021 pmid=33528085

</biblio>
[[Category:Definitions and explanations]]

Conformational nomenclature

2024-10-30T10:56:19Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Withers|Stephen Withers]]
* [[Responsible Curator]]: [[User:SpencerWilliams|Spencer Williams]]
----
The conformations adopted by a pyranose or furanose ring are denoted by a system in which a capital letter indicates the overall shape, ''C'' = chair, ''B'' = boat, ''H'' = half chair, ''S'' = skew boat, ''E'' = envelope <cite>IUPACNomenclature1981 IUPACNomenclature1980</cite>. The first four of these conformations has four atoms in a plane; the envelope conformation has five.

The particular conformation is then denoted by assigning the letter corresponding to the shape (''C'', ''B'', ''H'', ''S'', ''E''); determining the four (or five) atoms that define the plane; assigning a 'top' and 'bottom' face through the use of a left-hand rule counting in the order of increasing ring carbon; and then indicating the identities and relative positions (top face = superscript and prefix; bottom face = subscript and suffix) of the remaining two atoms on that capital letter. In the case of the envelope conformation, only a single atom is located outside of the plane. For a more detailed discussion see the excellent book by J. Fraser Stoddard <cite>Stoddart1971</cite>.

It should be noted that the conformational symbols for enantiomers are different. This is because the reference plane is the same, yet application of the left-hand rule results in a different 'top' and 'bottom' face. Thus the mirror image of α-D-glucose-4''C1 is α-L-glucose-1''C4. It is therefore important to state whether the D or L form is under consideration.

[[Image:Conformations.png|centre|800px]]
==References==
<biblio>
#IUPACNomenclature1981 Conformational nomenclature for five- and six-membered ring forms of monosaccharides and their derivatives, Pure and Appl. Chem., 1981, ''53'', 1901—1905. [http://dx.doi.org/10.1351/pac198153101901 DOI: 10.1351/pac198153101901]
#IUPACNomenclature1980 pmid=7460897
#Stoddart1971 Stereochemistry of Carbohydrates, J. Fraser Stoddart, John Wiley & Sons Inc, 1971, 264 pages.
</biblio>
[[Category:Definitions and explanations]]

Phosphorylases

2024-09-12T04:56:10Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]s: [[User:Spencer Williams|Spencer Williams]], [[User:Motomitsu Kitaoka|Motomitsu Kitaoka]]
* [[Responsible Curator]]: [[User:Spencer Williams|Spencer Williams]]
----
==Overview==
Phosphorylases are enzymes that catalyze the cleavage of a glycosidic bond through substitution with phosphate (formally referred to as phosphorolysis). Phosphorylases are usually named using a combination of the ‘substrate name’ and ‘phosphorylase’ <cite>Kitaoka2002 Nakai2013</cite>

[[Image:Phosphorylase.png|center|500px]]

Phosphorolysis of a glycosidic bond can occur with retention or inversion of configuration and always occurs in an ''[[exo]]''-fashion leading to formation of a monosaccharide or disaccharide 1-phosphate.

[[Image:Retaining&Inverting_phosphorylase.png|center|700px]]

The energy content of the sugar 1-phosphate product means that the cleavage reaction is often in a practical sense reversible and, in nature, these enzymes may be involved in either synthesis or cleavage of the glycosidic bond. As such there is a relatively fine distinction among sugar phosphorylases, [[glycoside hydrolase]]s and classical sugar nucleoside (di)phosphate dependent [[glycosyltransferase]]s. In the last case the synthetic reaction is normally, but not always, irreversible because of the higher energy of a sugar nucleoside (di)phosphate.
 
 
The phosphorylases are classified into various [[glycoside hydrolase]] (GH) and [[glycosyltransferase]] (GT) families on the basis of sequence similarity <cite>Nakai2013</cite>. Glycoside phosphorylases have been found in more than 8 GH and GT families (see below).

===Glycosyltransferase-like phosphorylases===
The classical example of phosphorylases are the glycogen/starch phosphorylases <cite>Lairsson2008 </cite>. These enzymes catalyze the cleavage of individual glucosyl residues from glycogen/amylopectin (up to five residues (or up to four for hyperthermophilic bacterial and archael forms) from a branchpoint), forming sequentially deglycosylated glycogen/amylopectin and glucose 1-phosphate. Glycogen/starch phosphorylases have a complex mechanism that is not fully understood and requires pyridoxal phosphate (PLP) as a cofactor. All glycogen/starch phosphorylases are classified into the same sequence-related [[glycosyltransferase]] family as starch phosphorylases ([[GT35]]), which also require a PLP cofactor. Trehalose phosphorylase (retaining) is classified as a glycosyltransferase and belongs to [[GT4]]. There is no evidence that trehalose phosphorylase (retaining) uses a PLP cofactor.

====Examples====
*Family [[GT4]] contains trehalose phosphorylase, a retaining enzyme.
*Family [[GT35]] contains glycogen and starch phosphorylases.
*Family [[GT108]] contains β-1,2-mannogen phosphorylases.

===Glycoside hydrolase-like phosphorylases===

Most sugar phosphorylases act on glucosides and many cleave simple disaccharides such as sucrose, trehalose, cellobiose and maltose leading to glucose-1-phosphate and the reducing end sugar (glucose or fructose in these specific cases). Other sugar phosphorylases are known that act on ''N'',''N'''-diacetylchitobiose, laminaribiose, 1,3-β-glucan and nucleosides. A phosphorylase from [[GH13]] has been reported that acts on α-1,4-glucans to release the disaccharide maltose 1-phosphate <cite>Elbein2010</cite>.

====Examples====
*Family [[GH3]] contains NagZ enzymes, which are ''N''-acetyl-β-D-glucosaminide/β-glucoside hydrolases/phosphorylases.
*Family [[GH13]] contains sucrose phosphorylase and α-1,4-glucan:maltose 1-phosphate maltosyltransferase.
*Family [[GH65]] contains maltose phosphorylase, trehalose phosphorylase, kojibiose (Glc-α-1,2-Glc) phosphorylase, nigerose (Glc-α-1,3-Glc) phosphorylase, and trehalose 6-phosphate phosphorylase.
*Family [[GH94]] contains cellobiose phosphorylase, cellodextrin phosphorylase, and chitobiose phosphorylase.
*Family [[GH112]] contains β-galactoside phosphorylases such as β-1,3-D-galactosyl-D-hexososamine phosphorylase, and β-1,4-D-galactosyl-L-rhamnose phosphorylase.
*Family [[GH130]] contains β-mannoside phosphorylases such as β-mannosyl-1,4-glucose phosphorylase and β-1,4-mannooligosaccharide phosphorylase.

==Mechanism of glycoside hydrolase-like phosphorylases==

Sequence and structural analysis of sugar phosphorylases reveal that some have sequences and structures (and likely mechanisms) similar to [[glycosyltransferases]], whereas others have sequences and structures that more closely resemble [[glycoside hydrolase]]s.

===Inverting phosphorylase mechanism===

All inverting phosphorylases are GH-like in their sequence classification. Phosphorolysis of a glycoside by a GH-like phosphorylase with net inversion of anomeric configuration is achieved via a one step, single-displacement mechanism involving [[oxocarbenium ion]]-like [[transition state]]s, as shown below. The reaction occurs with acid/base assistance from a suitably positioned carboxylate residue. This mechanism has clear parallels with the mechanism for [[inverting]] glycoside hydrolases.
 
[[Image:Inverting_b-glycoside_phosphorylase_mechanism.png|center|600px]]

===Retaining phosphorylase mechanism===
Phosphorolysis of a glycoside with net retention of configuration by the GH-like retaining phosphorylases is achieved via two step, double-displacement mechanisms involving a nucleophilic participation and a covalent intermediate. The mechanism of these enzymes involves a covalent glycosyl-enzyme intermediate, as shown in the figure below. Reaction occurs with acid/base and nucleophilic assistance provided by two amino acid side chains, typically glutamate or aspartate, but occasionally histidine (for family [[GH3]] NagZ enzymes). In the first step (called the glycosylation step), one residue plays the role of a nucleophile, attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme [[intermediate]]. At the same time the other residue functions as an acid catalyst and protonates the glycosidic oxygen as the bond cleaves. In the second step (termed the deglycosylation step), the glycosyl enzyme is cleaved by phosphate, with the other residue now acting as a base catalyst deprotonating the phosphate as it attacks. Each step passes through an [[oxocarbenium ion]]-like [[transition state]]. An alternative mechanism for retention of anomeric configuration has been proposed involving direct front-side nucleophilic displacement (SNi). Both of these mechanisms bear considerable analogy to those proposed for retaining glycosyltransferases <cite>Lairsson2004 Lairsson2008 </cite>.

[[Image:Retaining_a-glycoside_phosphorylase_mechanism.png|center|600px]]

==Mechanism of glycosyltransferase-like phosphorylases==

Both retaining ([[GT4]], [[GT35]]) and inverting ([[GT108]] enzymes have been identified within the glycosyltransferase-like phosphorylases

===Retaining phosphorylase mechanism===

As in the case for retaining [[glycosyltransferases]], the evidence supporting the specific molecular details are meagre. Two mechanistic proposals remain in contention. The first proposes a mechanism that is essentially identical to the two-step double displacement mechanism of a retaining GH-like phosphorylase drawn above. However, limited evidence is available to support the existence of a nucleophilic residue in the active site, and a front-side displacement mechanism termed an SNi (substitution, nucleophilic, internal return) has been posited as an alternative.

[[Image:Retaining_phosphorylase_mechanism.png|center|600px]]

== References ==
<biblio>
#Elbein2010 pmid=20118231
#Nakai2013 pmid=23403067
#Kitaoka2002 Kitaoka M, Hayashi K: Carbohydrate-processing phosphorolytic enzymes. ''Trends Glycosci. Glycotechnol.'' 2002, '''14''', 35-50.
#Lairsson2004 pmid=15489968
#Lairsson2008 pmid=18518825

</biblio>
[[Category:Definitions and explanations]]

Transglycosylases

2024-08-02T00:58:06Z

Spencer Williams:

{{CuratorApproved}}
* Author: [[User:SpencerWilliams|Spencer Williams]]
* Responsible Curator: [[User:SpencerWilliams|Spencer Williams]]
----
== Overview ==

'''Transglycosylases''' (also transglycosidases) are a class of GH enzymes that can catalyze the transformation of one glycoside to another. That is, these enzymes catalyze the intra- or intermolecular substitution of the anomeric position of a glycoside. Mechanistically, transglycosylases utilize the same mechanism as various [[retaining]] glycoside hydrolases <cite>Sinnott1990 Bissaro2015</cite>. Thus, reaction of the nucleophile of a retaining glycoside hydrolase with a substrate gives a glycosyl-enzyme [[intermediate]] that can be intercepted either by water to give the hydrolysis product, or by another acceptor (often another carbohydrate alcohol), to give a new glycoside or oligosaccharide <cite>Crout1998</cite>. Alternatively, transglycosylation can occur by [[neighboring group participation]], wherein a neighboring 2-acetamido group participates in the reaction to generate an [[oxazolinium ion]] intermediate, which again can react with another acceptor other than water. Some transglycosidases possess substantial [[glycoside hydrolase]] activity, and some glycoside hydrolases possess transglycosylase activity <cite>Vocadlo2000</cite>. Indeed, in many cases it is unclear what the major role of an enzyme that possesses both activities may be. Transglycosylases are [[sequence-based classification|classified]] as [[glycoside hydrolases]] into various GH families on the basis of sequence similarity.

[[Image:transglycosylation.png|thumb|center|600px|'''Figure. Generalized mechanism of a transglycosylase.''' Enzymatic cleavage of a substrate through a [[classical Koshland retaining mechanism]] results in formation of a glycosyl enzyme intermediate. This can partition to react with either water to cause hydrolysis ([[glycoside hydrolase]] activity) or to an alternative acceptor, often a sugar, to cause transglycosylation (transglycosylase activity).]]

===Families===
GH families with notable transglycosylase activity include: 
*[[GH2]], for example LacZ β-galactosidase converts lactose to allolactose <cite>Juers2012</cite>. 
*[[GH13]], for example cyclodextran glucanotransferases that convert linear amylose to cyclodextrins <cite>Uitdehaag1999</cite>; glycogen debranching enzyme, which transfers three glucose residues from the four-residue glycogen branch to a nearby branch <cite>Braun1996</cite>; and trehalose synthase, which catalyzes the interconversion of maltose and trehalose <cite>Zhang2011</cite>. 
*[[GH16]], for example xyloglucan endotransglycosylases, which cuts and rejoins xyloglucan chains in the plant cell wall <cite>Eklof2010</cite>.
*[[GH31]], for example α-transglucosidases, which catalyze the transfer of individual glucosyl residues between α-(1→4)-glucans <cite>Larsbrink2012</cite>.
*[[GH70]], for example glucansucrases, which catalyse the synthesis of high molecular weight glucans, from sucrose <cite>Hijum2006</cite>.
*[[GH77]], for examples amylomaltase, which catalyzes the synthesis of maltodextrins from maltose <cite>Maarel2013</cite>.
*[[GH23]], [[GH102]], [[GH103]], and [[GH104]] lytic transglycosylases, which convert peptidoglycan to 1,6-anhydrosugars <cite>Schuerwater2008</cite>.

===Inverting transglycosylases===
An anomer-inverting transglycosylase has been reported that is a member of Family [[GH186]], namely the cyclic glucohexadecaose-producing enzyme from ''Xanthomonas'' sp. <cite>#Motouchi2024</cite>. This enzyme converts linear β-1,2-glucan to cyclic-(β-1,2-Glc)15-1,6α. The reaction involves transglycosylation of a β-1,2-Glc bond to generate an α-1,6-Glc bond. Enzymes of this family are inverting glycosidases, and presumably, this product will be a substrate for the enzyme, possibly reversing the reaction or catalyzing hydrolysis, although this was not studied.

== References ==
<biblio>
#Crout1998 pmid=9667913
#Larsbrink2012 pmid=23132856
#Juers2012 pmid=23011886
#Eklof2010 pmid=20421457
#Braun1996 pmid=8611536
#Uitdehaag1999 pmid=10331869
#Hijum2006 pmid=16524921
#Zhang2011 pmid=21840994
#Schuerwater2008 pmid=17468031
#Maarel2013 pmid=23465909
#Vocadlo2000 Vocadlo, D. J. and Withers, S. G. (2008) Glycosidase-Catalysed Oligosaccharide Synthesis, Chapter 29 in ''Carbohydrates in Chemistry and Biology'', Ernst, B., Hart, G. W. and Sinaý, P., eds., Wiley-VCH Verlag GmbH, Weinheim, Germany. [http://dx.doi.org/10.1002/9783527618255.ch29 DOI:10.1002/9783527618255.ch29]
#Sinnott1990 Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. [http://dx.doi.org/10.1021/cr00105a006 DOI: 10.1021/cr00105a006]
#Bissaro2015 pmid=25793417
#Motouchi2024 pmid=38957137

</biblio>
[[Category:Definitions and explanations]]

Transglycosylases

2024-08-02T00:40:51Z

Spencer Williams:

{{CuratorApproved}}
* Author: [[User:SpencerWilliams|Spencer Williams]]
* Responsible Curator: [[User:SpencerWilliams|Spencer Williams]]
----
== Overview ==

'''Transglycosylases''' (also transglycosidases) are a class of GH enzymes that can catalyze the transformation of one glycoside to another. That is, these enzymes catalyze the intra- or intermolecular substitution of the anomeric position of a glycoside. Mechanistically, transglycosylases utilize the same mechanism as various [[retaining]] glycoside hydrolases <cite>Sinnott1990 Bissaro2015</cite>. Thus, reaction of the nucleophile of a retaining glycoside hydrolase with a substrate gives a glycosyl-enzyme [[intermediate]] that can be intercepted either by water to give the hydrolysis product, or by another acceptor (often another carbohydrate alcohol), to give a new glycoside or oligosaccharide <cite>Crout1998</cite>. Alternatively, transglycosylation can occur by [[neighboring group participation]], wherein a neighboring 2-acetamido group participates in the reaction to generate an [[oxazolinium ion]] intermediate, which again can react with another acceptor other than water. Some transglycosidases possess substantial [[glycoside hydrolase]] activity, and some glycoside hydrolases possess transglycosylase activity <cite>Vocadlo2000</cite>. Indeed, in many cases it is unclear what the major role of an enzyme that possesses both activities may be. Transglycosylases are [[sequence-based classification|classified]] as [[glycoside hydrolases]] into various GH families on the basis of sequence similarity.

[[Image:transglycosylation.png|thumb|center|600px|'''Figure. Generalized mechanism of a transglycosylase.''' Enzymatic cleavage of a substrate through a [[classical Koshland retaining mechanism]] results in formation of a glycosyl enzyme intermediate. This can partition to react with either water to cause hydrolysis ([[glycoside hydrolase]] activity) or to an alternative acceptor, often a sugar, to cause transglycosylation (transglycosylase activity).]]

===Families===
GH families with notable transglycosylase activity include: 
*[[GH2]], for example LacZ β-galactosidase converts lactose to allolactose <cite>Juers2012</cite>. 
*[[GH13]], for example cyclodextran glucanotransferases that convert linear amylose to cyclodextrins <cite>Uitdehaag1999</cite>; glycogen debranching enzyme, which transfers three glucose residues from the four-residue glycogen branch to a nearby branch <cite>Braun1996</cite>; and trehalose synthase, which catalyzes the interconversion of maltose and trehalose <cite>Zhang2011</cite>. 
*[[GH16]], for example xyloglucan endotransglycosylases, which cuts and rejoins xyloglucan chains in the plant cell wall <cite>Eklof2010</cite>.
*[[GH31]], for example α-transglucosidases, which catalyze the transfer of individual glucosyl residues between α-(1→4)-glucans <cite>Larsbrink2012</cite>.
*[[GH70]], for example glucansucrases, which catalyse the synthesis of high molecular weight glucans, from sucrose <cite>Hijum2006</cite>.
*[[GH77]], for examples amylomaltase, which catalyzes the synthesis of maltodextrins from maltose <cite>Maarel2013</cite>.
*[[GH23]], [[GH102]], [[GH103]], and [[GH104]] lytic transglycosylases, which convert peptidoglycan to 1,6-anhydrosugars <cite>Schuerwater2008</cite>.

== References ==
<biblio>
#Crout1998 pmid=9667913
#Larsbrink2012 pmid=23132856
#Juers2012 pmid=23011886
#Eklof2010 pmid=20421457
#Braun1996 pmid=8611536
#Uitdehaag1999 pmid=10331869
#Hijum2006 pmid=16524921
#Zhang2011 pmid=21840994
#Schuerwater2008 pmid=17468031
#Maarel2013 pmid=23465909
#Vocadlo2000 Vocadlo, D. J. and Withers, S. G. (2008) Glycosidase-Catalysed Oligosaccharide Synthesis, Chapter 29 in ''Carbohydrates in Chemistry and Biology'', Ernst, B., Hart, G. W. and Sinaý, P., eds., Wiley-VCH Verlag GmbH, Weinheim, Germany. [http://dx.doi.org/10.1002/9783527618255.ch29 DOI:10.1002/9783527618255.ch29]
#Sinnott1990 Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. [http://dx.doi.org/10.1021/cr00105a006 DOI: 10.1021/cr00105a006]
#Bissaro2015 pmid=25793417

</biblio>
[[Category:Definitions and explanations]]

Glycoside Hydrolase Family 188

2023-12-18T02:56:32Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Spencer Williams|Spencer Williams]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH188'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|NAD-dependent hydrolysis
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH188.html
|}
</div>


== Substrate specificities ==
The [[glycoside hydrolases]] of this family are found in bacteria, algae, plants and a small number of archaea <cite>#Kaur2024</cite>. The family contains enzymes with sulfoquinovosidase activity (EC 3.2.1.199), namely the ability to cleave glycosides of 6-deoxy-6-sulfoquinovose. Sulfoquinovosidases are also found in family [[GH31]] <cite>#Speciale2016</cite>. Enzymes of this family have the ability to cleave both α- and β-glycosides, and are dependent on an NAD+ cofactor. Sulfoquinovosidases hydrolyse glycosides of sulfoquinovose such as sulfoquinovosyl glycerol, and liberate free sulfoquinovose, which can be used as a substrate in bacterial sulfoglycolysis and sulfoquinovose sulfolysis pathways <cite>#Snow2021</cite>.

== Kinetics and Mechanism ==
GH188 enzymes utilize an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps. Exchange of the substrate C2 proton with solvent deuterium during the enzyme-catalyzed reaction was demonstrated by mass spectrometry <cite>#Kaur2024</cite>. The following chemical mechanism is proposed: (1) C3 hydride abstraction via the reduction of NAD+ cofactor to NADH and simultaneous oxidation of the C3 hydroxyl group; (2) α to the ketone functionality, the C2 proton is deprotonated by a general catalytic base residue; (3) cleavage of the C1-O1 bond occurs in an α,β-elimination, producing an α,β-unsaturated ketone [[intermediate]]; (4) 1,4-Michael-like addition of a water molecule at C1; and (5) reduction of the C3 carbonyl functionality by the enzyme-bound NADH generates the product. Similar mechanisms are used in families [[GH4]], [[GH109]], [[GH177]] and [[GH179]].

== Catalytic Residues ==
Catalytic residues can be inferred on the basis of X-ray crystallographic data for complexes of GH188 enzymes with NAD+ and sulfoquinovose <cite>#Kaur2024</cite>. Tyr136 in ''Arthrobacter'' sp. strain U41 SqgA is conserved in most family GH188 members and is located close to C2-OH, suggesting a possible role as a [[general base]]. His321 is located close to the C1-OH and may act as [[general acid]] (along with Tyr136) for the glycosidic oxygen facilitating glycosidic bond scission. The sulfonate group is recognized by a triad of amino acids: one oxygen H-bonds to Arg166 (2.6 Å), a second to Lys172 (2.9 Å), and a third to the backbone amide of Leu170 (2.8 Å).

== Three-dimensional structures ==
Crystallographic data is available for at least two GH188 enzymes, including as complexes with NADH, and NADH plus sulfoquinovose. The 3D X-ray crystal structures include those of ''Flavobacterium'' sp. strain K172 SqgA (PDB 8QC8, 8QC2) and ''Arthrobacter'' sp. strain U41 SqgA (PDB 8QC3, 8QC6. 8QC5) <cite>#Kaur2024</cite>. Each enzyme possesses an N-terminal dinucleotide-binding Rossman fold. The GH188 enzymes show structural similarities to inositol-2-dehydrogenase, glucose-fructose/IDH/MocA-like oxidoreductase, and NAD+-dependent ''N''-acetylgalactosaminidase of family [[GH109]].

== Family Firsts ==
;'''First stereochemistry determination
:not applicable
; '''First catalytic residue identification'''
:''Arthrobacter'' sp. strain U41 SqgA using 3D X-ray crystal structure <cite>#Kaur2024</cite>
; '''First 3-D structural determination'''
: ''Flavobacterium'' sp. strain K172 SqgA and ''Arthrobacter'' sp. strain U41 SqgA <cite>#Kaur2024</cite>
; '''First GH188 enzyme shown to hydrolyze both α- and β-substrates'''
:''Flavobacterium'' sp. strain K172 SqgA <cite>#Kaur2024</cite>

==References==
<biblio>
#Kaur2024 pmid=38100472
#Snow2021 pmid=34816844
#Speciale2016 pmid=26878550
</biblio>


[[Category:Glycoside Hydrolase Families|GH188]]

Glycoside Hydrolase Family 188

2023-12-17T22:53:09Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Spencer Williams|Spencer Williams]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH188'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|NAD-dependent hydrolysis
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH188.html
|}
</div>


== Substrate specificities ==
The [[glycoside hydrolases]] of this family are found in bacteria, algae, plants and a small number of archaea <cite>#Kaur2024</cite>. The family contains enzymes with sulfoquinovosidase activity (EC 3.2.1.199), namely the ability to cleave glycosides of 6-deoxy-6-sulfoquinovose. Sulfoquinovosidases are also found in family [[GH31]] <cite>#Speciale2016</cite>. Enzymes of this family have the ability to cleave both α- and β-glycosides, and are dependent on an NAD+ cofactor. Sulfoquinovosidases hydrolyse glycosides of sulfoquinovose such as sulfoquinovosyl glycerol, and liberate free sulfoquinovose, which can be used as a substrate in bacterial sulfoglycolysis and sulfoquinovose sulfolysis pathways <cite>#Snow2021</cite>.

== Kinetics and Mechanism ==
GH188 enzymes utilize an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps. Exchange of the substrate C2 proton with solvent deuterium during the enzyme-catalyzed reaction was demonstrated by mass spectrometry <cite>#Kaur2024</cite>. The following chemical mechanism is proposed: (1) C3 hydride abstraction via the reduction of NAD+ cofactor to NADH and simultaneous oxidation of the C3 hydroxyl group; (2) α to the ketone functionality, the C2 proton is deprotonated by a general catalytic base residue; (3) cleavage of the C1-O1 bond occurs in an α,β-elimination, producing an α,β-unsaturated ketone [[intermediate]]; (4) 1,4-Michael-like addition of a water molecule at C1; and (5) reduction of the C3 carbonyl functionality by the enzyme-bound NADH generates the product. Similar mechanisms are used in families [[GH4]], [[GH109]], [[GH177]] and [[GH179]].

== Catalytic Residues ==
Catalytic residues can be inferred on the basis of X-ray crystallographic data for complexes of GH188 enzymes with NAD+ and sulfoquinovose <cite>#Kaur2024</cite>. Tyr136 in ''Arthrobacter'' sp. strain U41 SqgA is conserved in all family GH188 members and is located close to C2-OH, suggesting a possible role as a [[general base]]. His321 is located close to the C1-OH and may act as [[general acid]] (along with Tyr136) for the glycosidic oxygen facilitating glycosidic bond scission. The sulfonate group is recognized by a triad of amino acids: one oxygen H-bonds to Arg166 (2.6 Å), a second to Lys172 (2.9 Å), and a third to the backbone amide of Leu170 (2.8 Å).

== Three-dimensional structures ==
Crystallographic data is available for at least two GH188 enzymes, including as complexes with NADH, and NADH plus sulfoquinovose. The 3D X-ray crystal structures include those of ''Flavobacterium'' sp. strain K172 SqgA (PDB 8QC8, 8QC2) and ''Arthrobacter'' sp. strain U41 SqgA (PDB 8QC3, 8QC6. 8QC5) <cite>#Kaur2024</cite>. Each enzyme possesses an N-terminal dinucleotide-binding Rossman fold. The GH188 enzymes show structural similarities to inositol-2-dehydrogenase, glucose-fructose/IDH/MocA-like oxidoreductase, and NAD+-dependent ''N''-acetylgalactosaminidase of family [[GH109]].

== Family Firsts ==
;'''First stereochemistry determination
:not applicable
; '''First catalytic residue identification'''
:''Arthrobacter'' sp. strain U41 SqgA using 3D X-ray crystal structure <cite>#Kaur2024</cite>
; '''First 3-D structural determination'''
: ''Flavobacterium'' sp. strain K172 SqgA and ''Arthrobacter'' sp. strain U41 SqgA <cite>#Kaur2024</cite>
; '''First GH188 enzyme shown to hydrolyze both α- and β-substrates'''
:''Flavobacterium'' sp. strain K172 SqgA <cite>#Kaur2024</cite>

==References==
<biblio>
#Kaur2024 pmid=38100472
#Snow2021 pmid=34816844
#Speciale2016 pmid=26878550
</biblio>


[[Category:Glycoside Hydrolase Families|GH188]]

Glycoside Hydrolase Family 188

2023-12-17T22:52:52Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]:
* [[Responsible Curator]]: [[User:Spencer Williams|Spencer Williams]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH188'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|NAD-dependent hydrolysis
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH188.html
|}
</div>


== Substrate specificities ==
The [[glycoside hydrolases]] of this family are found in bacteria, algae, plants and a small number of archaea <cite>#Kaur2024</cite>. The family contains enzymes with sulfoquinovosidase activity (EC 3.2.1.199), namely the ability to cleave glycosides of 6-deoxy-6-sulfoquinovose. Sulfoquinovosidases are also found in family [[GH31]] <cite>#Speciale2016</cite>. Enzymes of this family have the ability to cleave both α- and β-glycosides, and are dependent on an NAD+ cofactor. Sulfoquinovosidases hydrolyse glycosides of sulfoquinovose such as sulfoquinovosyl glycerol, and liberate free sulfoquinovose, which can be used as a substrate in bacterial sulfoglycolysis and sulfoquinovose sulfolysis pathways <cite>#Snow2021</cite>.

== Kinetics and Mechanism ==
GH188 enzymes utilize an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps. Exchange of the substrate C2 proton with solvent deuterium during the enzyme-catalyzed reaction was demonstrated by mass spectrometry <cite>#Kaur2024</cite>. The following chemical mechanism is proposed: (1) C3 hydride abstraction via the reduction of NAD+ cofactor to NADH and simultaneous oxidation of the C3 hydroxyl group; (2) α to the ketone functionality, the C2 proton is deprotonated by a general catalytic base residue; (3) cleavage of the C1-O1 bond occurs in an α,β-elimination, producing an α,β-unsaturated ketone [[intermediate]]; (4) 1,4-Michael-like addition of a water molecule at C1; and (5) reduction of the C3 carbonyl functionality by the enzyme-bound NADH generates the product. Similar mechanisms are used in families [[GH4]], [[GH109]], [[GH177]] and [[GH179]].

== Catalytic Residues ==
Catalytic residues can be inferred on the basis of X-ray crystallographic data for complexes of GH188 enzymes with NAD+ and sulfoquinovose <cite>#Kaur2024</cite>. Tyr136 in ''Arthrobacter'' sp. strain U41 SqgA is conserved in all family GH188 members and is located close to C2-OH, suggesting a possible role as a [[general base]]. His321 is located close to the C1-OH and may act as [[general acid]] (along with Tyr136) for the glycosidic oxygen facilitating glycosidic bond scission. The sulfonate group is recognized by a triad of amino acids: one oxygen H-bonds to Arg166 (2.6 Å), a second to Lys172 (2.9 Å), and a third to the backbone amide of Leu170 (2.8 Å).

== Three-dimensional structures ==
Crystallographic data is available for at least two GH188 enzymes, including as complexes with NADH, and NADH plus sulfoquinovose. The 3D X-ray crystal structures include those of ''Flavobacterium'' sp. strain K172 SqgA (PDB 8QC8, 8QC2) and ''Arthrobacter'' sp. strain U41 SqgA (PDB 8QC3, 8QC6. 8QC5) <cite>#Kaur2024</cite>. Each enzyme possesses an N-terminal dinucleotide-binding Rossman fold. The GH188 enzymes show structural similarities to inositol-2-dehydrogenase, glucose-fructose/IDH/MocA-like oxidoreductase, and NAD+-dependent ''N''-acetylgalactosaminidase of family [[GH109]].

== Family Firsts ==
;'''First stereochemistry determination
:not applicable
; '''First catalytic residue identification'''
:''Arthrobacter'' sp. strain U41 SqgA using 3D X-ray crystal structure <cite>#Kaur2024</cite>
; '''First 3-D structural determination'''
: ''Flavobacterium'' sp. strain K172 SqgA and ''Arthrobacter'' sp. strain U41 SqgA <cite>#Kaur2024</cite>
; '''First GH188 enzyme shown to hydrolyze both α- and β-substrates'''
:''Flavobacterium'' sp. strain K172 SqgA <cite>#Kaur2024</cite>

==References==
<biblio>
#Kaur2024 pmid=38100472
#Snow2021 pmid=34816844
#Speciale2016 pmid=26878550
</biblio>


[[Category:Glycoside Hydrolase Families|GH188]]

Glycoside hydrolases

2023-12-17T21:29:22Z

Spencer Williams: /* Alternative nucleophiles */

{{CuratorApproved}}
* Authors: [[User:Steve Withers|Steve Withers]], [[User:Spencer Williams|Spencer Williams]]
* Responsible Curator: [[User:Spencer Williams|Spencer Williams]]
----
== Overview ==
Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N- and S-linked glycosides.

[[Image:GHs.png|center|400px]]

== Classification ==
Glycoside hydrolases can be classified in many different ways. The following paragraphs list several different ways, the utility of which depends on the context in which the classification is made and used.

=== Endo/exo ===
''exo''- and ''endo''- refers to the ability of a glycoside hydrolase to cleave a substrate at the end (most frequently, but not always the non-reducing end) or within the middle of a chain <cite>DaviesHenrissat1995</cite>. For example, most cellulases are ''endo''-acting, whereas LacZ β-galactosidase from ''E. coli'' is ''exo''-acting. A general [[sub-site nomenclature]] exists to demarcate substrate binding in glycosidase active-sites.

[[Image:exo_endo.png|center|600px]]

=== Enzyme Commission (EC) number ===
EC numbers are codes representing the Enzyme Commission number. This is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. Every EC number is associated with a recommended name for the respective enzyme. EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number. A necessary consequence of the EC classification scheme is that codes can be applied only to enzymes for which a function has been biochemically identified. Additionally, certain enzymes can catalyze reactions that fall in more than one class. These enzymes must bear more than one EC number.

=== Mechanistic classification ===
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below.<cite>Gebler1992</cite> However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years, as discussed below.

[[Image:Retaining&inverting_GH.png|center|500px]]

=== Sequence-based classification ===
[[Sequence-based classification]] uses algorithmic methods to assign sequences to various families. The glycoside hydrolases have been classified into more than 100 families <cite>Henrissat1991</cite>; this is permanently available through the Carbohydrate Active enZyme database <cite>CAZyURL</cite>. Each family (GH family) contains proteins that are related by sequence, and by corollary, fold. This allows a number of useful predictions to be made since it has long been noted that the catalytic machinery and molecular mechanism is conserved for the vast majority of the glycosidase families <cite>Gebler1992</cite> as well as the geometry around the glycosidic bond (irrespective of naming conventions) <cite>Henrissat1995</cite>. Usually, the mechanism used (ie [[retaining]] or [[inverting]]) is conserved within a GH family. One notable exception is the glycoside hydrolases of family [[GH97]], which contains both retaining and inverting enzymes; a glutamate acts as a [[general base]] in inverting members, whereas an aspartate likely acts as a [[catalytic nucleophile]] in retaining members <cite>Gloster2008</cite>. Another mechanistic curiosity are the glycoside hydrolases of familes [[GH4]] and [[GH109]] which operate through an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s <cite>Yip2007</cite>. This allows a single enzyme to hydrolyze both α- and β-glycosides.

Classification of families into larger groups, termed '[[clans]]' has been proposed <cite>Henrissat1996</cite>. A '[[clan]]' is a group of families that possess significant similarity in their tertiary structure, catalytic residues and mechanism. Families within clans are thought to have a common evolutionary ancestry. For an updated table of glycoside hydrolase clans see the CAZy Database <cite>CAZyURL</cite>.

== Mechanism ==
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below <cite>Koshland1953</cite>. However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years.

===Inverting glycoside hydrolases===
Hydrolysis of a glycoside with net inversion of anomeric configuration is generally achieved via a one step, single-displacement mechanism involving [[oxocarbenium ion]]-like [[transition state]]s, as shown below. The reaction typically occurs with [[general acid]] and [[general base]] assistance from two amino acid side chains, normally glutamic or aspartic acids, that are typically located 6-11 A apart<cite>McCarter1994</cite>.

[[Image:Inverting glucosidase mechanism.png|centre|600px]]

====Glycosyl-phosphate cleaving enzymes that lack a general acid====
A subset of family [[GH92]] α-mannosidases catalyze the hydrolysis of mannose-1-phosphate linkages found in the mannose-1-phosphate-6-mannose groups of yeast mannoproteins. In these enzymes the usual [[general acid]] glutamic acid found in other members of this family is replaced by a glutamine. It has been suggested that the phosphate aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst <cite>Tiels2012</cite>. This replacement may also reduce charge repulsion between the glutamic acid residue and the anionic phosphate aglycon. A related example may be found in the case of the family [[GH1]] myrosinases.

===Retaining glycoside hydrolases===
====Classical Koshland retaining mechanism====
Hydrolysis with net retention of configuration is most commonly achieved via a two step, double-displacement mechanism involving a covalent glycosyl-enzyme [[intermediate]], as is shown in the figure below. Each step passes through an [[oxocarbenium ion]]-like [[transition state]]. Reaction occurs with acid/base and nucleophilic assistance provided by two amino acid side chains, typically glutamate or aspartate, located 5.5 A apart. In the first step (often called the glycosylation step), one residue plays the role of a nucleophile, attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme [[intermediate]]. At the same time the other residue functions as an acid catalyst and protonates the glycosidic oxygen as the bond cleaves. In the second step (known as the deglycosylation step), the glycosyl enzyme is hydrolyzed by water, with the other residue now acting as a base catalyst deprotonating the water molecule as it attacks. The pKa value of the acid/base group cycles between high and low values during catalysis to optimize it for its role at each step of catalysis <cite>McIntosh1996</cite>. In the case of sialidases, the catalytic nucleophile is a tyrosine residue (see below). This mechanism was originally proposed by Dan Koshland, although at the time the identities of the residues was unclear <cite>Koshland1953</cite>.

[[Image:Retaining_glycosidase_mechanism.png|centre|600px]]

====Neighboring group participation====
=====By a neighboring 2-acetamido group=====
Enzymes of [[GH18]], [[GH20]], [[GH25]], [[GH56]], [[GH84]], [[GH85]] and [[GH123]] hydrolyse substrates containing an ''N''-acetyl (acetamido) or ''N''-glycolyl group at the 2-position. These enzymes have no catalytic nucleophile: rather they utilize a mechanism in which the 2-acetamido group acts as an intramolecular nucleophile. Neighboring group participation by the 2-acetamido group leads to formation of an oxazoline (or more strictly an [[oxazolinium ion]]) [[intermediate]]. This mechanism was deduced from X-ray structures of complexes of chitinases with natural inhibitors <cite>Terwisscha1995</cite>, from the potent inhibition afforded by a stable thiazoline analogue of the oxazoline <cite>Knapp1996 Mark2001</cite>, and from detailed mechanistic analyses using substrates of modified reactivity <cite>Vocadlo2005</cite>. Typically, a stabilizing residue (a carboxylate) stabilizes the charge development in the [[transition state]]. Not all enzymes that cleave substrates possessing a 2-acetamido group utilize a neighboring groups participation mechanism; enzymes from [[GH3]] and [[GH22]] utilize a classical retaining mechanism with an enzymic nucleophile. Other hexosaminidases such as those of [[Glycoside Hydrolase Family 19]] utilize an inverting mechanism.

[[Image:Hex_neighboring_mechanism.png|centre|700px]]

=====By a neighboring 2-hydroxyl group=====
Enzymes of [[Glycoside Hydrolase Family 99]] hydrolyse α-mannoside substrates and lack a enzymic catalytic nucleophile. These enzymes use a mechanism in which the 2-hydroxyl group acts as an intramolecular nucleophile. Neighboring group participation by the 2-hydroxy group leads to formation of an epoxide (or more strictly a 1,2-anhydro sugar). This mechanism was implicated from 3D X-ray structures of complexes of bacterial endomannosidase/endomannanase with iminosugar inhibitors <cite>Thompson2012</cite>. Detailed mechanistic analyses using carbocyclic analogues of the 1,2-anhydro sugar, quantum mechanic/molecular mechanics computational modelling, and kinetic isotope effects <cite>Sobala2020</cite>.

====Myrosinases: Retaining glycoside hydrolases that lack a general acid and utilize an exogenous base====
Glycoside hydrolases termed myrosinases catalyze the hydrolysis of anionic thioglycosides (glucosinolates) found in plants. They are found in [[Glycoside Hydrolase Family 1]]. In these enzymes the usual acid/base glutamic acid found in other members of this family is replaced by a glutamine. This likely reduces charge repulsion between the anionic aglycon sulfate. The unusual aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst. However, since a base catalyst is required for the second step (hydrolysis or deglycosylation) these enzymes require an alternative basic group. This is provided by the co-enzyme L-ascorbate <cite>Burmeister2000</cite>.

[[Image:myrosinase.png|centre|550px]]

====Alternative nucleophiles====
Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of [[GH33]] and [[GH34]], and 2-keto-3-deoxy-D-lyxo-heptulosaric acid hydrolases of [[GH143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>. A cysteine nucleophile has been demonstrated for Zn2+-dependent arabinofuranosidases of families [[GH127]] and [[GH146]] through X-ray crystallography of the covalently labelled nucleophilic cysteine and MD and QM/MM simulations <cite>#McGregor2021</cite>.

[[Image:sialidase_mechanism.png|center|700px]]

====NAD-dependent hydrolysis====
The members of [[GH4]], [[GH109]], [[GH177]] and [[GH179]] use a mechanism that requires an NAD cofactor, which remains tightly bound throughout catalysis. The mechanism proceeds via anionic [[transition state]]s with elimination and redox steps rather than the classical mechanisms proceeding through[[oxocarbenium ion]]-like transition states. As shown below for a 6-phospho-β-glucosidase, the mechanism involves an initial oxidation of the 3-hydroxyl of the substrate by the enzyme-bound NAD cofactor. This increases the acidity of the C2 proton such that an E1cb elimination can occur with assistance from an enzymatic base. The α,β-unsaturated [[intermediate]] formed then undergoes addition of water at the anomeric centre and finally the ketone at C3 is reduced to generate the free sugar product. Thus, even though glycosidic bond cleavage occurred via an elimination mechanism, the overall reaction is hydrolysis. This mechanism was elucidated through a combination of stereochemical studies by NMR, kinetic isotope effects, linear free energy relationships, X-ray crystallography and UV/Vis spectrophotometry <cite>Yip2004 Rajan2004</cite>.

[[Image:Family_4_mechanism.png|center|700px]]

== References ==
<biblio>
#McCarter1994 pmid=7712292
#Koshland1953 Koshland, D. (1953) ''Biol. Rev.'' 28, 416.
#McIntosh1996 pmid=8756457
#Gebler1992 pmid=1618761
#Henrissat1995 pmid=7624375
#Terwisscha1995 pmid=7495789
#Mark2001 pmid=11124970
#Knapp1996 Knapp, S., Vocadlo, D., Gao, Z. N., Kirk, B., Lou, J. P., and Withers, S. G. (1996) ''Journal of the American Chemical Society 118'', 6804-6805.
#Vocadlo2005 pmid=16171396
#Burmeister2000 pmid=10978344
#Amaya2004 pmid=15130470
#Watts2003 pmid=12812490
#Watson2003 pmid=14580216
#Yip2004 pmid=15237973
#Rajan2004 pmid=15341727
#CAZyURL Carbohydrate Active Enzymes database; URL http://www.cazy.org/
#Henrissat1991 pmid=1747104
#Henrissat1996 pmid=8687420
#Gloster2008 pmid=18848471
#Yip2007 pmid=17676871
#Tiels2012 pmid=23159880
#DaviesHenrissat1995 pmid=8535779
#Ndeh2017 pmid=28329766
#Sobala2020 pmid=32490192
#McGregor2021 pmid=33528085

</biblio>
[[Category:Definitions and explanations]]

Glycoside hydrolases

2023-12-17T21:28:59Z

Spencer Williams: /* References */

{{CuratorApproved}}
* Authors: [[User:Steve Withers|Steve Withers]], [[User:Spencer Williams|Spencer Williams]]
* Responsible Curator: [[User:Spencer Williams|Spencer Williams]]
----
== Overview ==
Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N- and S-linked glycosides.

[[Image:GHs.png|center|400px]]

== Classification ==
Glycoside hydrolases can be classified in many different ways. The following paragraphs list several different ways, the utility of which depends on the context in which the classification is made and used.

=== Endo/exo ===
''exo''- and ''endo''- refers to the ability of a glycoside hydrolase to cleave a substrate at the end (most frequently, but not always the non-reducing end) or within the middle of a chain <cite>DaviesHenrissat1995</cite>. For example, most cellulases are ''endo''-acting, whereas LacZ β-galactosidase from ''E. coli'' is ''exo''-acting. A general [[sub-site nomenclature]] exists to demarcate substrate binding in glycosidase active-sites.

[[Image:exo_endo.png|center|600px]]

=== Enzyme Commission (EC) number ===
EC numbers are codes representing the Enzyme Commission number. This is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. Every EC number is associated with a recommended name for the respective enzyme. EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number. A necessary consequence of the EC classification scheme is that codes can be applied only to enzymes for which a function has been biochemically identified. Additionally, certain enzymes can catalyze reactions that fall in more than one class. These enzymes must bear more than one EC number.

=== Mechanistic classification ===
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below.<cite>Gebler1992</cite> However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years, as discussed below.

[[Image:Retaining&inverting_GH.png|center|500px]]

=== Sequence-based classification ===
[[Sequence-based classification]] uses algorithmic methods to assign sequences to various families. The glycoside hydrolases have been classified into more than 100 families <cite>Henrissat1991</cite>; this is permanently available through the Carbohydrate Active enZyme database <cite>CAZyURL</cite>. Each family (GH family) contains proteins that are related by sequence, and by corollary, fold. This allows a number of useful predictions to be made since it has long been noted that the catalytic machinery and molecular mechanism is conserved for the vast majority of the glycosidase families <cite>Gebler1992</cite> as well as the geometry around the glycosidic bond (irrespective of naming conventions) <cite>Henrissat1995</cite>. Usually, the mechanism used (ie [[retaining]] or [[inverting]]) is conserved within a GH family. One notable exception is the glycoside hydrolases of family [[GH97]], which contains both retaining and inverting enzymes; a glutamate acts as a [[general base]] in inverting members, whereas an aspartate likely acts as a [[catalytic nucleophile]] in retaining members <cite>Gloster2008</cite>. Another mechanistic curiosity are the glycoside hydrolases of familes [[GH4]] and [[GH109]] which operate through an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s <cite>Yip2007</cite>. This allows a single enzyme to hydrolyze both α- and β-glycosides.

Classification of families into larger groups, termed '[[clans]]' has been proposed <cite>Henrissat1996</cite>. A '[[clan]]' is a group of families that possess significant similarity in their tertiary structure, catalytic residues and mechanism. Families within clans are thought to have a common evolutionary ancestry. For an updated table of glycoside hydrolase clans see the CAZy Database <cite>CAZyURL</cite>.

== Mechanism ==
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below <cite>Koshland1953</cite>. However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years.

===Inverting glycoside hydrolases===
Hydrolysis of a glycoside with net inversion of anomeric configuration is generally achieved via a one step, single-displacement mechanism involving [[oxocarbenium ion]]-like [[transition state]]s, as shown below. The reaction typically occurs with [[general acid]] and [[general base]] assistance from two amino acid side chains, normally glutamic or aspartic acids, that are typically located 6-11 A apart<cite>McCarter1994</cite>.

[[Image:Inverting glucosidase mechanism.png|centre|600px]]

====Glycosyl-phosphate cleaving enzymes that lack a general acid====
A subset of family [[GH92]] α-mannosidases catalyze the hydrolysis of mannose-1-phosphate linkages found in the mannose-1-phosphate-6-mannose groups of yeast mannoproteins. In these enzymes the usual [[general acid]] glutamic acid found in other members of this family is replaced by a glutamine. It has been suggested that the phosphate aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst <cite>Tiels2012</cite>. This replacement may also reduce charge repulsion between the glutamic acid residue and the anionic phosphate aglycon. A related example may be found in the case of the family [[GH1]] myrosinases.

===Retaining glycoside hydrolases===
====Classical Koshland retaining mechanism====
Hydrolysis with net retention of configuration is most commonly achieved via a two step, double-displacement mechanism involving a covalent glycosyl-enzyme [[intermediate]], as is shown in the figure below. Each step passes through an [[oxocarbenium ion]]-like [[transition state]]. Reaction occurs with acid/base and nucleophilic assistance provided by two amino acid side chains, typically glutamate or aspartate, located 5.5 A apart. In the first step (often called the glycosylation step), one residue plays the role of a nucleophile, attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme [[intermediate]]. At the same time the other residue functions as an acid catalyst and protonates the glycosidic oxygen as the bond cleaves. In the second step (known as the deglycosylation step), the glycosyl enzyme is hydrolyzed by water, with the other residue now acting as a base catalyst deprotonating the water molecule as it attacks. The pKa value of the acid/base group cycles between high and low values during catalysis to optimize it for its role at each step of catalysis <cite>McIntosh1996</cite>. In the case of sialidases, the catalytic nucleophile is a tyrosine residue (see below). This mechanism was originally proposed by Dan Koshland, although at the time the identities of the residues was unclear <cite>Koshland1953</cite>.

[[Image:Retaining_glycosidase_mechanism.png|centre|600px]]

====Neighboring group participation====
=====By a neighboring 2-acetamido group=====
Enzymes of [[GH18]], [[GH20]], [[GH25]], [[GH56]], [[GH84]], [[GH85]] and [[GH123]] hydrolyse substrates containing an ''N''-acetyl (acetamido) or ''N''-glycolyl group at the 2-position. These enzymes have no catalytic nucleophile: rather they utilize a mechanism in which the 2-acetamido group acts as an intramolecular nucleophile. Neighboring group participation by the 2-acetamido group leads to formation of an oxazoline (or more strictly an [[oxazolinium ion]]) [[intermediate]]. This mechanism was deduced from X-ray structures of complexes of chitinases with natural inhibitors <cite>Terwisscha1995</cite>, from the potent inhibition afforded by a stable thiazoline analogue of the oxazoline <cite>Knapp1996 Mark2001</cite>, and from detailed mechanistic analyses using substrates of modified reactivity <cite>Vocadlo2005</cite>. Typically, a stabilizing residue (a carboxylate) stabilizes the charge development in the [[transition state]]. Not all enzymes that cleave substrates possessing a 2-acetamido group utilize a neighboring groups participation mechanism; enzymes from [[GH3]] and [[GH22]] utilize a classical retaining mechanism with an enzymic nucleophile. Other hexosaminidases such as those of [[Glycoside Hydrolase Family 19]] utilize an inverting mechanism.

[[Image:Hex_neighboring_mechanism.png|centre|700px]]

=====By a neighboring 2-hydroxyl group=====
Enzymes of [[Glycoside Hydrolase Family 99]] hydrolyse α-mannoside substrates and lack a enzymic catalytic nucleophile. These enzymes use a mechanism in which the 2-hydroxyl group acts as an intramolecular nucleophile. Neighboring group participation by the 2-hydroxy group leads to formation of an epoxide (or more strictly a 1,2-anhydro sugar). This mechanism was implicated from 3D X-ray structures of complexes of bacterial endomannosidase/endomannanase with iminosugar inhibitors <cite>Thompson2012</cite>. Detailed mechanistic analyses using carbocyclic analogues of the 1,2-anhydro sugar, quantum mechanic/molecular mechanics computational modelling, and kinetic isotope effects <cite>Sobala2020</cite>.

====Myrosinases: Retaining glycoside hydrolases that lack a general acid and utilize an exogenous base====
Glycoside hydrolases termed myrosinases catalyze the hydrolysis of anionic thioglycosides (glucosinolates) found in plants. They are found in [[Glycoside Hydrolase Family 1]]. In these enzymes the usual acid/base glutamic acid found in other members of this family is replaced by a glutamine. This likely reduces charge repulsion between the anionic aglycon sulfate. The unusual aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst. However, since a base catalyst is required for the second step (hydrolysis or deglycosylation) these enzymes require an alternative basic group. This is provided by the co-enzyme L-ascorbate <cite>Burmeister2000</cite>.

[[Image:myrosinase.png|centre|550px]]

====Alternative nucleophiles====
Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of [[GH33]] and [[GH34]], and 2-keto-3-deoxy-D-lyxo-heptulosaric acid hydrolases of [[GH143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>. A cysteine nucleophile has been demonstrated for Zn2+,/sup>-dependent arabinofuranosidases of families [[GH127]] and [[GH146]] through X-ray crystallography of the covalently labelled nucleophilic cysteine and MD and QM/MM simulations <cite>#McGregor2021</cite>.

[[Image:sialidase_mechanism.png|center|700px]]

====NAD-dependent hydrolysis====
The members of [[GH4]], [[GH109]], [[GH177]] and [[GH179]] use a mechanism that requires an NAD cofactor, which remains tightly bound throughout catalysis. The mechanism proceeds via anionic [[transition state]]s with elimination and redox steps rather than the classical mechanisms proceeding through[[oxocarbenium ion]]-like transition states. As shown below for a 6-phospho-β-glucosidase, the mechanism involves an initial oxidation of the 3-hydroxyl of the substrate by the enzyme-bound NAD cofactor. This increases the acidity of the C2 proton such that an E1cb elimination can occur with assistance from an enzymatic base. The α,β-unsaturated [[intermediate]] formed then undergoes addition of water at the anomeric centre and finally the ketone at C3 is reduced to generate the free sugar product. Thus, even though glycosidic bond cleavage occurred via an elimination mechanism, the overall reaction is hydrolysis. This mechanism was elucidated through a combination of stereochemical studies by NMR, kinetic isotope effects, linear free energy relationships, X-ray crystallography and UV/Vis spectrophotometry <cite>Yip2004 Rajan2004</cite>.

[[Image:Family_4_mechanism.png|center|700px]]

== References ==
<biblio>
#McCarter1994 pmid=7712292
#Koshland1953 Koshland, D. (1953) ''Biol. Rev.'' 28, 416.
#McIntosh1996 pmid=8756457
#Gebler1992 pmid=1618761
#Henrissat1995 pmid=7624375
#Terwisscha1995 pmid=7495789
#Mark2001 pmid=11124970
#Knapp1996 Knapp, S., Vocadlo, D., Gao, Z. N., Kirk, B., Lou, J. P., and Withers, S. G. (1996) ''Journal of the American Chemical Society 118'', 6804-6805.
#Vocadlo2005 pmid=16171396
#Burmeister2000 pmid=10978344
#Amaya2004 pmid=15130470
#Watts2003 pmid=12812490
#Watson2003 pmid=14580216
#Yip2004 pmid=15237973
#Rajan2004 pmid=15341727
#CAZyURL Carbohydrate Active Enzymes database; URL http://www.cazy.org/
#Henrissat1991 pmid=1747104
#Henrissat1996 pmid=8687420
#Gloster2008 pmid=18848471
#Yip2007 pmid=17676871
#Tiels2012 pmid=23159880
#DaviesHenrissat1995 pmid=8535779
#Ndeh2017 pmid=28329766
#Sobala2020 pmid=32490192
#McGregor2021 pmid=33528085

</biblio>
[[Category:Definitions and explanations]]

Glycoside hydrolases

2023-12-17T21:28:23Z

Spencer Williams: /* Alternative nucleophiles */

{{CuratorApproved}}
* Authors: [[User:Steve Withers|Steve Withers]], [[User:Spencer Williams|Spencer Williams]]
* Responsible Curator: [[User:Spencer Williams|Spencer Williams]]
----
== Overview ==
Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N- and S-linked glycosides.

[[Image:GHs.png|center|400px]]

== Classification ==
Glycoside hydrolases can be classified in many different ways. The following paragraphs list several different ways, the utility of which depends on the context in which the classification is made and used.

=== Endo/exo ===
''exo''- and ''endo''- refers to the ability of a glycoside hydrolase to cleave a substrate at the end (most frequently, but not always the non-reducing end) or within the middle of a chain <cite>DaviesHenrissat1995</cite>. For example, most cellulases are ''endo''-acting, whereas LacZ β-galactosidase from ''E. coli'' is ''exo''-acting. A general [[sub-site nomenclature]] exists to demarcate substrate binding in glycosidase active-sites.

[[Image:exo_endo.png|center|600px]]

=== Enzyme Commission (EC) number ===
EC numbers are codes representing the Enzyme Commission number. This is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. Every EC number is associated with a recommended name for the respective enzyme. EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number. A necessary consequence of the EC classification scheme is that codes can be applied only to enzymes for which a function has been biochemically identified. Additionally, certain enzymes can catalyze reactions that fall in more than one class. These enzymes must bear more than one EC number.

=== Mechanistic classification ===
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below.<cite>Gebler1992</cite> However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years, as discussed below.

[[Image:Retaining&inverting_GH.png|center|500px]]

=== Sequence-based classification ===
[[Sequence-based classification]] uses algorithmic methods to assign sequences to various families. The glycoside hydrolases have been classified into more than 100 families <cite>Henrissat1991</cite>; this is permanently available through the Carbohydrate Active enZyme database <cite>CAZyURL</cite>. Each family (GH family) contains proteins that are related by sequence, and by corollary, fold. This allows a number of useful predictions to be made since it has long been noted that the catalytic machinery and molecular mechanism is conserved for the vast majority of the glycosidase families <cite>Gebler1992</cite> as well as the geometry around the glycosidic bond (irrespective of naming conventions) <cite>Henrissat1995</cite>. Usually, the mechanism used (ie [[retaining]] or [[inverting]]) is conserved within a GH family. One notable exception is the glycoside hydrolases of family [[GH97]], which contains both retaining and inverting enzymes; a glutamate acts as a [[general base]] in inverting members, whereas an aspartate likely acts as a [[catalytic nucleophile]] in retaining members <cite>Gloster2008</cite>. Another mechanistic curiosity are the glycoside hydrolases of familes [[GH4]] and [[GH109]] which operate through an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s <cite>Yip2007</cite>. This allows a single enzyme to hydrolyze both α- and β-glycosides.

Classification of families into larger groups, termed '[[clans]]' has been proposed <cite>Henrissat1996</cite>. A '[[clan]]' is a group of families that possess significant similarity in their tertiary structure, catalytic residues and mechanism. Families within clans are thought to have a common evolutionary ancestry. For an updated table of glycoside hydrolase clans see the CAZy Database <cite>CAZyURL</cite>.

== Mechanism ==
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below <cite>Koshland1953</cite>. However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years.

===Inverting glycoside hydrolases===
Hydrolysis of a glycoside with net inversion of anomeric configuration is generally achieved via a one step, single-displacement mechanism involving [[oxocarbenium ion]]-like [[transition state]]s, as shown below. The reaction typically occurs with [[general acid]] and [[general base]] assistance from two amino acid side chains, normally glutamic or aspartic acids, that are typically located 6-11 A apart<cite>McCarter1994</cite>.

[[Image:Inverting glucosidase mechanism.png|centre|600px]]

====Glycosyl-phosphate cleaving enzymes that lack a general acid====
A subset of family [[GH92]] α-mannosidases catalyze the hydrolysis of mannose-1-phosphate linkages found in the mannose-1-phosphate-6-mannose groups of yeast mannoproteins. In these enzymes the usual [[general acid]] glutamic acid found in other members of this family is replaced by a glutamine. It has been suggested that the phosphate aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst <cite>Tiels2012</cite>. This replacement may also reduce charge repulsion between the glutamic acid residue and the anionic phosphate aglycon. A related example may be found in the case of the family [[GH1]] myrosinases.

===Retaining glycoside hydrolases===
====Classical Koshland retaining mechanism====
Hydrolysis with net retention of configuration is most commonly achieved via a two step, double-displacement mechanism involving a covalent glycosyl-enzyme [[intermediate]], as is shown in the figure below. Each step passes through an [[oxocarbenium ion]]-like [[transition state]]. Reaction occurs with acid/base and nucleophilic assistance provided by two amino acid side chains, typically glutamate or aspartate, located 5.5 A apart. In the first step (often called the glycosylation step), one residue plays the role of a nucleophile, attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme [[intermediate]]. At the same time the other residue functions as an acid catalyst and protonates the glycosidic oxygen as the bond cleaves. In the second step (known as the deglycosylation step), the glycosyl enzyme is hydrolyzed by water, with the other residue now acting as a base catalyst deprotonating the water molecule as it attacks. The pKa value of the acid/base group cycles between high and low values during catalysis to optimize it for its role at each step of catalysis <cite>McIntosh1996</cite>. In the case of sialidases, the catalytic nucleophile is a tyrosine residue (see below). This mechanism was originally proposed by Dan Koshland, although at the time the identities of the residues was unclear <cite>Koshland1953</cite>.

[[Image:Retaining_glycosidase_mechanism.png|centre|600px]]

====Neighboring group participation====
=====By a neighboring 2-acetamido group=====
Enzymes of [[GH18]], [[GH20]], [[GH25]], [[GH56]], [[GH84]], [[GH85]] and [[GH123]] hydrolyse substrates containing an ''N''-acetyl (acetamido) or ''N''-glycolyl group at the 2-position. These enzymes have no catalytic nucleophile: rather they utilize a mechanism in which the 2-acetamido group acts as an intramolecular nucleophile. Neighboring group participation by the 2-acetamido group leads to formation of an oxazoline (or more strictly an [[oxazolinium ion]]) [[intermediate]]. This mechanism was deduced from X-ray structures of complexes of chitinases with natural inhibitors <cite>Terwisscha1995</cite>, from the potent inhibition afforded by a stable thiazoline analogue of the oxazoline <cite>Knapp1996 Mark2001</cite>, and from detailed mechanistic analyses using substrates of modified reactivity <cite>Vocadlo2005</cite>. Typically, a stabilizing residue (a carboxylate) stabilizes the charge development in the [[transition state]]. Not all enzymes that cleave substrates possessing a 2-acetamido group utilize a neighboring groups participation mechanism; enzymes from [[GH3]] and [[GH22]] utilize a classical retaining mechanism with an enzymic nucleophile. Other hexosaminidases such as those of [[Glycoside Hydrolase Family 19]] utilize an inverting mechanism.

[[Image:Hex_neighboring_mechanism.png|centre|700px]]

=====By a neighboring 2-hydroxyl group=====
Enzymes of [[Glycoside Hydrolase Family 99]] hydrolyse α-mannoside substrates and lack a enzymic catalytic nucleophile. These enzymes use a mechanism in which the 2-hydroxyl group acts as an intramolecular nucleophile. Neighboring group participation by the 2-hydroxy group leads to formation of an epoxide (or more strictly a 1,2-anhydro sugar). This mechanism was implicated from 3D X-ray structures of complexes of bacterial endomannosidase/endomannanase with iminosugar inhibitors <cite>Thompson2012</cite>. Detailed mechanistic analyses using carbocyclic analogues of the 1,2-anhydro sugar, quantum mechanic/molecular mechanics computational modelling, and kinetic isotope effects <cite>Sobala2020</cite>.

====Myrosinases: Retaining glycoside hydrolases that lack a general acid and utilize an exogenous base====
Glycoside hydrolases termed myrosinases catalyze the hydrolysis of anionic thioglycosides (glucosinolates) found in plants. They are found in [[Glycoside Hydrolase Family 1]]. In these enzymes the usual acid/base glutamic acid found in other members of this family is replaced by a glutamine. This likely reduces charge repulsion between the anionic aglycon sulfate. The unusual aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst. However, since a base catalyst is required for the second step (hydrolysis or deglycosylation) these enzymes require an alternative basic group. This is provided by the co-enzyme L-ascorbate <cite>Burmeister2000</cite>.

[[Image:myrosinase.png|centre|550px]]

====Alternative nucleophiles====
Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of [[GH33]] and [[GH34]], and 2-keto-3-deoxy-D-lyxo-heptulosaric acid hydrolases of [[GH143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>. A cysteine nucleophile has been demonstrated for Zn2+,/sup>-dependent arabinofuranosidases of families [[GH127]] and [[GH146]] through X-ray crystallography of the covalently labelled nucleophilic cysteine and MD and QM/MM simulations <cite>#McGregor2021</cite>.

[[Image:sialidase_mechanism.png|center|700px]]

====NAD-dependent hydrolysis====
The members of [[GH4]], [[GH109]], [[GH177]] and [[GH179]] use a mechanism that requires an NAD cofactor, which remains tightly bound throughout catalysis. The mechanism proceeds via anionic [[transition state]]s with elimination and redox steps rather than the classical mechanisms proceeding through[[oxocarbenium ion]]-like transition states. As shown below for a 6-phospho-β-glucosidase, the mechanism involves an initial oxidation of the 3-hydroxyl of the substrate by the enzyme-bound NAD cofactor. This increases the acidity of the C2 proton such that an E1cb elimination can occur with assistance from an enzymatic base. The α,β-unsaturated [[intermediate]] formed then undergoes addition of water at the anomeric centre and finally the ketone at C3 is reduced to generate the free sugar product. Thus, even though glycosidic bond cleavage occurred via an elimination mechanism, the overall reaction is hydrolysis. This mechanism was elucidated through a combination of stereochemical studies by NMR, kinetic isotope effects, linear free energy relationships, X-ray crystallography and UV/Vis spectrophotometry <cite>Yip2004 Rajan2004</cite>.

[[Image:Family_4_mechanism.png|center|700px]]

== References ==
<biblio>
#McCarter1994 pmid=7712292
#Koshland1953 Koshland, D. (1953) ''Biol. Rev.'' 28, 416.
#McIntosh1996 pmid=8756457
#Gebler1992 pmid=1618761
#Henrissat1995 pmid=7624375
#Terwisscha1995 pmid=7495789
#Mark2001 pmid=11124970
#Knapp1996 Knapp, S., Vocadlo, D., Gao, Z. N., Kirk, B., Lou, J. P., and Withers, S. G. (1996) ''Journal of the American Chemical Society 118'', 6804-6805.
#Vocadlo2005 pmid=16171396
#Burmeister2000 pmid=10978344
#Amaya2004 pmid=15130470
#Watts2003 pmid=12812490
#Watson2003 pmid=14580216
#Yip2004 pmid=15237973
#Rajan2004 pmid=15341727
#CAZyURL Carbohydrate Active Enzymes database; URL http://www.cazy.org/
#Henrissat1991 pmid=1747104
#Henrissat1996 pmid=8687420
#Gloster2008 pmid=18848471
#Yip2007 pmid=17676871
#Tiels2012 pmid=23159880
#DaviesHenrissat1995 pmid=8535779
#Ndeh2017 pmid=28329766
#Sobala2020 pmid=32490192
#Thompson2012 pmid=22219371

</biblio>
[[Category:Definitions and explanations]]

Glycoside Hydrolase Families

2023-12-17T21:23:34Z

Spencer Williams: updated list of NAD-dependent hydrolysis families

''This page lists all the Glycoside Hydrolase (GH) Family pages in ''CAZypedia'' that have been given [[:Category:Curator approved|Curator Approved]] status, as well as those that are currently [[:Category:Under construction|under construction]], [[:Category:Unassigned pages|unassigned]] (''i.e.'' lacking a [[Responsible Curator]] and [[Author]]), or deleted.''

== Overview ==
The term [[glycoside hydrolase|glycoside hydrolase (GH)]] (''alternatively, [[glycoside hydrolase|glycosidase]]'') formally refers to enzymes that catalyze the hydrolytic cleavage of the glycosidic bond to give the carbohydrate hemiacetal. Detailed explanations of the distinct catalytic mechanisms employed by these enzymes can be found on the [[glycoside hydrolase]] lexicon page. Since the seminal [[sequence-based classification]] of GHs into families, it has subsequently been observed that some of these families also group non-hydrolytic enzymes and proteins, due to sequence and structural similarity <cite>Henrissat1989 Henrissat1991 Henrissat1993 Henrissat1996 Henrissat1997 DaviesSinnott2008 Davies1995 VocadloDavies2008 YipWithers2006</cite>. In many cases, these alternative activities bear some degree of mechanistic similarity (''e.g.'', conserved catalytic residues or enzyme intermediates) to the eponymous enzymes:

* [[Transglycosylases]] are mechanistically related to [[retaining]] [[glycoside hydrolases]], with the exception that a sugar (or another nucleophile), rather than water, acts as the acceptor substrate to yield glycosidic bond exchange.

* [[Phosphorylases]] cleave glycosidic bonds using phosphate as a nucleophile to yield sugar-1-phosphates; this reaction is readily reversible, allowing the synthesis of glycosidic linkages. Sequence classifies many, but not all (see [[glycosyltransferases]] for exceptions) [[phosphorylases]] with [[retaining]] or [[inverting]] [[glycoside hydrolases]].

* [[Alpha-glucan lyases]] are found within family [[GH31]] and degrade α-(1-4)-linked glucans (''e.g.'' starch) and oligosaccharides via an elimination mechanism that yields an enol (unsaturated) product that tautomerises to its keto form, 1,5-anhydro fructose.

* [[NAD-dependent hydrolysis|NAD-dependent glycoside hydrolases]] of families [[GH4]], [[GH109]], [[GH177]], [[GH179]] and [[GH188]] use nicotinamide adenine dinucleotide as redox cofactor to activate the sugar ring for glycosidic bond cleavage by elimination.

== Curator Approved ==
<blockquote class="toccolours" style="float:none; padding: 10px 15px 10px 15px; display:table;">
[[Image:Approve_icon-50px.png|left]] These pages have been approved by the [[Responsible Curator]] as essentially complete. ''CAZypedia'' is a living document, so further improvement of these pages is still possible; please see the individual pages for more information.
</blockquote>
{{#dpl:
|category=Glycoside Hydrolase Families
|category=Curator approved
|mode=userformat
|resultsheader=\nThere are currently %PAGES% [[:Category:Curator approved|Curator approved]] Glycoside Hydrolase (GH) Family pages in ''CAZypedia''.\n
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== Under construction ==
<blockquote class="toccolours" style="float:none; padding: 10px 15px 10px 15px; display:table;">
[[Image:Under_construction_icon-blue-48px.png|left]]These pages are currently [[:Category:Under construction|under construction]] in ''CAZypedia''. As such, the [[Responsible Curator]] has deemed that the page's content is not quite up to ''CAZypedia's'' standards for full public consumption. All information on these pages should therefore be considered to be under revision and may be subject to major changes.
</blockquote>
{{#dpl:
|category=Glycoside Hydrolase Families
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|mode=userformat
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== Unassigned pages ==
<blockquote class="toccolours" style="float:none; padding: 10px 15px 10px 15px; display:table;">
[[File:Blank user-200px.png|left|40px]]The following [[:Category:Unassigned pages|Unassigned pages]] are currently lacking a [[Responsible Curator]] and one or more [[Author]]s. If you are an expert on any of these families and would like to help us improve ''CAZypedia'' by getting involved with the production and maintenance of the corresponding page(s), please contact a member of the [[Board of Curators]]. ''Undergraduate students, (post)graduate students, post-doctoral researchers, research associates, and professors are all welcomed to contribute!''

</blockquote>
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== Deleted families ==
<blockquote class="toccolours" style="float:none; padding: 10px 15px 10px 15px; display:table;">
[[File:Nuvola_apps_important.png|left|40px]]The following families have been deleted from the CAZy database. Please see the individual ''CAZypedia'' pages and links to the corresponding CAZy DB pages for specific explanations.
</blockquote>
{{#dpl:
|category=Glycoside Hydrolase Families
|category=Deleted families
|mode=userformat
|resultsheader=\nThere are currently %PAGES% pages in ''CAZypedia'' that describe Glycoside Hydrolase families deleted from the CAZy DB.\n
|noresultsheader=\nThere are currently no pages in this category.\n
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<div style="-webkit-column-count:10;column-count:10;">{{#dpl:
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</div>

== References ==
<biblio>
#Henrissat1989 pmid=2806912
#Henrissat1991 pmid=1747104

#Henrissat1993 pmid=8352747
#Henrissat1996 pmid=8687420
#Davies1995 pmid=8535779
#Henrissat1997 pmid=9345621
#DaviesSinnott2008 Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. ''The Biochemist'', vol. 30, no. 4., pp. 26-32. [https://doi.org/10.1042/BIO03004026 DOI:10.1042/BIO03004026].
#VocadloDavies2008 pmid=18558099
#YipWithers2006 pmid=16495121
</biblio>

Glycoside Hydrolase Family 188

2023-12-17T21:22:22Z

Spencer Williams:

{{UnderConstruction}}
* [[Author]]:
* [[Responsible Curator]]: [[User:Spencer Williams|Spencer Williams]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH188'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|NAD-dependent hydrolysis
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH188.html
|}
</div>


== Substrate specificities ==
The [[glycoside hydrolases]] of this family are found in bacteria, algae, plants and a small number of archaea <cite>#Kaur2024</cite>. The family contains enzymes with sulfoquinovosidase activity (EC 3.2.1.199), namely the ability to cleave glycosides of 6-deoxy-6-sulfoquinovose. Sulfoquinovosidases are also found in family [[GH31]] <cite>#Speciale2016</cite>. Enzymes of this family have the ability to cleave both α- and β-glycosides, and are dependent on an NAD+ cofactor.

== Kinetics and Mechanism ==
GH188 enzymes utilize an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps. Exchange of the substrate C2 proton with solvent deuterium during the enzyme-catalyzed reaction was demonstrated by mass spectrometry <cite>#Kaur2024</cite>. The following chemical mechanism is proposed: (1) C3 hydride abstraction via the reduction of NAD+ cofactor to NADH and simultaneous oxidation of the C3 hydroxyl group; (2) α to the ketone functionality, the C2 proton is deprotonated by a general catalytic base residue; (3) cleavage of the C1-O1 bond occurs in an α,β-elimination, producing an α,β-unsaturated ketone [[intermediate]]; (4) 1,4-Michael-like addition of a water molecule at C1; and (5) reduction of the C3 carbonyl functionality by the enzyme-bound NADH generates the product. Similar mechanisms are used in families [[GH4]], [[GH109]], [[GH177]] and [[GH179]].

== Catalytic Residues ==
Catalytic residues can be inferred on the basis of X-ray crystallographic data for complexes of GH188 enzymes with NAD+ and sulfoquinovose <cite>#Kaur2024</cite>. Tyr136 in ''Arthrobacter'' sp. strain U41 SqgA is conserved in all family GH188 members and is located close to C2-OH, suggesting a possible role as a [[general base]]. His321 is located close to the C1-OH and may act as [[general acid]] (along with Tyr136) for the glycosidic oxygen facilitating glycosidic bond scission. The sulfonate group is recognized by a triad of amino acids: one oxygen H-bonds to Arg166 (2.6 Å), a second to Lys172 (2.9 Å), and a third to the backbone amide of Leu170 (2.8 Å).

== Three-dimensional structures ==
Crystallographic data is available for at least two GH188 enzymes, including as complexes with NADH, and NADH plus sulfoquinovose. The 3D X-ray crystal structures include those of ''Flavobacterium'' sp. strain K172 SqgA (PDB 8QC8, 8QC2) and ''Arthrobacter'' sp. strain U41 SqgA (PDB 8QC3, 8QC6. 8QC5) <cite>#Kaur2024</cite>. Each enzyme possesses an N-terminal dinucleotide-binding Rossman fold. The GH188 enzymes show structural similarities to inositol-2-dehydrogenase, glucose-fructose/IDH/MocA-like oxidoreductase, and NAD+-dependent ''N''-acetylgalactosaminidase of family [[GH109]].

== Family Firsts ==
;'''First stereochemistry determination
:not applicable
; '''First catalytic residue identification'''
:''Arthrobacter'' sp. strain U41 SqgA using 3D X-ray crystal structure <cite>#Kaur2024</cite>
; '''First 3-D structural determination'''
: ''Flavobacterium'' sp. strain K172 SqgA and ''Arthrobacter'' sp. strain U41 SqgA <cite>#Kaur2024</cite>
; '''First GH188 enzyme shown to hydrolyze both α- and β-substrates'''
:''Flavobacterium'' sp. strain K172 SqgA <cite>#Kaur2024</cite>

==References==
<biblio>
#Kaur2024 pmid=38100472

#Speciale2016 pmid=26878550
</biblio>


[[Category:Glycoside Hydrolase Families|GH188]]

Glycoside Hydrolase Family 188

2023-12-17T21:18:30Z

Spencer Williams:

{{UnderConstruction}}
* [[Author]]:
* [[Responsible Curator]]: [[User:Spencer Williams|Spencer Williams]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH188'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|NAD-dependent hydrolysis
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH188.html
|}
</div>


== Substrate specificities ==
The [[glycoside hydrolases]] of this family are found in bacteria, algae, plants and a small number of archaea <cite>#Kaur2024</cite>. The family contains enzymes with sulfoquinovosidase activity (EC 3.2.1.199), namely the ability to cleave glycosides of 6-deoxy-6-sulfoquinovose. Sulfoquinovosidases are also found in family [[GH31]] <cite>#Speciale2016</cite>. Enzymes of this family have the ability to cleave both α- and β-glycosides, and are dependent on an NAD+ cofactor.

== Kinetics and Mechanism ==
GH188 enzymes utilize an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps. Exchange of the substrate C2 proton with solvent deuterium during the enzyme-catalyzed reaction was demonstrated by mass spectrometry <cite>#Kaur2024</cite>. The following chemical mechanism is proposed: (1) C3 hydride abstraction via the reduction of NAD+ cofactor to NADH and simultaneous oxidation of the C3 hydroxyl group; (2) α to the ketone functionality, the C2 proton is deprotonated by a general catalytic base residue; (3) cleavage of the C1-O1 bond occurs in an α,β-elimination, producing an α,β-unsaturated ketone [[intermediate]]; (4) 1,4-Michael-like addition of a water molecule at C1; and (5) reduction of the C3 carbonyl functionality by the enzyme-bound NADH generates the product.

== Catalytic Residues ==
Catalytic residues can be inferred on the basis of X-ray crystallographic data for complexes of GH188 enzymes with NAD+ and sulfoquinovose <cite>#Kaur2024</cite>. Tyr136 in ''Arthrobacter'' sp. strain U41 SqgA is conserved in all family GH188 members and is located close to C2-OH, suggesting a possible role as a [[general base]]. His321 is located close to the C1-OH and may act as [[general acid]] (along with Tyr136) for the glycosidic oxygen facilitating glycosidic bond scission. The sulfonate group is recognized by a triad of amino acids: one oxygen H-bonds to Arg166 (2.6 Å), a second to Lys172 (2.9 Å), and a third to the backbone amide of Leu170 (2.8 Å).

== Three-dimensional structures ==
Crystallographic data is available for at least two GH188 enzymes, including as complexes with NADH, and NADH plus sulfoquinovose. The 3D X-ray crystal structures include those of ''Flavobacterium'' sp. strain K172 SqgA (PDB 8QC8, 8QC2) and ''Arthrobacter'' sp. strain U41 SqgA (PDB 8QC3, 8QC6. 8QC5) <cite>#Kaur2024</cite>. Each enzyme possesses an N-terminal dinucleotide-binding Rossman fold. The GH188 enzymes show structural similarities to inositol-2-dehydrogenase, glucose-fructose/IDH/MocA-like oxidoreductase, and NAD+-dependent ''N''-acetylgalactosaminidase of family [[GH109]].

== Family Firsts ==
;'''First stereochemistry determination
:not applicable
; '''First catalytic residue identification'''
:''Arthrobacter'' sp. strain U41 SqgA using 3D X-ray crystal structure <cite>#Kaur2024</cite>
; '''First 3-D structural determination'''
: ''Flavobacterium'' sp. strain K172 SqgA and ''Arthrobacter'' sp. strain U41 SqgA <cite>#Kaur2024</cite>
; '''First GH188 enzyme shown to hydrolyze both α- and β-substrates'''
:''Flavobacterium'' sp. strain K172 SqgA <cite>#Kaur2024</cite>

==References==
<biblio>
#Kaur2024 pmid=38100472

#Speciale2016 pmid=26878550
</biblio>


[[Category:Glycoside Hydrolase Families|GH188]]

Glycoside Hydrolase Family 188

2023-12-17T21:17:24Z

Spencer Williams:

{{UnderConstruction}}
* [[Author]]:
* [[Responsible Curator]]: [[User:Spencer Williams|Spencer Williams]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH188'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|NAD-dependent hydrolysis
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH188.html
|}
</div>


== Substrate specificities ==
The [[glycoside hydrolases]] of this family are found in bacteria, algae, plants and a small number of archaea <cite>#Kaur2024</cite>. The family contains enzymes with sulfoquinovosidase activity (EC 3.2.1.199), namely the ability to cleave glycosides of 6-deoxy-6-sulfoquinovose. Sulfoquinovosidases are also found in family [[GH31]] <cite>#Speciale2016</cite>. Enzymes of this family have the ability to cleave both α- and β-glycosides, and are dependent on an NAD+ cofactor.

== Kinetics and Mechanism ==
GH188 enzymes utilize an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps. Exchange of the substrate C2 proton with solvent deuterium during the enzyme-catalyzed reaction was demonstrated by mass spectrometry <cite>#Kaur2024</cite>. The following chemical mechanism is proposed: (1) C3 hydride abstraction via the reduction of NAD+ cofactor to NADH and simultaneous oxidation of the C3 hydroxyl group; (2) α to the ketone functionality, the C2 proton is deprotonated by a general catalytic base residue; (3) cleavage of the C1-O1 bond occurs in an α,β-elimination, producing an α,β-unsaturated ketone [[intermediate]]; (4) 1,4-Michael-like addition of a water molecule at C1; and (5) reduction of the C3 carbonyl functionality by the enzyme-bound NADH generates the product.

== Catalytic Residues ==
Catalytic residues can be inferred on the basis of X-ray crystallographic data for complexes of GH188 enzymes with NAD+ and sulfoquinovose <cite>#Kaur2024</cite>. Tyr136 in ''Arthrobacter'' sp. strain U41 SqgA is conserved in all family GH188 members and is located close to C2-OH, suggesting a possible role as a [[general base]]. His321 is located close to the C1-OH and may act as [[general acid]] (along with Tyr136) for the glycosidic oxygen facilitating glycosidic bond scission. The sulfonate group is recognized by a triad of amino acids: one oxygen H-bonds to Arg166 (2.6 Å), a second to Lys172 (2.9 Å), and a third to the backbone amide of Leu170 (2.8 Å).

== Three-dimensional structures ==
Crystallographic data is available for at least two GH188 enzymes, including as complexes with NADH, and NADH plus sulfoquinovose. The 3D X-ray crystal structures include those of ''Flavobacterium'' sp. strain K172 SqgA (PDB 8QC8, 8QC2) and ''Arthrobacter'' sp. strain U41 SqgA (PDB 8QC3, 8QC6. 8QC5) <cite>#Kaur2024</cite>. Each enzyme possesses an N-terminal dinucleotide-binding Rossman fold. The GH188 enzymes show structural similarities to inositol-2-dehydrogenase, glucose-fructose/IDH/MocA-like oxidoreductase, and NAD+-dependent ''N''-acetylgalactosaminidase of family [[GH109]].

== Family Firsts ==
;'''First stereochemistry determination
:not applicable
; '''First catalytic residue identification'''
:''Arthrobacter'' sp. strain U41 SqgA using 3D X-ray crystal structure <cite>#Kaur2024</cite>
; '''First 3-D structural determination'''
: ''Flavobacterium'' sp. strain K172 SqgA and ''Arthrobacter'' sp. strain U41 SqgA <cite>#Kaur2024</cite>
; '''First GH188 enzyme shown to hydrolyze both α- and β-substrates'''
:''Flavobacterium'' sp. strain K172 SqgA <cite>#Kaur2024</cite>

#Kaur2024 pmid=38100472

#Speciale2016 pmid=26878550
</biblio>


[[Category:Glycoside Hydrolase Families|GH188]]

Glycoside Hydrolase Family 31

2023-12-17T05:18:42Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Ran Zhang|Ran Zhang]], [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Steve Withers|Steve Withers]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''
|-
|'''Clan'''
|GH-D
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH31.html
|}
</div>

== Substrate specificities ==
CAZy Family GH31 has been reviewed and classified on the basis of sequence and structures into subfamilies <cite>#Arumapperuma2023</cite>. Family GH13 is one of the two major families of [[glycoside hydrolases]], along with GH13, that contain α-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal α-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to α-glucosidases, GH31 also contains α-xylosidases, isomaltosyltransferases, maltase/glucoamylases and sulfoquinovosidases. Sulfoquinovosidases (SQases) cleave the α-glycosidic linkage of α-sulfoquinovosides (glycosides of 6-sulfoglucose), present within sulfoquinovosyl diacylglyceride, a sulfolipid produced by photosynthetic organisms including plants <cite>Speciale2016</cite>. SQases are also present in family [[GH188]]. Another mechanistically interesting activity is the non-hydrolytic [[Alpha-glucan lyases|α-glucan lyases]], which cleave α-glucans to give 1,5-anhydro-fructose. GH31 enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.

== Kinetics and Mechanism ==
Enzymes of family GH31 are [[retaining]] α-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement <cite>Chiba1979</cite>. GH31 enzymes (except for the [[α-glucan lyases]]) are believed to follow the classical [[Koshland double-displacement mechanism]]. <cite>Frandsen1998</cite> This has been strongly supported by labeling of the [[catalytic nucleophile]] of several GH31 enzymes using conduritol B epoxide <cite>Iwanami1995</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>Quaroni1976</cite> and human lysosomal α-glucosidase <cite>Hermans1991</cite>. Later studies on an α-glucosidase from ''Aspergillus niger'' <cite>Lee2001</cite>, an α-xylosidase from ''Escherichia coli'' <cite>Lovering2005</cite>, YihQ sulfoquinovosidase from ''Escherichia coli'' <cite>Speciale2016</cite>, and an α-xylosidase from ''Cellvibrio japonicus'' <cite>Larsbrink2011</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent [[intermediate]]s. Subsequently, retention of the anomeric configuration was directly observed by H-1 NMR during the hydrolysis of a natural xylogluco-oligosaccharide substrate by ''C. japonicus'' Xyl31A <cite>Larsbrink2012</cite>, and of a synthetic α-sulfoquinovoside by ''E. coli'' YihQ sulfoquinovosidase <cite>Speciale2016</cite>.

The [[α-glucan lyases]] from GH31 cleave α-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose (see [[Alpha-glucan lyases|Lexicon]]). Detailed mechanistic studies have been carried out on ''Gracilariopsis'' α-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>Lee2002 Lee2003</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme [[intermediate]] by 5-fluoro-β-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two α-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-α-D-glucopyranosyl fluoride.

== Catalytic Residues ==
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>Frandsen1998 Lovering2005 Lee2003</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the [[catalytic nucleophile]], corresponding to Asp224 of ''Aspergillus niger'' α-glucosidase within the sequence IDM <cite>Iwanami1995 Hermans1991</cite>. This was confirmed by using the more reliable 5-fluoro-α-D-glucopyranosyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>Lee2001</cite>.

The [[general acid/base]] residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' α-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>Okuyama2001</cite>. This was subsequently confirmed by the crystallographic studies on α-xylosidase (YicI) from ''Escherichia coli'' <cite>Lovering2005</cite> and successfully engineering YicI into the first α-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>Kim2006</cite>.

The [[catalytic nucleophile]] in ''Gracilariopsis'' α-1,4-glucan lyase has been identified as Asp553 in the sequence QDM, through the use of 5-fluoro-β-L-idopyranosyl fluoride <cite>Lee2002</cite>. This corresponds precisely to the position of the [[catalytic nucleophile]] in the GH31 glycosidases. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the [[catalytic nucleophile]] as it departs <cite>Lee2003</cite>.

== Three-dimensional structures ==
The first crystal structure of a GH31 enzyme was that of the α-xylosidase YicI from ''Escherichia coli'', published in 2005 <cite>Lovering2005</cite>. Since that time, a number of structures have continued to emerge. Among these, the crystallographic study of the ''Sulfolobus solfataricus'' α-glucosidase (MalA) is notable as the first experimental report to highlight the 3-D structural relationship of GH31 enzymes to those of [[GH27]] and [[GH36]] <cite>Ernst2006</cite>; these three families now compose clan [http://www.cazy.org/fam/acc_GH.html#table GH-D]. The structure of the N-terminal domain of human intestinal maltase-glucoamylase was the first from a eukaryotic member of GH31 <cite>Sim2008</cite>. These structures reveal a common (β/α)8 barrel catalytic domain. Most GH31 members are multi-domain proteins, while the specific function (if any) of these accessory domains is generally unknown. An exception is the α-xylosidase, ''Cj''Xyl31A of ''C. japonicus'', in which a PA-14 domain that is rare among GH31 members is suggested to confer increased catalytic specificity toward large oligosaccharide substrates <cite>Larsbrink2011 Larsbrink2012</cite>. The X-ray structure of an inactive mutant of ''E. coli'' YihQ sulfoquinovosidase in complex with a substrate revealed that sulfonate recognition was achieved by a triad of W304, R301 and Y508 (the latter through a bridging water molecule) <cite>Speciale2016</cite>.

== Family Firsts ==

;'''First stereochemical outcome'''
:Determined for several α-glucosidases by a combination of polarimetric and reducing end measurements <cite>Chiba1979</cite>

;'''First [[catalytic nucleophile]] identification'''
:Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling <cite>Quaroni1976</cite>

;'''First [[general acid/base]] residue identification'''
:''Schizosaccharomyces pombe'' α-glucosidase by sequence comparison and kinetic studies of the mutants <cite>Okuyama2001</cite>

;'''First three-dimensional structure of GH31 enzymes'''
:''Escherichia coli'' α-xylosidase (YicI) <cite>Lovering2005</cite>

== References ==
<biblio>
#Arumapperuma2023 pmid=36806678

#Chiba1979 pmid=376499
#Frandsen1998 pmid=9620260
#Iwanami1995 pmid=7766184
#Quaroni1976 pmid=776963
#Hermans1991 pmid=1856189
#Lee2001 pmid=11583585
#Lovering2005 pmid=15501829
#Lee2002 pmid=11982345
#Lee2003 pmid=14596624
#Okuyama2001 pmid=11298744
#Kim2006 pmid=16478160
#Ernst2006 pmid=16580018
#Sim2008 pmid=18036614
#Larsbrink2011 pmid=21426303
#Larsbrink2012 pmid=22961810
#Speciale2016 pmid=26878550
</biblio>


[[Category:Glycoside Hydrolase Families|GH031]]

Glycoside Hydrolase Family 31

2023-12-16T06:13:36Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Ran Zhang|Ran Zhang]], [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Steve Withers|Steve Withers]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''
|-
|'''Clan'''
|GH-D
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH31.html
|}
</div>

== Substrate specificities ==
CAZy Family GH31 has been reviewed and classified on the basis of sequence and structures into subfamilies <cite>#Arumapperuma2023</cite>. Family GH13 is one of the two major families of [[glycoside hydrolases]], along with GH13, that contain α-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal α-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to α-glucosidases, GH31 also contains α-xylosidases, isomaltosyltransferases, maltase/glucoamylases and sulfoquinovosidases. Sulfoquinovosidases (SQases) cleave the α-glycosidic linkage of α-sulfoquinovosides (glycosides of 6-sulfoglucose), present within sulfoquinovosyl diacylglyceride, a sulfolipid produced by photosynthetic organisms including plants <cite>Speciale2016</cite>. SQases are also present in family [[GH188]]. Another mechanistically interesting activity is the non-hydrolytic [[Alpha-glucan lyases|α-glucan lyases]]. GH31 enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.

== Kinetics and Mechanism ==
Enzymes of family GH31 are [[retaining]] α-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement <cite>Chiba1979</cite>. GH31 enzymes (except for the [[α-glucan lyases]]) are believed to follow the classical [[Koshland double-displacement mechanism]]. <cite>Frandsen1998</cite> This has been strongly supported by labeling of the [[catalytic nucleophile]] of several GH31 enzymes using conduritol B epoxide <cite>Iwanami1995</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>Quaroni1976</cite> and human lysosomal α-glucosidase <cite>Hermans1991</cite>. Later studies on an α-glucosidase from ''Aspergillus niger'' <cite>Lee2001</cite>, an α-xylosidase from ''Escherichia coli'' <cite>Lovering2005</cite>, YihQ sulfoquinovosidase from ''Escherichia coli'' <cite>Speciale2016</cite>, and an α-xylosidase from ''Cellvibrio japonicus'' <cite>Larsbrink2011</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent [[intermediate]]s. Subsequently, retention of the anomeric configuration was directly observed by H-1 NMR during the hydrolysis of a natural xylogluco-oligosaccharide substrate by ''C. japonicus'' Xyl31A <cite>Larsbrink2012</cite>, and of a synthetic α-sulfoquinovoside by ''E. coli'' YihQ sulfoquinovosidase <cite>Speciale2016</cite>.

The [[α-glucan lyases]] from GH31 cleave α-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose (see [[Alpha-glucan lyases|Lexicon]]). Detailed mechanistic studies have been carried out on ''Gracilariopsis'' α-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>Lee2002 Lee2003</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme [[intermediate]] by 5-fluoro-β-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two α-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-α-D-glucopyranosyl fluoride.

== Catalytic Residues ==
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>Frandsen1998 Lovering2005 Lee2003</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the [[catalytic nucleophile]], corresponding to Asp224 of ''Aspergillus niger'' α-glucosidase within the sequence IDM <cite>Iwanami1995 Hermans1991</cite>. This was confirmed by using the more reliable 5-fluoro-α-D-glucopyranosyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>Lee2001</cite>.

The [[general acid/base]] residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' α-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>Okuyama2001</cite>. This was subsequently confirmed by the crystallographic studies on α-xylosidase (YicI) from ''Escherichia coli'' <cite>Lovering2005</cite> and successfully engineering YicI into the first α-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>Kim2006</cite>.

The [[catalytic nucleophile]] in ''Gracilariopsis'' α-1,4-glucan lyase has been identified as Asp553 in the sequence QDM, through the use of 5-fluoro-β-L-idopyranosyl fluoride <cite>Lee2002</cite>. This corresponds precisely to the position of the [[catalytic nucleophile]] in the GH31 glycosidases. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the [[catalytic nucleophile]] as it departs <cite>Lee2003</cite>.

== Three-dimensional structures ==
The first crystal structure of a GH31 enzyme was that of the α-xylosidase YicI from ''Escherichia coli'', published in 2005 <cite>Lovering2005</cite>. Since that time, a number of structures have continued to emerge. Among these, the crystallographic study of the ''Sulfolobus solfataricus'' α-glucosidase (MalA) is notable as the first experimental report to highlight the 3-D structural relationship of GH31 enzymes to those of [[GH27]] and [[GH36]] <cite>Ernst2006</cite>; these three families now compose clan [http://www.cazy.org/fam/acc_GH.html#table GH-D]. The structure of the N-terminal domain of human intestinal maltase-glucoamylase was the first from a eukaryotic member of GH31 <cite>Sim2008</cite>. These structures reveal a common (β/α)8 barrel catalytic domain. Most GH31 members are multi-domain proteins, while the specific function (if any) of these accessory domains is generally unknown. An exception is the α-xylosidase, ''Cj''Xyl31A of ''C. japonicus'', in which a PA-14 domain that is rare among GH31 members is suggested to confer increased catalytic specificity toward large oligosaccharide substrates <cite>Larsbrink2011 Larsbrink2012</cite>. The X-ray structure of an inactive mutant of ''E. coli'' YihQ sulfoquinovosidase in complex with a substrate revealed that sulfonate recognition was achieved by a triad of W304, R301 and Y508 (the latter through a bridging water molecule) <cite>Speciale2016</cite>.

== Family Firsts ==

;'''First stereochemical outcome'''
:Determined for several α-glucosidases by a combination of polarimetric and reducing end measurements <cite>Chiba1979</cite>

;'''First [[catalytic nucleophile]] identification'''
:Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling <cite>Quaroni1976</cite>

;'''First [[general acid/base]] residue identification'''
:''Schizosaccharomyces pombe'' α-glucosidase by sequence comparison and kinetic studies of the mutants <cite>Okuyama2001</cite>

;'''First three-dimensional structure of GH31 enzymes'''
:''Escherichia coli'' α-xylosidase (YicI) <cite>Lovering2005</cite>

== References ==
<biblio>
#Arumapperuma2023 pmid=36806678

#Chiba1979 pmid=376499
#Frandsen1998 pmid=9620260
#Iwanami1995 pmid=7766184
#Quaroni1976 pmid=776963
#Hermans1991 pmid=1856189
#Lee2001 pmid=11583585
#Lovering2005 pmid=15501829
#Lee2002 pmid=11982345
#Lee2003 pmid=14596624
#Okuyama2001 pmid=11298744
#Kim2006 pmid=16478160
#Ernst2006 pmid=16580018
#Sim2008 pmid=18036614
#Larsbrink2011 pmid=21426303
#Larsbrink2012 pmid=22961810
#Speciale2016 pmid=26878550
</biblio>


[[Category:Glycoside Hydrolase Families|GH031]]

Glycoside Hydrolase Family 31

2023-12-16T05:50:15Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Ran Zhang|Ran Zhang]], [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Steve Withers|Steve Withers]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''
|-
|'''Clan'''
|GH-D
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH31.html
|}
</div>

== Substrate specificities ==
CAZy Family GH31 is one of the two major families of [[glycoside hydrolases]], along with GH13, that contain α-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal α-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to α-glucosidases, GH31 also contains α-xylosidases, isomaltosyltransferases, maltase/glucoamylases and sulfoquinovosidases [{{EClink}}3.2.1.199 3.2.1.199]. Sulfoquinovosidases (SQases) cleave the α-glycosidic linkage of α-sulfoquinovosides (glycosides of 6-sulfoglucose), present within sulfoquinovosyl diacylglyceride, a sulfolipid produced by photosynthetic organisms including plants <cite>Speciale2016</cite>. SQases are also present in family [[GH188]]. Another mechanistically interesting activity is the non-hydrolytic [[Alpha-glucan lyases|α-glucan lyases]]. GH31 enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.

== Kinetics and Mechanism ==
Enzymes of family GH31 are [[retaining]] α-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement <cite>Chiba1979</cite>. GH31 enzymes (except for the [[α-glucan lyases]]) are believed to follow the classical [[Koshland double-displacement mechanism]]. <cite>Frandsen1998</cite> This has been strongly supported by labeling of the [[catalytic nucleophile]] of several GH31 enzymes using conduritol B epoxide <cite>Iwanami1995</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>Quaroni1976</cite> and human lysosomal α-glucosidase <cite>Hermans1991</cite>. Later studies on an α-glucosidase from ''Aspergillus niger'' <cite>Lee2001</cite>, an α-xylosidase from ''Escherichia coli'' <cite>Lovering2005</cite>, YihQ sulfoquinovosidase from ''Escherichia coli'' <cite>Speciale2016</cite>, and an α-xylosidase from ''Cellvibrio japonicus'' <cite>Larsbrink2011</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent [[intermediate]]s. Subsequently, retention of the anomeric configuration was directly observed by H-1 NMR during the hydrolysis of a natural xylogluco-oligosaccharide substrate by ''C. japonicus'' Xyl31A <cite>Larsbrink2012</cite>, and of a synthetic α-sulfoquinovoside by ''E. coli'' YihQ sulfoquinovosidase <cite>Speciale2016</cite>.

The [[α-glucan lyases]] from GH31 cleave α-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose (see [[Alpha-glucan lyases|Lexicon]]). Detailed mechanistic studies have been carried out on ''Gracilariopsis'' α-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>Lee2002 Lee2003</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme [[intermediate]] by 5-fluoro-β-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two α-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-α-D-glucopyranosyl fluoride.

== Catalytic Residues ==
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>Frandsen1998 Lovering2005 Lee2003</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the [[catalytic nucleophile]], corresponding to Asp224 of ''Aspergillus niger'' α-glucosidase within the sequence IDM <cite>Iwanami1995 Hermans1991</cite>. This was confirmed by using the more reliable 5-fluoro-α-D-glucopyranosyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>Lee2001</cite>.

The [[general acid/base]] residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' α-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>Okuyama2001</cite>. This was subsequently confirmed by the crystallographic studies on α-xylosidase (YicI) from ''Escherichia coli'' <cite>Lovering2005</cite> and successfully engineering YicI into the first α-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>Kim2006</cite>.

The [[catalytic nucleophile]] in ''Gracilariopsis'' α-1,4-glucan lyase has been identified as Asp553 in the sequence QDM, through the use of 5-fluoro-β-L-idopyranosyl fluoride <cite>Lee2002</cite>. This corresponds precisely to the position of the [[catalytic nucleophile]] in the GH31 glycosidases. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the [[catalytic nucleophile]] as it departs <cite>Lee2003</cite>.

== Three-dimensional structures ==
The first crystal structure of a GH31 enzyme was that of the α-xylosidase YicI from ''Escherichia coli'', published in 2005 <cite>Lovering2005</cite>. Since that time, a number of structures have continued to emerge. Among these, the crystallographic study of the ''Sulfolobus solfataricus'' α-glucosidase (MalA) is notable as the first experimental report to highlight the 3-D structural relationship of GH31 enzymes to those of [[GH27]] and [[GH36]] <cite>Ernst2006</cite>; these three families now compose clan [http://www.cazy.org/fam/acc_GH.html#table GH-D]. The structure of the N-terminal domain of human intestinal maltase-glucoamylase was the first from a eukaryotic member of GH31 <cite>Sim2008</cite>. These structures reveal a common (β/α)8 barrel catalytic domain. Most GH31 members are multi-domain proteins, while the specific function (if any) of these accessory domains is generally unknown. An exception is the α-xylosidase, ''Cj''Xyl31A of ''C. japonicus'', in which a PA-14 domain that is rare among GH31 members is suggested to confer increased catalytic specificity toward large oligosaccharide substrates <cite>Larsbrink2011 Larsbrink2012</cite>. The X-ray structure of an inactive mutant of ''E. coli'' YihQ sulfoquinovosidase in complex with a substrate revealed that sulfonate recognition was achieved by a triad of W304, R301 and Y508 (the latter through a bridging water molecule) <cite>Speciale2016</cite>.

== Family Firsts ==

;'''First stereochemical outcome'''
:Determined for several α-glucosidases by a combination of polarimetric and reducing end measurements <cite>Chiba1979</cite>

;'''First [[catalytic nucleophile]] identification'''
:Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling <cite>Quaroni1976</cite>

;'''First [[general acid/base]] residue identification'''
:''Schizosaccharomyces pombe'' α-glucosidase by sequence comparison and kinetic studies of the mutants <cite>Okuyama2001</cite>

;'''First three-dimensional structure of GH31 enzymes'''
:''Escherichia coli'' α-xylosidase (YicI) <cite>Lovering2005</cite>

== References ==
<biblio>
#Chiba1979 pmid=376499
#Frandsen1998 pmid=9620260
#Iwanami1995 pmid=7766184
#Quaroni1976 pmid=776963
#Hermans1991 pmid=1856189
#Lee2001 pmid=11583585
#Lovering2005 pmid=15501829
#Lee2002 pmid=11982345
#Lee2003 pmid=14596624
#Okuyama2001 pmid=11298744
#Kim2006 pmid=16478160
#Ernst2006 pmid=16580018
#Sim2008 pmid=18036614
#Larsbrink2011 pmid=21426303
#Larsbrink2012 pmid=22961810
#Speciale2016 pmid=26878550
</biblio>


[[Category:Glycoside Hydrolase Families|GH031]]

Glycoside Hydrolase Family 4

2023-12-16T02:35:44Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Vivian Yip|Vivian Yip]]
* [[Responsible Curator]]: [[User:Steve Withers|Steve Withers]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family 4'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|NAD-dependent hydrolysis
|-
|'''Active site residues'''
|not known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" | {{CAZyDBlink}}GH4.html
|}
</div>

==Substrate specificities==
The majority of the [[glycoside hydrolases]] of this family are of bacterial origin, but they are also found in the archaeal taxa <cite>#1</cite>. Unlike other families of glycosidases, some of the enzymes in Family 4 possess distinct substrate specificities from each other<cite>#1 #2</cite>. This family contains α-glucosidases, α-galactosidases, α-glucuronidases, 6-phospho-α-glucosidases, and 6-phospho-β-glucosidases. Similar to [[GH1]], some enzymes prefer phosphorylated substrates over non-phosphorylated substrates <cite>#2 #3</cite>. Members of this family include both α- and β-glycosidases. The ability of a single enzyme to hydrolyze natural substrates of different anomeric configurations was first discovered in family GH4, and they were the first glycosidases that exhibited an absolute requirement for NAD+ and a divalent metal ion and in some instances reducing environments for catalytic activity <cite>#4 #5 #6 #7 #8 #9 #10 #11</cite>. The cofactors are proposed to play key mechanistic roles in the atypical glycosidase mechanism. Unlike GH4, metal ions are required by some glycosidases for structural integrity <cite>#12 #13</cite> or proper configuration of an active enzyme <cite>#14 #15 #16 #17</cite>. Meanwhile, GH4 is the first family of glycoside hydrolases reported to require a NAD+ cofactor for catalytic activity <cite>#4 #5 #6 #7 #8 #10 #11 #18 #19 #20 #21 #22</cite>. Subsequently, enzymes of families [[GH109]], [[GH177]], [[GH179]], and [[GH188]] has been shown to also require a NAD+ cofactor. The hydrolysis of thioglycosides with activated leaving groups has been documented in GH84 <cite>#23</cite>, GH1 <cite>#24 #25 #26 #27 #28 #29</cite>. However, GH4 is currently the only family that has been shown to catalyze the hydrolysis of unactivated thioglycoside substrates <cite>#30</cite>. The remarkable substrate specificities are all feasible due to the β-elimination mechanism discussed below.

==Kinetics and Mechanism==
GH4 enzymes have been shown to be [[retaining]] glycosidases <cite>#31 #32 #33 #34</cite>. However, the enzymes do not utilize the classical [[Koshland double-displacement mechanism]] <cite>#35</cite>. BglT (6-phospho-β-glucosidase) from ''Thermotoga maritima'' <cite>#36</cite> and GlvA (6-phospho-α-glucosidase) from ''Bacillus subtilis'' <cite>#32</cite> are two of the most thoroughly studied enzymes of this family, and both are proposed to utilize an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s. Exchange of the substrate C2 proton with solvent deuterium during the enzyme-catalyzed reaction was the first indication of a C2-H2 bond cleavage in the substrate, which is inconsistent with the double-displacement mechanism <cite>#31 #33</cite>. This fact coupled with the proximity of the NAD+ cofactor to C3 of the glucose 6-phosphate product and the high structural similarity to lactate/malate dehydrogenases as garnered from the x-ray crystallographic data <cite>#9 #31 #33 #34</cite>, led to the following proposed mechanism <cite>#31 #33</cite>: 1) C3 hydride abstraction via the reduction of NAD+ cofactor to NADH and consequent oxidation of the C3 hydroxyl group; 2) adjacent to the ketone functionality, the C2 proton is activated to deprotonation by a near-by catalytic base residue; 3) cleavage of the C1-O1 linkage occurs via an α,β-elimination mechanism, producing an α,β-unsaturated [[intermediate]]; 5) a 1,4-Michael-like addition of a water molecule at C1 takes place; and 6) reduction of the C3 carbonyl functionality by the "on-board" NADH generates the final hydrolysis product.

Primary kinetic isotope effects and the determination of Brønsted relationships revealed that both the C3 hydride and C2 proton abstractions are partially rate-limiting and that cleavage of the C1-O1 linkage is not rate-limiting <cite>#32 #33 #36</cite>. This led to the proposed E1cb mechanism for both BglT and GlvA <cite>#32 #36</cite>.

==Catalytic Residues==
The proposed catalytic residues were mostly derived from x-ray crystallographic data and pH-dependent activity profiles obtained for BglT and GlvA <cite>#31 #32 #33 #36</cite>. A Tyr residue that is conserved in all 6-phospho-α- and 6-phospho-β-glycosidases was found to be approximately 4 Å away from C2 of the reaction product, which can potentially act as a [[general base]] <cite>#31 #32 #33 #34 #36</cite>. The pH-dependent activity profiles were that of double ionization curves with pHopt at approximately 8 <cite>#32 #36</cite>. Two p''K''a values of approximately 7 and 9 were determined <cite>#32 #36</cite>. The p''K''a value of approximately 7 was proposed to correspond to that of the Tyr catalytic base, since this residue would need to be deprotonated for enzyme activity. The position of the Tyr in the enzyme active site was used as a possible explanation for the relatively low p''K''a compared to that of a free Tyr (p''K''a = 10). In the case of BglT, the Tyr residue is located at a distance of 3.4 Å from the glycosidic oxygen, making it ideal to assume a second role in providing [[general acid]] catalysis to the departing oxygen as well as C2 deprotonation <cite>#31 #36</cite>. The p''K''a value of approximately 9 was proposed to correspond to the ionization of the conserved Arg residue(s) that were found to be within hydrogen bonding distance of the phosphate group of the substrate in these two 6-phospho-glycosidases <cite>#31 #34 #36</cite>. Again, the normal p''K''a of 12 for Arg could be lowered to 9, and this matches the expectation that protonated Arg residue(s) would be needed to form electrostatic interactions with the substrate phosphate moiety. Two other conserved residues, Cys and His, were shown to be responsible for chelating the metal ion in BglT and GlvA. In the AglA structure, an Asp residue occupies the same position as the catalytic Tyr base, so the Asp is proposed to take on this role in AglA and possibly other GH4 enzymes that prefer non-phosphorylated substrates.

==Three-dimensional structures==
Crystallographic data is available for a number of GH4 enzymes, including α- and β-members as well as those enzymes specific for phosphorylated and non-phosphorylated substrates. The x-ray crystal structures currently available for this family are those of the ''Thermotoga maritima'' α-glucosidase AglA (PDB 1OBB) <cite>#9</cite>, the ''T. maritima'' α-glucosidase Agu4B (PDB 1VJT) <cite>#37</cite>, the ''Bacillus subtilis'' 6-phospo-α-glucosidase GlvA (PDB 1U8X) <cite>#34</cite>, and two 6-phospho-β-glucosidases: one from ''Geobacillus stearothermophilus'' (PDB 1S6Y) <cite>#37</cite> and other BglT from ''T. maritima'' (PDB 1UP6) <cite>#31 #33</cite>. Each enzyme possesses the characteristic dinucleotide-binding Rossman fold <cite>#38</cite>. The crystal structures of Agu4B (PDB 1VJT) and the 6-phospho-β-glucosidase (PDB 1SY6) from do not provide useful information about the active sites. No ligands are bound in the structure of the 6-phospho-β-glucosidase from ''G. stearothermophilus''. In the Agu4B structure, only the NAD+ cofactor is found at the enzyme active site, but no data for the nicotinamide ring is provided, possibly because it is not well-defined in the crystal structure. The AglA structure is that of an inactive enzyme with no divalent metal ion. The GH4 enzymes show some structural similarities to lactate/malate dehydrogenases as well as dehydratases <cite>#9 #31 #33 #34 #37</cite>.

==Family Firsts==
;'''First stereochemistry determination
:''Thermotoga maritima'' BglT (6-phospho-β-glucosidase) via NMR and HPLC analysis of methyl-glycoside product <cite>#33</cite>
; '''First catalytic residue identification'''
:''Thermotoga maritima'' BglT (6-phospho-β-glucosidase) via x-ray crystal structure <cite>#33</cite>
; '''First 3-D structural determination'''
:''Thermotoga maritima'' AlgA (α-glucosidase) (inactive enzyme) <cite>#9</cite>
:''Thermotoga maritima'' BglT (6-phospho-β-glucosidase) (active enzyme with all cofactors present) <cite>#31 #33</cite>
; '''First GH4 enzyme shown to hydrolyze both α- and β-substrates'''
:''Fusobacterium mortiferum'' MalH (6-phosph-α-glucosidase) <cite>#19</cite>
; '''First β-glycosidase'''
:''Escherichia coli'' CelF (6-phospho-β-glucosidase) <cite>#22</cite>

==References==
<biblio>
#1 pmid=18838391
#2 pmid=9345621
#3 pmid=8687420
#4 pmid=10972187
#5 pmid=9765262
#6 pmid=7730284
#7 pmid=9209025
#8 pmid=11473129
#9 pmid=12588867
#10 pmid=5543331
#11 pmid=2831880
#12 pmid=16712774
#13 pmid=2548811
#14 pmid=4625429
#15 pmid=4721625
#16 pmid=27358
#17 pmid=6774745
#18 pmid=11322729
#19 pmid=11882720
#20 pmid=12062428
#21 pmid=5329906
#22 pmid=10572139
#23 pmid=16332065
#24 pmid=3096349
#25 pmid=9195886
#26 pmid=10978344
#27 pmid=8952475
#28 pmid=3278958
#29 pmid=1731996
#30 pmid=16917793
#31 pmid=15670594
#32 pmid=17676871
#33 pmid=15237973
#34 pmid=15341727
#35 Koshland DE Jr: ''Stereochemistry and the mechanism of enzyme reactions.'' Biol Rev 1953, 28:416-436. [https://doi.org/10.1111/j.1469-185X.1953.tb01386.x DOI:10.1111/j.1469-185X.1953.tb01386.x]
#36 pmid=16401086
#37 pmid=10592235
#38 pmid=4375720
</biblio>

[[Category:Glycoside Hydrolase Families|GH004]]

Glycoside Hydrolase Family 4

2023-12-16T02:35:23Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Vivian Yip|Vivian Yip]]
* [[Responsible Curator]]: [[User:Steve Withers|Steve Withers]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family 4'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|NAD-dependent hydrolysis
|-
|'''Active site residues'''
|not known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" | {{CAZyDBlink}}GH4.html
|}
</div>

==Substrate specificities==
The majority of the [[glycoside hydrolases]] of this family are of bacterial origin, but they have recently been found in the archaeal taxa <cite>#1</cite>. Unlike other families of glycosidases, some of the enzymes in Family 4 possess distinct substrate specificities from each other<cite>#1 #2</cite>. This family contains α-glucosidases, α-galactosidases, α-glucuronidases, 6-phospho-α-glucosidases, and 6-phospho-β-glucosidases. Similar to [[GH1]], some enzymes prefer phosphorylated substrates over non-phosphorylated substrates <cite>#2 #3</cite>. Members of this family include both α- and β-glycosidases. The ability of a single enzyme to hydrolyze natural substrates of different anomeric configurations was first discovered in family GH4, and they were the first glycosidases that exhibited an absolute requirement for NAD+ and a divalent metal ion and in some instances reducing environments for catalytic activity <cite>#4 #5 #6 #7 #8 #9 #10 #11</cite>. The cofactors are proposed to play key mechanistic roles in the atypical glycosidase mechanism. Unlike GH4, metal ions are required by some glycosidases for structural integrity <cite>#12 #13</cite> or proper configuration of an active enzyme <cite>#14 #15 #16 #17</cite>. Meanwhile, GH4 is the first family of glycoside hydrolases reported to require a NAD+ cofactor for catalytic activity <cite>#4 #5 #6 #7 #8 #10 #11 #18 #19 #20 #21 #22</cite>. Subsequently, enzymes of families [[GH109]], [[GH177]], [[GH179]], and [[GH188]] has been shown to also require a NAD+ cofactor. The hydrolysis of thioglycosides with activated leaving groups has been documented in GH84 <cite>#23</cite>, GH1 <cite>#24 #25 #26 #27 #28 #29</cite>. However, GH4 is currently the only family that has been shown to catalyze the hydrolysis of unactivated thioglycoside substrates <cite>#30</cite>. The remarkable substrate specificities are all feasible due to the β-elimination mechanism discussed below.

==Kinetics and Mechanism==
GH4 enzymes have been shown to be [[retaining]] glycosidases <cite>#31 #32 #33 #34</cite>. However, the enzymes do not utilize the classical [[Koshland double-displacement mechanism]] <cite>#35</cite>. BglT (6-phospho-β-glucosidase) from ''Thermotoga maritima'' <cite>#36</cite> and GlvA (6-phospho-α-glucosidase) from ''Bacillus subtilis'' <cite>#32</cite> are two of the most thoroughly studied enzymes of this family, and both are proposed to utilize an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s. Exchange of the substrate C2 proton with solvent deuterium during the enzyme-catalyzed reaction was the first indication of a C2-H2 bond cleavage in the substrate, which is inconsistent with the double-displacement mechanism <cite>#31 #33</cite>. This fact coupled with the proximity of the NAD+ cofactor to C3 of the glucose 6-phosphate product and the high structural similarity to lactate/malate dehydrogenases as garnered from the x-ray crystallographic data <cite>#9 #31 #33 #34</cite>, led to the following proposed mechanism <cite>#31 #33</cite>: 1) C3 hydride abstraction via the reduction of NAD+ cofactor to NADH and consequent oxidation of the C3 hydroxyl group; 2) adjacent to the ketone functionality, the C2 proton is activated to deprotonation by a near-by catalytic base residue; 3) cleavage of the C1-O1 linkage occurs via an α,β-elimination mechanism, producing an α,β-unsaturated [[intermediate]]; 5) a 1,4-Michael-like addition of a water molecule at C1 takes place; and 6) reduction of the C3 carbonyl functionality by the "on-board" NADH generates the final hydrolysis product.

Primary kinetic isotope effects and the determination of Brønsted relationships revealed that both the C3 hydride and C2 proton abstractions are partially rate-limiting and that cleavage of the C1-O1 linkage is not rate-limiting <cite>#32 #33 #36</cite>. This led to the proposed E1cb mechanism for both BglT and GlvA <cite>#32 #36</cite>.

==Catalytic Residues==
The proposed catalytic residues were mostly derived from x-ray crystallographic data and pH-dependent activity profiles obtained for BglT and GlvA <cite>#31 #32 #33 #36</cite>. A Tyr residue that is conserved in all 6-phospho-α- and 6-phospho-β-glycosidases was found to be approximately 4 Å away from C2 of the reaction product, which can potentially act as a [[general base]] <cite>#31 #32 #33 #34 #36</cite>. The pH-dependent activity profiles were that of double ionization curves with pHopt at approximately 8 <cite>#32 #36</cite>. Two p''K''a values of approximately 7 and 9 were determined <cite>#32 #36</cite>. The p''K''a value of approximately 7 was proposed to correspond to that of the Tyr catalytic base, since this residue would need to be deprotonated for enzyme activity. The position of the Tyr in the enzyme active site was used as a possible explanation for the relatively low p''K''a compared to that of a free Tyr (p''K''a = 10). In the case of BglT, the Tyr residue is located at a distance of 3.4 Å from the glycosidic oxygen, making it ideal to assume a second role in providing [[general acid]] catalysis to the departing oxygen as well as C2 deprotonation <cite>#31 #36</cite>. The p''K''a value of approximately 9 was proposed to correspond to the ionization of the conserved Arg residue(s) that were found to be within hydrogen bonding distance of the phosphate group of the substrate in these two 6-phospho-glycosidases <cite>#31 #34 #36</cite>. Again, the normal p''K''a of 12 for Arg could be lowered to 9, and this matches the expectation that protonated Arg residue(s) would be needed to form electrostatic interactions with the substrate phosphate moiety. Two other conserved residues, Cys and His, were shown to be responsible for chelating the metal ion in BglT and GlvA. In the AglA structure, an Asp residue occupies the same position as the catalytic Tyr base, so the Asp is proposed to take on this role in AglA and possibly other GH4 enzymes that prefer non-phosphorylated substrates.

==Three-dimensional structures==
Crystallographic data is available for a number of GH4 enzymes, including α- and β-members as well as those enzymes specific for phosphorylated and non-phosphorylated substrates. The x-ray crystal structures currently available for this family are those of the ''Thermotoga maritima'' α-glucosidase AglA (PDB 1OBB) <cite>#9</cite>, the ''T. maritima'' α-glucosidase Agu4B (PDB 1VJT) <cite>#37</cite>, the ''Bacillus subtilis'' 6-phospo-α-glucosidase GlvA (PDB 1U8X) <cite>#34</cite>, and two 6-phospho-β-glucosidases: one from ''Geobacillus stearothermophilus'' (PDB 1S6Y) <cite>#37</cite> and other BglT from ''T. maritima'' (PDB 1UP6) <cite>#31 #33</cite>. Each enzyme possesses the characteristic dinucleotide-binding Rossman fold <cite>#38</cite>. The crystal structures of Agu4B (PDB 1VJT) and the 6-phospho-β-glucosidase (PDB 1SY6) from do not provide useful information about the active sites. No ligands are bound in the structure of the 6-phospho-β-glucosidase from ''G. stearothermophilus''. In the Agu4B structure, only the NAD+ cofactor is found at the enzyme active site, but no data for the nicotinamide ring is provided, possibly because it is not well-defined in the crystal structure. The AglA structure is that of an inactive enzyme with no divalent metal ion. The GH4 enzymes show some structural similarities to lactate/malate dehydrogenases as well as dehydratases <cite>#9 #31 #33 #34 #37</cite>.

==Family Firsts==
;'''First stereochemistry determination
:''Thermotoga maritima'' BglT (6-phospho-β-glucosidase) via NMR and HPLC analysis of methyl-glycoside product <cite>#33</cite>
; '''First catalytic residue identification'''
:''Thermotoga maritima'' BglT (6-phospho-β-glucosidase) via x-ray crystal structure <cite>#33</cite>
; '''First 3-D structural determination'''
:''Thermotoga maritima'' AlgA (α-glucosidase) (inactive enzyme) <cite>#9</cite>
:''Thermotoga maritima'' BglT (6-phospho-β-glucosidase) (active enzyme with all cofactors present) <cite>#31 #33</cite>
; '''First GH4 enzyme shown to hydrolyze both α- and β-substrates'''
:''Fusobacterium mortiferum'' MalH (6-phosph-α-glucosidase) <cite>#19</cite>
; '''First β-glycosidase'''
:''Escherichia coli'' CelF (6-phospho-β-glucosidase) <cite>#22</cite>

==References==
<biblio>
#1 pmid=18838391
#2 pmid=9345621
#3 pmid=8687420
#4 pmid=10972187
#5 pmid=9765262
#6 pmid=7730284
#7 pmid=9209025
#8 pmid=11473129
#9 pmid=12588867
#10 pmid=5543331
#11 pmid=2831880
#12 pmid=16712774
#13 pmid=2548811
#14 pmid=4625429
#15 pmid=4721625
#16 pmid=27358
#17 pmid=6774745
#18 pmid=11322729
#19 pmid=11882720
#20 pmid=12062428
#21 pmid=5329906
#22 pmid=10572139
#23 pmid=16332065
#24 pmid=3096349
#25 pmid=9195886
#26 pmid=10978344
#27 pmid=8952475
#28 pmid=3278958
#29 pmid=1731996
#30 pmid=16917793
#31 pmid=15670594
#32 pmid=17676871
#33 pmid=15237973
#34 pmid=15341727
#35 Koshland DE Jr: ''Stereochemistry and the mechanism of enzyme reactions.'' Biol Rev 1953, 28:416-436. [https://doi.org/10.1111/j.1469-185X.1953.tb01386.x DOI:10.1111/j.1469-185X.1953.tb01386.x]
#36 pmid=16401086
#37 pmid=10592235
#38 pmid=4375720
</biblio>

[[Category:Glycoside Hydrolase Families|GH004]]

Glycoside Hydrolase Family 179

2023-12-16T02:22:40Z

Spencer Williams:

{{UnderConstruction}}
* [[Author]]: [[User:Andrea Strazzulli|Andrea Strazzulli]]
* [[Responsible Curator]]: [[User:Marco Moracci|Marco Moracci]]
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH179'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|NAD-dependent hydrolysis
|-
|'''Active site residues'''
|known/not known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH179.html
|}
</div>


== Substrate specificities ==
Content is to be added here.

Authors may get an idea of what to put in each field from ''Curator Approved'' [[Glycoside Hydrolase Families]]. ''(TIP: Right click with your mouse and open this link in a new browser window...)''

In the meantime, please see these references for an essential introduction to the CAZy classification system: <cite>DaviesSinnott2008 Cantarel2009</cite>.

== Kinetics and Mechanism ==
Content is to be added here.

== Catalytic Residues ==
Content is to be added here.

== Three-dimensional structures ==
Content is to be added here.

== Family Firsts ==
;First stereochemistry determination: Content is to be added here.
;First catalytic nucleophile identification: Content is to be added here.
;First general acid/base residue identification: Content is to be added here.
;First 3-D structure: Content is to be added here.

== References ==
<biblio>
#Cantarel2009 pmid=18838391
#DaviesSinnott2008 Davies, G.J. and Sinnott, M.L. (2008) Sorting the diverse: the sequence-based classifications of carbohydrate-active enzymes. ''The Biochemist'', vol. 30, no. 4., pp. 26-32. [https://doi.org/10.1042/BIO03004026 Download PDF version].
</biblio>


[[Category:Glycoside Hydrolase Families|GH179]]

Glycoside Hydrolase Family 109

2023-12-16T02:22:27Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Bernie|Bernard Henrissat]]
* [[Responsible Curator]]: [[User:Bernie|Bernard Henrissat]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH109'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|NAD-dependent hydrolysis
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH109.html
|}
</div>

== Substrate specificities ==
The only activity so far identified in this recently discovered family of [[glycoside hydrolases]] is that of α-''N''-acetylgalactosaminidase, although the lack of activity of several family members on GalNAc substrates suggests that other substrates might exist. The most characterized member of this family is the enzyme from ''Elizabethkingia meningosepticum''. Because it operates at neutral pH optimum, this enzyme was used succesfully for the removal of the A antigen on red blood cells thus opening the possibility of blood group conversion to universal group O <cite>1</cite>. The enzyme clearly prefers GalNAc over Gal, as indicated by a 2,000-fold reduction in kcat for the hydrolysis of p-nitrophenyl α-galactoside compared with p-nitrophenyl α-''N''-acetyl-galactosaminide and by a more than tenfold increase in ''K''m <cite>1</cite>.

== Kinetics and Mechanism ==
Family GH109 enzymes operate via the unusual [[NAD-dependent hydrolysis]] mechanism involving an NAD+ cofactor, that so far has been seen only in [[Glycoside Hydrolase Family 4]] ([[GH4]]), despite different overall folds between these families (see below). NMR monitoring of the reaction catalyzed by α-''N''-acetylgalactosaminidase indicated that the enzyme proceeds with retention of the anomeric configuration and concomitant exchange of the GalNAc H-2 atom for a solvent proton [1]. This, and the indispensable presence of NAD+, indicate that GH109 enzymes most likely operate by a similar [[retaining]] mechanism. In this mechanism, the NAD+ molecule oxidizes the substrate at C-3, thereby acidifying the proton at C-2 and producing NADH. Deprotonation of C-2 by an enzymatic base with concomitant elimination of the glycosidic oxygen generates a 1,2-unsaturated [[intermediate]]. The reaction is completed by addition of water to the Michael-like acceptor and reduction of the resulting ketone by the NADH molecule, which returns to the initial NAD+ state, ready for another catalytic cycle. The mechanism of GH109 enzymes allows cleavage of thioglycosides and of glycosides of the opposite anomeric configuration (both at a comparatively slow rate), two features that are extremely rare among 'classical' glycosidases <cite>1</cite>.

== Catalytic Residues ==
A stated above the enzymes of this family do not use a classical acid/base catalysis, but instead use a rare catalytic mechanism involving [[NAD-dependent hydrolysis]] with an NAD+ cofactor, highly similar to that seen in family [[Glycoside Hydrolase Family 4]]. The catalytic machinery therefore comprises NAD+ and Tyr-179, which abstracts H-2 to form the unsaturated [[intermediate]]. Subtle differences with family [[GH4]] exist, however, such as the absence in GH109 of an identifiable acid to assist glycosidic bond cleavage. It is believed that this enables the hydrolysis of the 'wrong' anomer.

== Three-dimensional structures ==
The three-dimensional structure of ''Elizabethkingia meningosepticum'' α-''N''-acetylgalactosaminidase has been reported in 2007 <cite>1</cite>. The closest structural relatives belong to the Gfo/Idh/MocA oxidoreductase family (Z-score of 29.4 and r.m.s. deviation of 3.0 A for 329 equivalent Ca-atoms for ''Zymomonas mobilis'' glucose-fructose oxidoreductase ([{{PDBlink}}1ofg PDB 1ofg]). More distant structural homologs are identified by means of the classical Rossmann fold. The structural similarity includes the active-site architecture, where the spatial arrangement of NAD+ and several other residues is conserved, suggesting a common ancestor that has evolved its NAD+-based molecular mechanism to adapt to diverse metabolic requirements <cite>1</cite>.

== Family Firsts ==
;First sterochemistry determination: ''Elizabethkingia meningosepticum'' α-''N''-acetylgalactosaminidase by 1H NMR <cite>1</cite>.
;First mechanistic identification: ''Elizabethkingia meningosepticum'' α-''N''-acetylgalactosaminidase, by deuterium exchange of H-2, involvement of NAD+ and structural similarity with GH4 enzymes <cite>1</cite>.
;First 3-D structure: ''Elizabethkingia meningosepticum'' α-''N''-acetylgalactosaminidase <cite>1</cite> ([{{PDBlink}}2ixa PDB 2ixa]) and ([{{PDBlink}}2ixb PDB 2ixb]).

== References ==
<biblio>
#1 pmid=17401360

</biblio>

[[Category:Glycoside Hydrolase Families|GH109]]

Glycoside Hydrolase Family 4

2023-12-16T02:22:02Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Vivian Yip|Vivian Yip]]
* [[Responsible Curator]]: [[User:Steve Withers|Steve Withers]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family 4'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|NAD-dependent hydrolysis
|-
|'''Active site residues'''
|not known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" | {{CAZyDBlink}}GH4.html
|}
</div>

==Substrate specificities==
The majority of the [[glycoside hydrolases]] of this family are of bacterial origin, but they have recently been found in the archaeal taxa <cite>#1</cite>. Unlike other families of glycosidases, some of the enzymes in Family 4 possess distinct substrate specificities from each other<cite>#1 #2</cite>. This family contains α-glucosidases, α-galactosidases, α-glucuronidases, 6-phospho-α-glucosidases, and 6-phospho-β-glucosidases. Similar to [[GH1]], some enzymes prefer phosphorylated substrates over non-phosphorylated substrates <cite>#2 #3</cite>. Unique to GH4 is the presence of both α- and β-glycosidases. The ability of a single enzyme to hydrolyze natural substrates of different anomeric configurations has not been discovered in other [[glycoside hydrolase]] families to-date. GH4 enzymes were the first glycosidases shown to demonstrate an absolute requirement for NAD+ and a divalent metal ion and in some instances reducing environments for catalytic activity <cite>#4 #5 #6 #7 #8 #9 #10 #11</cite>. The cofactors are proposed to play key mechanistic roles in the atypical glycosidase mechanism. Unlike GH4, metal ions are required by some glycosidases for structural integrity <cite>#12 #13</cite> or proper configuration of an active enzyme <cite>#14 #15 #16 #17</cite>. Meanwhile, GH4 is the first family of glycoside hydrolases reported to require a NAD+ cofactor for catalytic activity <cite>#4 #5 #6 #7 #8 #10 #11 #18 #19 #20 #21 #22</cite>. Subsequently, GH109 has more recently been shown to demonstrate the same use of a NAD+ cofactor, but this family does not require metal ions or reducing conditions for activity. The hydrolysis of thioglycosides with activated leaving groups has been documented in GH84 <cite>#23</cite>, GH1 <cite>#24 #25 #26 #27 #28 #29</cite>. However, GH4 is currently the only family that has been shown to catalyze the hydrolysis of unactivated thioglycoside substrates <cite>#30</cite>. The remarkable substrate specificities are all feasible due to the β-elimination mechanism discussed below.

==Kinetics and Mechanism==
GH4 enzymes have been shown to be [[retaining]] glycosidases <cite>#31 #32 #33 #34</cite>. However, the enzymes do not utilize the classical [[Koshland double-displacement mechanism]] <cite>#35</cite>. BglT (6-phospho-β-glucosidase) from ''Thermotoga maritima'' <cite>#36</cite> and GlvA (6-phospho-α-glucosidase) from ''Bacillus subtilis'' <cite>#32</cite> are two of the most thoroughly studied enzymes of this family, and both are proposed to utilize an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s. Exchange of the substrate C2 proton with solvent deuterium during the enzyme-catalyzed reaction was the first indication of a C2-H2 bond cleavage in the substrate, which is inconsistent with the double-displacement mechanism <cite>#31 #33</cite>. This fact coupled with the proximity of the NAD+ cofactor to C3 of the glucose 6-phosphate product and the high structural similarity to lactate/malate dehydrogenases as garnered from the x-ray crystallographic data <cite>#9 #31 #33 #34</cite>, led to the following proposed mechanism <cite>#31 #33</cite>: 1) C3 hydride abstraction via the reduction of NAD+ cofactor to NADH and consequent oxidation of the C3 hydroxyl group; 2) adjacent to the ketone functionality, the C2 proton is activated to deprotonation by a near-by catalytic base residue; 3) cleavage of the C1-O1 linkage occurs via an α,β-elimination mechanism, producing an α,β-unsaturated [[intermediate]]; 5) a 1,4-Michael-like addition of a water molecule at C1 takes place; and 6) reduction of the C3 carbonyl functionality by the "on-board" NADH generates the final hydrolysis product.

Primary kinetic isotope effects and the determination of Brønsted relationships revealed that both the C3 hydride and C2 proton abstractions are partially rate-limiting and that cleavage of the C1-O1 linkage is not rate-limiting <cite>#32 #33 #36</cite>. This led to the proposed E1cb mechanism for both BglT and GlvA <cite>#32 #36</cite>.

==Catalytic Residues==
The proposed catalytic residues were mostly derived from x-ray crystallographic data and pH-dependent activity profiles obtained for BglT and GlvA <cite>#31 #32 #33 #36</cite>. A Tyr residue that is conserved in all 6-phospho-α- and 6-phospho-β-glycosidases was found to be approximately 4 Å away from C2 of the reaction product, which can potentially act as a [[general base]] <cite>#31 #32 #33 #34 #36</cite>. The pH-dependent activity profiles were that of double ionization curves with pHopt at approximately 8 <cite>#32 #36</cite>. Two p''K''a values of approximately 7 and 9 were determined <cite>#32 #36</cite>. The p''K''a value of approximately 7 was proposed to correspond to that of the Tyr catalytic base, since this residue would need to be deprotonated for enzyme activity. The position of the Tyr in the enzyme active site was used as a possible explanation for the relatively low p''K''a compared to that of a free Tyr (p''K''a = 10). In the case of BglT, the Tyr residue is located at a distance of 3.4 Å from the glycosidic oxygen, making it ideal to assume a second role in providing [[general acid]] catalysis to the departing oxygen as well as C2 deprotonation <cite>#31 #36</cite>. The p''K''a value of approximately 9 was proposed to correspond to the ionization of the conserved Arg residue(s) that were found to be within hydrogen bonding distance of the phosphate group of the substrate in these two 6-phospho-glycosidases <cite>#31 #34 #36</cite>. Again, the normal p''K''a of 12 for Arg could be lowered to 9, and this matches the expectation that protonated Arg residue(s) would be needed to form electrostatic interactions with the substrate phosphate moiety. Two other conserved residues, Cys and His, were shown to be responsible for chelating the metal ion in BglT and GlvA. In the AglA structure, an Asp residue occupies the same position as the catalytic Tyr base, so the Asp is proposed to take on this role in AglA and possibly other GH4 enzymes that prefer non-phosphorylated substrates.

==Three-dimensional structures==
Crystallographic data is available for a number of GH4 enzymes, including α- and β-members as well as those enzymes specific for phosphorylated and non-phosphorylated substrates. The x-ray crystal structures currently available for this family are those of the ''Thermotoga maritima'' α-glucosidase AglA (PDB 1OBB) <cite>#9</cite>, the ''T. maritima'' α-glucosidase Agu4B (PDB 1VJT) <cite>#37</cite>, the ''Bacillus subtilis'' 6-phospo-α-glucosidase GlvA (PDB 1U8X) <cite>#34</cite>, and two 6-phospho-β-glucosidases: one from ''Geobacillus stearothermophilus'' (PDB 1S6Y) <cite>#37</cite> and other BglT from ''T. maritima'' (PDB 1UP6) <cite>#31 #33</cite>. Each enzyme possesses the characteristic dinucleotide-binding Rossman fold <cite>#38</cite>. The crystal structures of Agu4B (PDB 1VJT) and the 6-phospho-β-glucosidase (PDB 1SY6) from do not provide useful information about the active sites. No ligands are bound in the structure of the 6-phospho-β-glucosidase from ''G. stearothermophilus''. In the Agu4B structure, only the NAD+ cofactor is found at the enzyme active site, but no data for the nicotinamide ring is provided, possibly because it is not well-defined in the crystal structure. The AglA structure is that of an inactive enzyme with no divalent metal ion. The GH4 enzymes show some structural similarities to lactate/malate dehydrogenases as well as dehydratases <cite>#9 #31 #33 #34 #37</cite>.

==Family Firsts==
;'''First stereochemistry determination
:''Thermotoga maritima'' BglT (6-phospho-β-glucosidase) via NMR and HPLC analysis of methyl-glycoside product <cite>#33</cite>
; '''First catalytic residue identification'''
:''Thermotoga maritima'' BglT (6-phospho-β-glucosidase) via x-ray crystal structure <cite>#33</cite>
; '''First 3-D structural determination'''
:''Thermotoga maritima'' AlgA (α-glucosidase) (inactive enzyme) <cite>#9</cite>
:''Thermotoga maritima'' BglT (6-phospho-β-glucosidase) (active enzyme with all cofactors present) <cite>#31 #33</cite>
; '''First GH4 enzyme shown to hydrolyze both α- and β-substrates'''
:''Fusobacterium mortiferum'' MalH (6-phosph-α-glucosidase) <cite>#19</cite>
; '''First β-glycosidase'''
:''Escherichia coli'' CelF (6-phospho-β-glucosidase) <cite>#22</cite>

==References==
<biblio>
#1 pmid=18838391
#2 pmid=9345621
#3 pmid=8687420
#4 pmid=10972187
#5 pmid=9765262
#6 pmid=7730284
#7 pmid=9209025
#8 pmid=11473129
#9 pmid=12588867
#10 pmid=5543331
#11 pmid=2831880
#12 pmid=16712774
#13 pmid=2548811
#14 pmid=4625429
#15 pmid=4721625
#16 pmid=27358
#17 pmid=6774745
#18 pmid=11322729
#19 pmid=11882720
#20 pmid=12062428
#21 pmid=5329906
#22 pmid=10572139
#23 pmid=16332065
#24 pmid=3096349
#25 pmid=9195886
#26 pmid=10978344
#27 pmid=8952475
#28 pmid=3278958
#29 pmid=1731996
#30 pmid=16917793
#31 pmid=15670594
#32 pmid=17676871
#33 pmid=15237973
#34 pmid=15341727
#35 Koshland DE Jr: ''Stereochemistry and the mechanism of enzyme reactions.'' Biol Rev 1953, 28:416-436. [https://doi.org/10.1111/j.1469-185X.1953.tb01386.x DOI:10.1111/j.1469-185X.1953.tb01386.x]
#36 pmid=16401086
#37 pmid=10592235
#38 pmid=4375720
</biblio>

[[Category:Glycoside Hydrolase Families|GH004]]

Glycoside hydrolases

2023-06-28T23:57:04Z

Spencer Williams:

{{CuratorApproved}}
* Authors: [[User:Steve Withers|Steve Withers]], [[User:Spencer Williams|Spencer Williams]]
* Responsible Curator: [[User:Spencer Williams|Spencer Williams]]
----
== Overview ==
Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N- and S-linked glycosides.

[[Image:GHs.png|center|400px]]

== Classification ==
Glycoside hydrolases can be classified in many different ways. The following paragraphs list several different ways, the utility of which depends on the context in which the classification is made and used.

=== Endo/exo ===
''exo''- and ''endo''- refers to the ability of a glycoside hydrolase to cleave a substrate at the end (most frequently, but not always the non-reducing end) or within the middle of a chain <cite>DaviesHenrissat1995</cite>. For example, most cellulases are ''endo''-acting, whereas LacZ β-galactosidase from ''E. coli'' is ''exo''-acting. A general [[sub-site nomenclature]] exists to demarcate substrate binding in glycosidase active-sites.

[[Image:exo_endo.png|center|600px]]

=== Enzyme Commission (EC) number ===
EC numbers are codes representing the Enzyme Commission number. This is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. Every EC number is associated with a recommended name for the respective enzyme. EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number. A necessary consequence of the EC classification scheme is that codes can be applied only to enzymes for which a function has been biochemically identified. Additionally, certain enzymes can catalyze reactions that fall in more than one class. These enzymes must bear more than one EC number.

=== Mechanistic classification ===
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below.<cite>Gebler1992</cite> However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years, as discussed below.

[[Image:Retaining&inverting_GH.png|center|500px]]

=== Sequence-based classification ===
[[Sequence-based classification]] uses algorithmic methods to assign sequences to various families. The glycoside hydrolases have been classified into more than 100 families <cite>Henrissat1991</cite>; this is permanently available through the Carbohydrate Active enZyme database <cite>CAZyURL</cite>. Each family (GH family) contains proteins that are related by sequence, and by corollary, fold. This allows a number of useful predictions to be made since it has long been noted that the catalytic machinery and molecular mechanism is conserved for the vast majority of the glycosidase families <cite>Gebler1992</cite> as well as the geometry around the glycosidic bond (irrespective of naming conventions) <cite>Henrissat1995</cite>. Usually, the mechanism used (ie [[retaining]] or [[inverting]]) is conserved within a GH family. One notable exception is the glycoside hydrolases of family [[GH97]], which contains both retaining and inverting enzymes; a glutamate acts as a [[general base]] in inverting members, whereas an aspartate likely acts as a [[catalytic nucleophile]] in retaining members <cite>Gloster2008</cite>. Another mechanistic curiosity are the glycoside hydrolases of familes [[GH4]] and [[GH109]] which operate through an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s <cite>Yip2007</cite>. This allows a single enzyme to hydrolyze both α- and β-glycosides.

Classification of families into larger groups, termed '[[clans]]' has been proposed <cite>Henrissat1996</cite>. A '[[clan]]' is a group of families that possess significant similarity in their tertiary structure, catalytic residues and mechanism. Families within clans are thought to have a common evolutionary ancestry. For an updated table of glycoside hydrolase clans see the CAZy Database <cite>CAZyURL</cite>.

== Mechanism ==
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below <cite>Koshland1953</cite>. However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years.

===Inverting glycoside hydrolases===
Hydrolysis of a glycoside with net inversion of anomeric configuration is generally achieved via a one step, single-displacement mechanism involving [[oxocarbenium ion]]-like [[transition state]]s, as shown below. The reaction typically occurs with [[general acid]] and [[general base]] assistance from two amino acid side chains, normally glutamic or aspartic acids, that are typically located 6-11 A apart<cite>McCarter1994</cite>.

[[Image:Inverting glucosidase mechanism.png|centre|600px]]

====Glycosyl-phosphate cleaving enzymes that lack a general acid====
A subset of family [[GH92]] α-mannosidases catalyze the hydrolysis of mannose-1-phosphate linkages found in the mannose-1-phosphate-6-mannose groups of yeast mannoproteins. In these enzymes the usual [[general acid]] glutamic acid found in other members of this family is replaced by a glutamine. It has been suggested that the phosphate aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst <cite>Tiels2012</cite>. This replacement may also reduce charge repulsion between the glutamic acid residue and the anionic phosphate aglycon. A related example may be found in the case of the family [[GH1]] myrosinases.

===Retaining glycoside hydrolases===
====Classical Koshland retaining mechanism====
Hydrolysis with net retention of configuration is most commonly achieved via a two step, double-displacement mechanism involving a covalent glycosyl-enzyme [[intermediate]], as is shown in the figure below. Each step passes through an [[oxocarbenium ion]]-like [[transition state]]. Reaction occurs with acid/base and nucleophilic assistance provided by two amino acid side chains, typically glutamate or aspartate, located 5.5 A apart. In the first step (often called the glycosylation step), one residue plays the role of a nucleophile, attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme [[intermediate]]. At the same time the other residue functions as an acid catalyst and protonates the glycosidic oxygen as the bond cleaves. In the second step (known as the deglycosylation step), the glycosyl enzyme is hydrolyzed by water, with the other residue now acting as a base catalyst deprotonating the water molecule as it attacks. The pKa value of the acid/base group cycles between high and low values during catalysis to optimize it for its role at each step of catalysis <cite>McIntosh1996</cite>. In the case of sialidases, the catalytic nucleophile is a tyrosine residue (see below). This mechanism was originally proposed by Dan Koshland, although at the time the identities of the residues was unclear <cite>Koshland1953</cite>.

[[Image:Retaining_glycosidase_mechanism.png|centre|600px]]

====Neighboring group participation====
'''1. By a neighboring 2-acetamido group''' 
Enzymes of glycoside hydrolase families [[Glycoside Hydrolase Family 18|18]], [[Glycoside Hydrolase Family 20|20]], [[Glycoside Hydrolase Family 25|25]], [[Glycoside Hydrolase Family 56|56]], [[Glycoside Hydrolase Family 84|84]], [[Glycoside Hydrolase Family 85|85]] and [[Glycoside Hydrolase Family 123|123]] hydrolyse substrates containing an ''N''-acetyl (acetamido) or ''N''-glycolyl group at the 2-position. These enzymes have no catalytic nucleophile: rather they utilize a mechanism in which the 2-acetamido group acts as an intramolecular nucleophile. Neighboring group participation by the 2-acetamido group leads to formation of an oxazoline (or more strictly an [[oxazolinium ion]]) [[intermediate]]. This mechanism was deduced from X-ray structures of complexes of chitinases with natural inhibitors <cite>Terwisscha1995</cite>, from the potent inhibition afforded by a stable thiazoline analogue of the oxazoline <cite>Knapp1996 Mark2001</cite>, and from detailed mechanistic analyses using substrates of modified reactivity <cite>Vocadlo2005</cite>. Typically, a stabilizing residue (a carboxylate) stabilizes the charge development in the [[transition state]]. Not all enzymes that cleave substrates possessing a 2-acetamido group utilize a neighboring groups participation mechanism; enzyme of glycoside hydrolase families [[Glycoside Hydrolase Family 3|3]] and [[Glycoside Hydrolase Family 22|22]] utilize a classical retaining mechanism with an enzymic nucleophile. Other hexosaminidases such as those of [[Glycoside Hydrolase Family 19]] utilize an inverting mechanism.

[[Image:Hex_neighboring_mechanism.png|centre|700px]]

'''2. By a neighboring 2-hydroxyl group''' 
Enzymes of glycoside hydrolase family [[Glycoside Hydrolase Family 99|99]] hydrolyse α-mannoside substrates and lack a enzymic catalytic nucleophile. These enzymes use a mechanism in which the 2-hydroxyl group acts as an intramolecular nucleophile. Neighboring group participation by the 2-hydroxy group leads to formation of an epoxide (or more strictly a 1,2-anhydro sugar). This mechanism was implicated from 3D X-ray structures of complexes of bacterial endomannosidase/endomannanase with iminosugar inhibitors <cite>Thompson2012</cite>. Detailed mechanistic analyses using carbocyclic analogues of the 1,2-anhydro sugar, quantum mechanic/molecular mechanics computational modelling, and kinetic isotope effects <cite>Sobala2020</cite>.

====Myrosinases: Retaining glycoside hydrolases that lack a general acid and utilize an exogenous base====
Glycoside hydrolases termed myrosinases catalyze the hydrolysis of anionic thioglycosides (glucosinolates) found in plants. They are found in [[Glycoside Hydrolase Family 1]]. In these enzymes the usual acid/base glutamic acid found in other members of this family is replaced by a glutamine. This likely reduces charge repulsion between the anionic aglycon sulfate. The unusual aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst. However, since a base catalyst is required for the second step (hydrolysis or deglycosylation) these enzymes require an alternative basic group. This is provided by the co-enzyme L-ascorbate <cite>Burmeister2000</cite>.

[[Image:myrosinase.png|centre|550px]]

====Alternative nucleophiles====
Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of glycoside hydrolase families [[Glycoside Hydrolase Family 33|33]] and [[Glycoside Hydrolase Family 34|34]], and 2-keto-3-deoxy-D-lyxo-heptulosaric acid hydrolases of [[glycoside hydrolase family 143|143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>.

[[Image:sialidase_mechanism.png|center|700px]]

====NAD-dependent hydrolysis====
The glycoside hydrolases of family [[Glycoside Hydrolase Family 4|4]], [[Glycoside Hydrolase Family 109|109]], [[Glycoside Hydrolase Family 177|177]] and [[Glycoside Hydrolase Family 179|179]] use a mechanism that requires an NAD cofactor, which remains tightly bound throughout catalysis. The mechanism proceeds via anionic [[transition state]]s with elimination and redox steps rather than the classical mechanisms proceeding through[[oxocarbenium ion]]-like transition states. As shown below for a 6-phospho-β-glucosidase, the mechanism involves an initial oxidation of the 3-hydroxyl of the substrate by the enzyme-bound NAD cofactor. This increases the acidity of the C2 proton such that an E1cb elimination can occur with assistance from an enzymatic base. The α,β-unsaturated [[intermediate]] formed then undergoes addition of water at the anomeric centre and finally the ketone at C3 is reduced to generate the free sugar product. Thus, even though glycosidic bond cleavage occurred via an elimination mechanism, the overall reaction is hydrolysis. This mechanism was elucidated through a combination of stereochemical studies by NMR, kinetic isotope effects, linear free energy relationships, X-ray crystallography and UV/Vis spectrophotometry <cite>Yip2004 Rajan2004</cite>.

[[Image:Family_4_mechanism.png|center|700px]]

== References ==
<biblio>
#McCarter1994 pmid=7712292
#Koshland1953 Koshland, D. (1953) ''Biol. Rev.'' 28, 416.
#McIntosh1996 pmid=8756457
#Gebler1992 pmid=1618761
#Henrissat1995 pmid=7624375
#Terwisscha1995 pmid=7495789
#Mark2001 pmid=11124970
#Knapp1996 Knapp, S., Vocadlo, D., Gao, Z. N., Kirk, B., Lou, J. P., and Withers, S. G. (1996) ''Journal of the American Chemical Society 118'', 6804-6805.
#Vocadlo2005 pmid=16171396
#Burmeister2000 pmid=10978344
#Amaya2004 pmid=15130470
#Watts2003 pmid=12812490
#Watson2003 pmid=14580216
#Yip2004 pmid=15237973
#Rajan2004 pmid=15341727
#CAZyURL Carbohydrate Active Enzymes database; URL http://www.cazy.org/
#Henrissat1991 pmid=1747104
#Henrissat1996 pmid=8687420
#Gloster2008 pmid=18848471
#Yip2007 pmid=17676871
#Tiels2012 pmid=23159880
#DaviesHenrissat1995 pmid=8535779
#Ndeh2017 pmid=28329766
#Sobala2020 pmid=32490192
#Thompson2012 pmid=22219371

</biblio>
[[Category:Definitions and explanations]]

Glycoside hydrolases

2023-06-28T23:55:25Z

Spencer Williams: /* References */

{{CuratorApproved}}
* Authors: [[User:Steve Withers|Steve Withers]], [[User:Spencer Williams|Spencer Williams]]
* Responsible Curator: [[User:Spencer Williams|Spencer Williams]]
----
== Overview ==
Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N- and S-linked glycosides.

[[Image:GHs.png|center|400px]]

== Classification ==
Glycoside hydrolases can be classified in many different ways. The following paragraphs list several different ways, the utility of which depends on the context in which the classification is made and used.

=== Endo/exo ===
''exo''- and ''endo''- refers to the ability of a glycoside hydrolase to cleave a substrate at the end (most frequently, but not always the non-reducing end) or within the middle of a chain <cite>DaviesHenrissat1995</cite>. For example, most cellulases are ''endo''-acting, whereas LacZ β-galactosidase from ''E. coli'' is ''exo''-acting. A general [[sub-site nomenclature]] exists to demarcate substrate binding in glycosidase active-sites.

[[Image:exo_endo.png|center|600px]]

=== Enzyme Commission (EC) number ===
EC numbers are codes representing the Enzyme Commission number. This is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. Every EC number is associated with a recommended name for the respective enzyme. EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number. A necessary consequence of the EC classification scheme is that codes can be applied only to enzymes for which a function has been biochemically identified. Additionally, certain enzymes can catalyze reactions that fall in more than one class. These enzymes must bear more than one EC number.

=== Mechanistic classification ===
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below.<cite>Gebler1992</cite> However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years, as discussed below.

[[Image:Retaining&inverting_GH.png|center|500px]]

=== Sequence-based classification ===
[[Sequence-based classification]] uses algorithmic methods to assign sequences to various families. The glycoside hydrolases have been classified into more than 100 families <cite>Henrissat1991</cite>; this is permanently available through the Carbohydrate Active enZyme database <cite>CAZyURL</cite>. Each family (GH family) contains proteins that are related by sequence, and by corollary, fold. This allows a number of useful predictions to be made since it has long been noted that the catalytic machinery and molecular mechanism is conserved for the vast majority of the glycosidase families <cite>Gebler1992</cite> as well as the geometry around the glycosidic bond (irrespective of naming conventions) <cite>Henrissat1995</cite>. Usually, the mechanism used (ie [[retaining]] or [[inverting]]) is conserved within a GH family. One notable exception is the glycoside hydrolases of family [[GH97]], which contains both retaining and inverting enzymes; a glutamate acts as a [[general base]] in inverting members, whereas an aspartate likely acts as a [[catalytic nucleophile]] in retaining members <cite>Gloster2008</cite>. Another mechanistic curiosity are the glycoside hydrolases of familes [[GH4]] and [[GH109]] which operate through an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s <cite>Yip2007</cite>. This allows a single enzyme to hydrolyze both α- and β-glycosides.

Classification of families into larger groups, termed '[[clans]]' has been proposed <cite>Henrissat1996</cite>. A '[[clan]]' is a group of families that possess significant similarity in their tertiary structure, catalytic residues and mechanism. Families within clans are thought to have a common evolutionary ancestry. For an updated table of glycoside hydrolase clans see the CAZy Database <cite>CAZyURL</cite>.

== Mechanism ==
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below <cite>Koshland1953</cite>. However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years.

===Inverting glycoside hydrolases===
Hydrolysis of a glycoside with net inversion of anomeric configuration is generally achieved via a one step, single-displacement mechanism involving [[oxocarbenium ion]]-like [[transition state]]s, as shown below. The reaction typically occurs with [[general acid]] and [[general base]] assistance from two amino acid side chains, normally glutamic or aspartic acids, that are typically located 6-11 A apart<cite>McCarter1994</cite>.

[[Image:Inverting glucosidase mechanism.png|centre|600px]]

====Glycosyl-phosphate cleaving enzymes that lack a general acid====
A subset of family [[GH92]] α-mannosidases catalyze the hydrolysis of mannose-1-phosphate linkages found in the mannose-1-phosphate-6-mannose groups of yeast mannoproteins. In these enzymes the usual [[general acid]] glutamic acid found in other members of this family is replaced by a glutamine. It has been suggested that the phosphate aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst <cite>Tiels2012</cite>. This replacement may also reduce charge repulsion between the glutamic acid residue and the anionic phosphate aglycon. A related example may be found in the case of the family [[GH1]] myrosinases.

===Retaining glycoside hydrolases===
====Classical Koshland retaining mechanism====
Hydrolysis with net retention of configuration is most commonly achieved via a two step, double-displacement mechanism involving a covalent glycosyl-enzyme [[intermediate]], as is shown in the figure below. Each step passes through an [[oxocarbenium ion]]-like [[transition state]]. Reaction occurs with acid/base and nucleophilic assistance provided by two amino acid side chains, typically glutamate or aspartate, located 5.5 A apart. In the first step (often called the glycosylation step), one residue plays the role of a nucleophile, attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme [[intermediate]]. At the same time the other residue functions as an acid catalyst and protonates the glycosidic oxygen as the bond cleaves. In the second step (known as the deglycosylation step), the glycosyl enzyme is hydrolyzed by water, with the other residue now acting as a base catalyst deprotonating the water molecule as it attacks. The pKa value of the acid/base group cycles between high and low values during catalysis to optimize it for its role at each step of catalysis <cite>McIntosh1996</cite>. In the case of sialidases, the catalytic nucleophile is a tyrosine residue (see below). This mechanism was originally proposed by Dan Koshland, although at the time the identities of the residues was unclear <cite>Koshland1953</cite>.

[[Image:Retaining_glycosidase_mechanism.png|centre|600px]]

====Neighboring group participation====
'''1. By a neighboring 2-acetamido group''' 
Enzymes of glycoside hydrolase families [[Glycoside Hydrolase Family 18|18]], [[Glycoside Hydrolase Family 20|20]], [[Glycoside Hydrolase Family 25|25]], [[Glycoside Hydrolase Family 56|56]], [[Glycoside Hydrolase Family 84|84]], [[Glycoside Hydrolase Family 85|85]] and [[Glycoside Hydrolase Family 123|123]] hydrolyse substrates containing an ''N''-acetyl (acetamido) or ''N''-glycolyl group at the 2-position. These enzymes have no catalytic nucleophile: rather they utilize a mechanism in which the 2-acetamido group acts as an intramolecular nucleophile. Neighboring group participation by the 2-acetamido group leads to formation of an oxazoline (or more strictly an [[oxazolinium ion]]) [[intermediate]]. This mechanism was deduced from X-ray structures of complexes of chitinases with natural inhibitors <cite>Terwisscha1995</cite>, from the potent inhibition afforded by a stable thiazoline analogue of the oxazoline <cite>Knapp1996 Mark2001</cite>, and from detailed mechanistic analyses using substrates of modified reactivity <cite>Vocadlo2005</cite>. Typically, a stabilizing residue (a carboxylate) stabilizes the charge development in the [[transition state]]. Not all enzymes that cleave substrates possessing a 2-acetamido group utilize a neighboring groups participation mechanism; enzyme of glycoside hydrolase families [[Glycoside Hydrolase Family 3|3]] and [[Glycoside Hydrolase Family 22|22]] utilize a classical retaining mechanism with an enzymic nucleophile. Other hexosaminidases such as those of [[Glycoside Hydrolase Family 19]] utilize an inverting mechanism.

[[Image:Hex_neighboring_mechanism.png|centre|700px]]

'''2. By a neighboring 2-hydroxyl group''' 
Enzymes of glycoside hydrolase family [[Glycoside Hydrolase Family 99|99]] hydrolyse a-mannoside substrates and lack a enzymic catalytic nucleophile. These enzymes use a mechanism in which the 2-hydroxyl group acts as an intramolecular nucleophile. Neighboring group participation by the 2-hydroxy group leads to formation of an epoxide (or more strictly a 1,2-anhydro sugar). This mechanism was implicated from 3D X-ray structures of complexes of bacterial endomannosidase/endomannanase with iminosugar inhibitors <cite>Thompson2012</cite>. Detailed mechanistic analyses using carbocyclic analogues of the 1,2-anhydro sugar, quantum mechanic/molecular mechanics computational modelling, and kinetic isotope effects <cite>Sobala2020</cite>.

====Myrosinases: Retaining glycoside hydrolases that lack a general acid and utilize an exogenous base====
Glycoside hydrolases termed myrosinases catalyze the hydrolysis of anionic thioglycosides (glucosinolates) found in plants. They are found in [[Glycoside Hydrolase Family 1]]. In these enzymes the usual acid/base glutamic acid found in other members of this family is replaced by a glutamine. This likely reduces charge repulsion between the anionic aglycon sulfate. The unusual aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst. However, since a base catalyst is required for the second step (hydrolysis or deglycosylation) these enzymes require an alternative basic group. This is provided by the co-enzyme L-ascorbate <cite>Burmeister2000</cite>.

[[Image:myrosinase.png|centre|550px]]

====Alternative nucleophiles====
Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of glycoside hydrolase families [[Glycoside Hydrolase Family 33|33]] and [[Glycoside Hydrolase Family 34|34]], and 2-keto-3-deoxy-d-lyxo-heptulosaric acid hydrolases of [[glycoside hydrolase family 143|143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>.

[[Image:sialidase_mechanism.png|center|700px]]

====NAD-dependent hydrolysis====
The glycoside hydrolases of family [[Glycoside Hydrolase Family 4|4]], [[Glycoside Hydrolase Family 109|109]], [[Glycoside Hydrolase Family 177|177]] and [[Glycoside Hydrolase Family 179|179]] use a mechanism that requires an NAD cofactor, which remains tightly bound throughout catalysis. The mechanism proceeds via anionic [[transition state]]s with elimination and redox steps rather than the classical mechanisms proceeding through[[oxocarbenium ion]]-like transition states. As shown below for a 6-phospho-β-glucosidase, the mechanism involves an initial oxidation of the 3-hydroxyl of the substrate by the enzyme-bound NAD cofactor. This increases the acidity of the C2 proton such that an E1cb elimination can occur with assistance from an enzymatic base. The α,β-unsaturated [[intermediate]] formed then undergoes addition of water at the anomeric centre and finally the ketone at C3 is reduced to generate the free sugar product. Thus, even though glycosidic bond cleavage occurred via an elimination mechanism, the overall reaction is hydrolysis. This mechanism was elucidated through a combination of stereochemical studies by NMR, kinetic isotope effects, linear free energy relationships, X-ray crystallography and UV/Vis spectrophotometry <cite>Yip2004 Rajan2004</cite>.

[[Image:Family_4_mechanism.png|center|700px]]

== References ==
<biblio>
#McCarter1994 pmid=7712292
#Koshland1953 Koshland, D. (1953) ''Biol. Rev.'' 28, 416.
#McIntosh1996 pmid=8756457
#Gebler1992 pmid=1618761
#Henrissat1995 pmid=7624375
#Terwisscha1995 pmid=7495789
#Mark2001 pmid=11124970
#Knapp1996 Knapp, S., Vocadlo, D., Gao, Z. N., Kirk, B., Lou, J. P., and Withers, S. G. (1996) ''Journal of the American Chemical Society 118'', 6804-6805.
#Vocadlo2005 pmid=16171396
#Burmeister2000 pmid=10978344
#Amaya2004 pmid=15130470
#Watts2003 pmid=12812490
#Watson2003 pmid=14580216
#Yip2004 pmid=15237973
#Rajan2004 pmid=15341727
#CAZyURL Carbohydrate Active Enzymes database; URL http://www.cazy.org/
#Henrissat1991 pmid=1747104
#Henrissat1996 pmid=8687420
#Gloster2008 pmid=18848471
#Yip2007 pmid=17676871
#Tiels2012 pmid=23159880
#DaviesHenrissat1995 pmid=8535779
#Ndeh2017 pmid=28329766
#Sobala2020 pmid=32490192
#Thompson2012 pmid=22219371

</biblio>
[[Category:Definitions and explanations]]

Glycoside hydrolases

2023-06-28T23:54:52Z

Spencer Williams: /* Neighboring group participation */

{{CuratorApproved}}
* Authors: [[User:Steve Withers|Steve Withers]], [[User:Spencer Williams|Spencer Williams]]
* Responsible Curator: [[User:Spencer Williams|Spencer Williams]]
----
== Overview ==
Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N- and S-linked glycosides.

[[Image:GHs.png|center|400px]]

== Classification ==
Glycoside hydrolases can be classified in many different ways. The following paragraphs list several different ways, the utility of which depends on the context in which the classification is made and used.

=== Endo/exo ===
''exo''- and ''endo''- refers to the ability of a glycoside hydrolase to cleave a substrate at the end (most frequently, but not always the non-reducing end) or within the middle of a chain <cite>DaviesHenrissat1995</cite>. For example, most cellulases are ''endo''-acting, whereas LacZ β-galactosidase from ''E. coli'' is ''exo''-acting. A general [[sub-site nomenclature]] exists to demarcate substrate binding in glycosidase active-sites.

[[Image:exo_endo.png|center|600px]]

=== Enzyme Commission (EC) number ===
EC numbers are codes representing the Enzyme Commission number. This is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. Every EC number is associated with a recommended name for the respective enzyme. EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number. A necessary consequence of the EC classification scheme is that codes can be applied only to enzymes for which a function has been biochemically identified. Additionally, certain enzymes can catalyze reactions that fall in more than one class. These enzymes must bear more than one EC number.

=== Mechanistic classification ===
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below.<cite>Gebler1992</cite> However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years, as discussed below.

[[Image:Retaining&inverting_GH.png|center|500px]]

=== Sequence-based classification ===
[[Sequence-based classification]] uses algorithmic methods to assign sequences to various families. The glycoside hydrolases have been classified into more than 100 families <cite>Henrissat1991</cite>; this is permanently available through the Carbohydrate Active enZyme database <cite>CAZyURL</cite>. Each family (GH family) contains proteins that are related by sequence, and by corollary, fold. This allows a number of useful predictions to be made since it has long been noted that the catalytic machinery and molecular mechanism is conserved for the vast majority of the glycosidase families <cite>Gebler1992</cite> as well as the geometry around the glycosidic bond (irrespective of naming conventions) <cite>Henrissat1995</cite>. Usually, the mechanism used (ie [[retaining]] or [[inverting]]) is conserved within a GH family. One notable exception is the glycoside hydrolases of family [[GH97]], which contains both retaining and inverting enzymes; a glutamate acts as a [[general base]] in inverting members, whereas an aspartate likely acts as a [[catalytic nucleophile]] in retaining members <cite>Gloster2008</cite>. Another mechanistic curiosity are the glycoside hydrolases of familes [[GH4]] and [[GH109]] which operate through an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s <cite>Yip2007</cite>. This allows a single enzyme to hydrolyze both α- and β-glycosides.

Classification of families into larger groups, termed '[[clans]]' has been proposed <cite>Henrissat1996</cite>. A '[[clan]]' is a group of families that possess significant similarity in their tertiary structure, catalytic residues and mechanism. Families within clans are thought to have a common evolutionary ancestry. For an updated table of glycoside hydrolase clans see the CAZy Database <cite>CAZyURL</cite>.

== Mechanism ==
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below <cite>Koshland1953</cite>. However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years.

===Inverting glycoside hydrolases===
Hydrolysis of a glycoside with net inversion of anomeric configuration is generally achieved via a one step, single-displacement mechanism involving [[oxocarbenium ion]]-like [[transition state]]s, as shown below. The reaction typically occurs with [[general acid]] and [[general base]] assistance from two amino acid side chains, normally glutamic or aspartic acids, that are typically located 6-11 A apart<cite>McCarter1994</cite>.

[[Image:Inverting glucosidase mechanism.png|centre|600px]]

====Glycosyl-phosphate cleaving enzymes that lack a general acid====
A subset of family [[GH92]] α-mannosidases catalyze the hydrolysis of mannose-1-phosphate linkages found in the mannose-1-phosphate-6-mannose groups of yeast mannoproteins. In these enzymes the usual [[general acid]] glutamic acid found in other members of this family is replaced by a glutamine. It has been suggested that the phosphate aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst <cite>Tiels2012</cite>. This replacement may also reduce charge repulsion between the glutamic acid residue and the anionic phosphate aglycon. A related example may be found in the case of the family [[GH1]] myrosinases.

===Retaining glycoside hydrolases===
====Classical Koshland retaining mechanism====
Hydrolysis with net retention of configuration is most commonly achieved via a two step, double-displacement mechanism involving a covalent glycosyl-enzyme [[intermediate]], as is shown in the figure below. Each step passes through an [[oxocarbenium ion]]-like [[transition state]]. Reaction occurs with acid/base and nucleophilic assistance provided by two amino acid side chains, typically glutamate or aspartate, located 5.5 A apart. In the first step (often called the glycosylation step), one residue plays the role of a nucleophile, attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme [[intermediate]]. At the same time the other residue functions as an acid catalyst and protonates the glycosidic oxygen as the bond cleaves. In the second step (known as the deglycosylation step), the glycosyl enzyme is hydrolyzed by water, with the other residue now acting as a base catalyst deprotonating the water molecule as it attacks. The pKa value of the acid/base group cycles between high and low values during catalysis to optimize it for its role at each step of catalysis <cite>McIntosh1996</cite>. In the case of sialidases, the catalytic nucleophile is a tyrosine residue (see below). This mechanism was originally proposed by Dan Koshland, although at the time the identities of the residues was unclear <cite>Koshland1953</cite>.

[[Image:Retaining_glycosidase_mechanism.png|centre|600px]]

====Neighboring group participation====
'''1. By a neighboring 2-acetamido group''' 
Enzymes of glycoside hydrolase families [[Glycoside Hydrolase Family 18|18]], [[Glycoside Hydrolase Family 20|20]], [[Glycoside Hydrolase Family 25|25]], [[Glycoside Hydrolase Family 56|56]], [[Glycoside Hydrolase Family 84|84]], [[Glycoside Hydrolase Family 85|85]] and [[Glycoside Hydrolase Family 123|123]] hydrolyse substrates containing an ''N''-acetyl (acetamido) or ''N''-glycolyl group at the 2-position. These enzymes have no catalytic nucleophile: rather they utilize a mechanism in which the 2-acetamido group acts as an intramolecular nucleophile. Neighboring group participation by the 2-acetamido group leads to formation of an oxazoline (or more strictly an [[oxazolinium ion]]) [[intermediate]]. This mechanism was deduced from X-ray structures of complexes of chitinases with natural inhibitors <cite>Terwisscha1995</cite>, from the potent inhibition afforded by a stable thiazoline analogue of the oxazoline <cite>Knapp1996 Mark2001</cite>, and from detailed mechanistic analyses using substrates of modified reactivity <cite>Vocadlo2005</cite>. Typically, a stabilizing residue (a carboxylate) stabilizes the charge development in the [[transition state]]. Not all enzymes that cleave substrates possessing a 2-acetamido group utilize a neighboring groups participation mechanism; enzyme of glycoside hydrolase families [[Glycoside Hydrolase Family 3|3]] and [[Glycoside Hydrolase Family 22|22]] utilize a classical retaining mechanism with an enzymic nucleophile. Other hexosaminidases such as those of [[Glycoside Hydrolase Family 19]] utilize an inverting mechanism.

[[Image:Hex_neighboring_mechanism.png|centre|700px]]

'''2. By a neighboring 2-hydroxyl group''' 
Enzymes of glycoside hydrolase family [[Glycoside Hydrolase Family 99|99]] hydrolyse a-mannoside substrates and lack a enzymic catalytic nucleophile. These enzymes use a mechanism in which the 2-hydroxyl group acts as an intramolecular nucleophile. Neighboring group participation by the 2-hydroxy group leads to formation of an epoxide (or more strictly a 1,2-anhydro sugar). This mechanism was implicated from 3D X-ray structures of complexes of bacterial endomannosidase/endomannanase with iminosugar inhibitors <cite>Thompson2012</cite>. Detailed mechanistic analyses using carbocyclic analogues of the 1,2-anhydro sugar, quantum mechanic/molecular mechanics computational modelling, and kinetic isotope effects <cite>Sobala2020</cite>.

====Myrosinases: Retaining glycoside hydrolases that lack a general acid and utilize an exogenous base====
Glycoside hydrolases termed myrosinases catalyze the hydrolysis of anionic thioglycosides (glucosinolates) found in plants. They are found in [[Glycoside Hydrolase Family 1]]. In these enzymes the usual acid/base glutamic acid found in other members of this family is replaced by a glutamine. This likely reduces charge repulsion between the anionic aglycon sulfate. The unusual aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst. However, since a base catalyst is required for the second step (hydrolysis or deglycosylation) these enzymes require an alternative basic group. This is provided by the co-enzyme L-ascorbate <cite>Burmeister2000</cite>.

[[Image:myrosinase.png|centre|550px]]

====Alternative nucleophiles====
Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of glycoside hydrolase families [[Glycoside Hydrolase Family 33|33]] and [[Glycoside Hydrolase Family 34|34]], and 2-keto-3-deoxy-d-lyxo-heptulosaric acid hydrolases of [[glycoside hydrolase family 143|143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>.

[[Image:sialidase_mechanism.png|center|700px]]

====NAD-dependent hydrolysis====
The glycoside hydrolases of family [[Glycoside Hydrolase Family 4|4]], [[Glycoside Hydrolase Family 109|109]], [[Glycoside Hydrolase Family 177|177]] and [[Glycoside Hydrolase Family 179|179]] use a mechanism that requires an NAD cofactor, which remains tightly bound throughout catalysis. The mechanism proceeds via anionic [[transition state]]s with elimination and redox steps rather than the classical mechanisms proceeding through[[oxocarbenium ion]]-like transition states. As shown below for a 6-phospho-β-glucosidase, the mechanism involves an initial oxidation of the 3-hydroxyl of the substrate by the enzyme-bound NAD cofactor. This increases the acidity of the C2 proton such that an E1cb elimination can occur with assistance from an enzymatic base. The α,β-unsaturated [[intermediate]] formed then undergoes addition of water at the anomeric centre and finally the ketone at C3 is reduced to generate the free sugar product. Thus, even though glycosidic bond cleavage occurred via an elimination mechanism, the overall reaction is hydrolysis. This mechanism was elucidated through a combination of stereochemical studies by NMR, kinetic isotope effects, linear free energy relationships, X-ray crystallography and UV/Vis spectrophotometry <cite>Yip2004 Rajan2004</cite>.

[[Image:Family_4_mechanism.png|center|700px]]

== References ==
<biblio>
#McCarter1994 pmid=7712292
#Koshland1953 Koshland, D. (1953) ''Biol. Rev.'' 28, 416.
#McIntosh1996 pmid=8756457
#Gebler1992 pmid=1618761
#Henrissat1995 pmid=7624375
#Terwisscha1995 pmid=7495789
#Mark2001 pmid=11124970
#Knapp1996 Knapp, S., Vocadlo, D., Gao, Z. N., Kirk, B., Lou, J. P., and Withers, S. G. (1996) ''Journal of the American Chemical Society 118'', 6804-6805.
#Vocadlo2005 pmid=16171396
#Burmeister2000 pmid=10978344
#Amaya2004 pmid=15130470
#Watts2003 pmid=12812490
#Watson2003 pmid=14580216
#Yip2004 pmid=15237973
#Rajan2004 pmid=15341727
#CAZyURL Carbohydrate Active Enzymes database; URL http://www.cazy.org/
#Henrissat1991 pmid=1747104
#Henrissat1996 pmid=8687420
#Gloster2008 pmid=18848471
#Yip2007 pmid=17676871
#Tiels2012 pmid=23159880
#DaviesHenrissat1995 pmid=8535779
#Ndeh2017 pmid=28329766
</biblio>
[[Category:Definitions and explanations]]

Glycoside hydrolases

2023-06-28T23:54:13Z

Spencer Williams: /* Neighboring group participation */

{{CuratorApproved}}
* Authors: [[User:Steve Withers|Steve Withers]], [[User:Spencer Williams|Spencer Williams]]
* Responsible Curator: [[User:Spencer Williams|Spencer Williams]]
----
== Overview ==
Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N- and S-linked glycosides.

[[Image:GHs.png|center|400px]]

== Classification ==
Glycoside hydrolases can be classified in many different ways. The following paragraphs list several different ways, the utility of which depends on the context in which the classification is made and used.

=== Endo/exo ===
''exo''- and ''endo''- refers to the ability of a glycoside hydrolase to cleave a substrate at the end (most frequently, but not always the non-reducing end) or within the middle of a chain <cite>DaviesHenrissat1995</cite>. For example, most cellulases are ''endo''-acting, whereas LacZ β-galactosidase from ''E. coli'' is ''exo''-acting. A general [[sub-site nomenclature]] exists to demarcate substrate binding in glycosidase active-sites.

[[Image:exo_endo.png|center|600px]]

=== Enzyme Commission (EC) number ===
EC numbers are codes representing the Enzyme Commission number. This is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. Every EC number is associated with a recommended name for the respective enzyme. EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number. A necessary consequence of the EC classification scheme is that codes can be applied only to enzymes for which a function has been biochemically identified. Additionally, certain enzymes can catalyze reactions that fall in more than one class. These enzymes must bear more than one EC number.

=== Mechanistic classification ===
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below.<cite>Gebler1992</cite> However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years, as discussed below.

[[Image:Retaining&inverting_GH.png|center|500px]]

=== Sequence-based classification ===
[[Sequence-based classification]] uses algorithmic methods to assign sequences to various families. The glycoside hydrolases have been classified into more than 100 families <cite>Henrissat1991</cite>; this is permanently available through the Carbohydrate Active enZyme database <cite>CAZyURL</cite>. Each family (GH family) contains proteins that are related by sequence, and by corollary, fold. This allows a number of useful predictions to be made since it has long been noted that the catalytic machinery and molecular mechanism is conserved for the vast majority of the glycosidase families <cite>Gebler1992</cite> as well as the geometry around the glycosidic bond (irrespective of naming conventions) <cite>Henrissat1995</cite>. Usually, the mechanism used (ie [[retaining]] or [[inverting]]) is conserved within a GH family. One notable exception is the glycoside hydrolases of family [[GH97]], which contains both retaining and inverting enzymes; a glutamate acts as a [[general base]] in inverting members, whereas an aspartate likely acts as a [[catalytic nucleophile]] in retaining members <cite>Gloster2008</cite>. Another mechanistic curiosity are the glycoside hydrolases of familes [[GH4]] and [[GH109]] which operate through an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s <cite>Yip2007</cite>. This allows a single enzyme to hydrolyze both α- and β-glycosides.

Classification of families into larger groups, termed '[[clans]]' has been proposed <cite>Henrissat1996</cite>. A '[[clan]]' is a group of families that possess significant similarity in their tertiary structure, catalytic residues and mechanism. Families within clans are thought to have a common evolutionary ancestry. For an updated table of glycoside hydrolase clans see the CAZy Database <cite>CAZyURL</cite>.

== Mechanism ==
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below <cite>Koshland1953</cite>. However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years.

===Inverting glycoside hydrolases===
Hydrolysis of a glycoside with net inversion of anomeric configuration is generally achieved via a one step, single-displacement mechanism involving [[oxocarbenium ion]]-like [[transition state]]s, as shown below. The reaction typically occurs with [[general acid]] and [[general base]] assistance from two amino acid side chains, normally glutamic or aspartic acids, that are typically located 6-11 A apart<cite>McCarter1994</cite>.

[[Image:Inverting glucosidase mechanism.png|centre|600px]]

====Glycosyl-phosphate cleaving enzymes that lack a general acid====
A subset of family [[GH92]] α-mannosidases catalyze the hydrolysis of mannose-1-phosphate linkages found in the mannose-1-phosphate-6-mannose groups of yeast mannoproteins. In these enzymes the usual [[general acid]] glutamic acid found in other members of this family is replaced by a glutamine. It has been suggested that the phosphate aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst <cite>Tiels2012</cite>. This replacement may also reduce charge repulsion between the glutamic acid residue and the anionic phosphate aglycon. A related example may be found in the case of the family [[GH1]] myrosinases.

===Retaining glycoside hydrolases===
====Classical Koshland retaining mechanism====
Hydrolysis with net retention of configuration is most commonly achieved via a two step, double-displacement mechanism involving a covalent glycosyl-enzyme [[intermediate]], as is shown in the figure below. Each step passes through an [[oxocarbenium ion]]-like [[transition state]]. Reaction occurs with acid/base and nucleophilic assistance provided by two amino acid side chains, typically glutamate or aspartate, located 5.5 A apart. In the first step (often called the glycosylation step), one residue plays the role of a nucleophile, attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme [[intermediate]]. At the same time the other residue functions as an acid catalyst and protonates the glycosidic oxygen as the bond cleaves. In the second step (known as the deglycosylation step), the glycosyl enzyme is hydrolyzed by water, with the other residue now acting as a base catalyst deprotonating the water molecule as it attacks. The pKa value of the acid/base group cycles between high and low values during catalysis to optimize it for its role at each step of catalysis <cite>McIntosh1996</cite>. In the case of sialidases, the catalytic nucleophile is a tyrosine residue (see below). This mechanism was originally proposed by Dan Koshland, although at the time the identities of the residues was unclear <cite>Koshland1953</cite>.

[[Image:Retaining_glycosidase_mechanism.png|centre|600px]]

====Neighboring group participation====
1. By a neighboring 2-acetamido group
Enzymes of glycoside hydrolase families [[Glycoside Hydrolase Family 18|18]], [[Glycoside Hydrolase Family 20|20]], [[Glycoside Hydrolase Family 25|25]], [[Glycoside Hydrolase Family 56|56]], [[Glycoside Hydrolase Family 84|84]], [[Glycoside Hydrolase Family 85|85]] and [[Glycoside Hydrolase Family 123|123]] hydrolyse substrates containing an ''N''-acetyl (acetamido) or ''N''-glycolyl group at the 2-position. These enzymes have no catalytic nucleophile: rather they utilize a mechanism in which the 2-acetamido group acts as an intramolecular nucleophile. Neighboring group participation by the 2-acetamido group leads to formation of an oxazoline (or more strictly an [[oxazolinium ion]]) [[intermediate]]. This mechanism was deduced from X-ray structures of complexes of chitinases with natural inhibitors <cite>Terwisscha1995</cite>, from the potent inhibition afforded by a stable thiazoline analogue of the oxazoline <cite>Knapp1996 Mark2001</cite>, and from detailed mechanistic analyses using substrates of modified reactivity <cite>Vocadlo2005</cite>. Typically, a stabilizing residue (a carboxylate) stabilizes the charge development in the [[transition state]]. Not all enzymes that cleave substrates possessing a 2-acetamido group utilize a neighboring groups participation mechanism; enzyme of glycoside hydrolase families [[Glycoside Hydrolase Family 3|3]] and [[Glycoside Hydrolase Family 22|22]] utilize a classical retaining mechanism with an enzymic nucleophile. Other hexosaminidases such as those of [[Glycoside Hydrolase Family 19]] utilize an inverting mechanism.

[[Image:Hex_neighboring_mechanism.png|centre|700px]]

2. By a neighboring 2-hydroxyl group
Enzymes of glycoside hydrolase family [[Glycoside Hydrolase Family 99|99]] hydrolyse a-mannoside substrates and lack a enzymic catalytic nucleophile. These enzymes use a mechanism in which the 2-hydroxyl group acts as an intramolecular nucleophile. Neighboring group participation by the 2-hydroxy group leads to formation of an epoxide (or more strictly a 1,2-anhydro sugar). This mechanism was implicated from 3D X-ray structures of complexes of bacterial endomannosidase/endomannanase with iminosugar inhibitors <cite>Thompson2012</cite>. Detailed mechanistic analyses using carbocyclic analogues of the 1,2-anhydro sugar, quantum mechanic/molecular mechanics computational modelling, and kinetic isotope effects <cite>Sobala2020</cite>.

====Myrosinases: Retaining glycoside hydrolases that lack a general acid and utilize an exogenous base====
Glycoside hydrolases termed myrosinases catalyze the hydrolysis of anionic thioglycosides (glucosinolates) found in plants. They are found in [[Glycoside Hydrolase Family 1]]. In these enzymes the usual acid/base glutamic acid found in other members of this family is replaced by a glutamine. This likely reduces charge repulsion between the anionic aglycon sulfate. The unusual aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst. However, since a base catalyst is required for the second step (hydrolysis or deglycosylation) these enzymes require an alternative basic group. This is provided by the co-enzyme L-ascorbate <cite>Burmeister2000</cite>.

[[Image:myrosinase.png|centre|550px]]

====Alternative nucleophiles====
Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of glycoside hydrolase families [[Glycoside Hydrolase Family 33|33]] and [[Glycoside Hydrolase Family 34|34]], and 2-keto-3-deoxy-d-lyxo-heptulosaric acid hydrolases of [[glycoside hydrolase family 143|143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>.

[[Image:sialidase_mechanism.png|center|700px]]

====NAD-dependent hydrolysis====
The glycoside hydrolases of family [[Glycoside Hydrolase Family 4|4]], [[Glycoside Hydrolase Family 109|109]], [[Glycoside Hydrolase Family 177|177]] and [[Glycoside Hydrolase Family 179|179]] use a mechanism that requires an NAD cofactor, which remains tightly bound throughout catalysis. The mechanism proceeds via anionic [[transition state]]s with elimination and redox steps rather than the classical mechanisms proceeding through[[oxocarbenium ion]]-like transition states. As shown below for a 6-phospho-β-glucosidase, the mechanism involves an initial oxidation of the 3-hydroxyl of the substrate by the enzyme-bound NAD cofactor. This increases the acidity of the C2 proton such that an E1cb elimination can occur with assistance from an enzymatic base. The α,β-unsaturated [[intermediate]] formed then undergoes addition of water at the anomeric centre and finally the ketone at C3 is reduced to generate the free sugar product. Thus, even though glycosidic bond cleavage occurred via an elimination mechanism, the overall reaction is hydrolysis. This mechanism was elucidated through a combination of stereochemical studies by NMR, kinetic isotope effects, linear free energy relationships, X-ray crystallography and UV/Vis spectrophotometry <cite>Yip2004 Rajan2004</cite>.

[[Image:Family_4_mechanism.png|center|700px]]

== References ==
<biblio>
#McCarter1994 pmid=7712292
#Koshland1953 Koshland, D. (1953) ''Biol. Rev.'' 28, 416.
#McIntosh1996 pmid=8756457
#Gebler1992 pmid=1618761
#Henrissat1995 pmid=7624375
#Terwisscha1995 pmid=7495789
#Mark2001 pmid=11124970
#Knapp1996 Knapp, S., Vocadlo, D., Gao, Z. N., Kirk, B., Lou, J. P., and Withers, S. G. (1996) ''Journal of the American Chemical Society 118'', 6804-6805.
#Vocadlo2005 pmid=16171396
#Burmeister2000 pmid=10978344
#Amaya2004 pmid=15130470
#Watts2003 pmid=12812490
#Watson2003 pmid=14580216
#Yip2004 pmid=15237973
#Rajan2004 pmid=15341727
#CAZyURL Carbohydrate Active Enzymes database; URL http://www.cazy.org/
#Henrissat1991 pmid=1747104
#Henrissat1996 pmid=8687420
#Gloster2008 pmid=18848471
#Yip2007 pmid=17676871
#Tiels2012 pmid=23159880
#DaviesHenrissat1995 pmid=8535779
#Ndeh2017 pmid=28329766
</biblio>
[[Category:Definitions and explanations]]

Glycoside hydrolases

2023-06-28T23:21:07Z

Spencer Williams: /* NAD-dependent hydrolysis */

{{CuratorApproved}}
* Authors: [[User:Steve Withers|Steve Withers]], [[User:Spencer Williams|Spencer Williams]]
* Responsible Curator: [[User:Spencer Williams|Spencer Williams]]
----
== Overview ==
Glycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N- and S-linked glycosides.

[[Image:GHs.png|center|400px]]

== Classification ==
Glycoside hydrolases can be classified in many different ways. The following paragraphs list several different ways, the utility of which depends on the context in which the classification is made and used.

=== Endo/exo ===
''exo''- and ''endo''- refers to the ability of a glycoside hydrolase to cleave a substrate at the end (most frequently, but not always the non-reducing end) or within the middle of a chain <cite>DaviesHenrissat1995</cite>. For example, most cellulases are ''endo''-acting, whereas LacZ β-galactosidase from ''E. coli'' is ''exo''-acting. A general [[sub-site nomenclature]] exists to demarcate substrate binding in glycosidase active-sites.

[[Image:exo_endo.png|center|600px]]

=== Enzyme Commission (EC) number ===
EC numbers are codes representing the Enzyme Commission number. This is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze. Every EC number is associated with a recommended name for the respective enzyme. EC numbers do not specify enzymes, but enzyme-catalyzed reactions. If different enzymes (for instance from different organisms) catalyze the same reaction, then they receive the same EC number. A necessary consequence of the EC classification scheme is that codes can be applied only to enzymes for which a function has been biochemically identified. Additionally, certain enzymes can catalyze reactions that fall in more than one class. These enzymes must bear more than one EC number.

=== Mechanistic classification ===
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below.<cite>Gebler1992</cite> However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years, as discussed below.

[[Image:Retaining&inverting_GH.png|center|500px]]

=== Sequence-based classification ===
[[Sequence-based classification]] uses algorithmic methods to assign sequences to various families. The glycoside hydrolases have been classified into more than 100 families <cite>Henrissat1991</cite>; this is permanently available through the Carbohydrate Active enZyme database <cite>CAZyURL</cite>. Each family (GH family) contains proteins that are related by sequence, and by corollary, fold. This allows a number of useful predictions to be made since it has long been noted that the catalytic machinery and molecular mechanism is conserved for the vast majority of the glycosidase families <cite>Gebler1992</cite> as well as the geometry around the glycosidic bond (irrespective of naming conventions) <cite>Henrissat1995</cite>. Usually, the mechanism used (ie [[retaining]] or [[inverting]]) is conserved within a GH family. One notable exception is the glycoside hydrolases of family [[GH97]], which contains both retaining and inverting enzymes; a glutamate acts as a [[general base]] in inverting members, whereas an aspartate likely acts as a [[catalytic nucleophile]] in retaining members <cite>Gloster2008</cite>. Another mechanistic curiosity are the glycoside hydrolases of familes [[GH4]] and [[GH109]] which operate through an [[NAD-dependent hydrolysis]] mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic [[transition state]]s <cite>Yip2007</cite>. This allows a single enzyme to hydrolyze both α- and β-glycosides.

Classification of families into larger groups, termed '[[clans]]' has been proposed <cite>Henrissat1996</cite>. A '[[clan]]' is a group of families that possess significant similarity in their tertiary structure, catalytic residues and mechanism. Families within clans are thought to have a common evolutionary ancestry. For an updated table of glycoside hydrolase clans see the CAZy Database <cite>CAZyURL</cite>.

== Mechanism ==
Two reaction mechanisms are most commonly found for the retaining and inverting enzymes, as first outlined by Koshland and as described below <cite>Koshland1953</cite>. However several interesting variations on these mechanisms have been found, and one fundamentally different mechanism, catalyzed by an NADH cofactor, has been discovered in recent years.

===Inverting glycoside hydrolases===
Hydrolysis of a glycoside with net inversion of anomeric configuration is generally achieved via a one step, single-displacement mechanism involving [[oxocarbenium ion]]-like [[transition state]]s, as shown below. The reaction typically occurs with [[general acid]] and [[general base]] assistance from two amino acid side chains, normally glutamic or aspartic acids, that are typically located 6-11 A apart<cite>McCarter1994</cite>.

[[Image:Inverting glucosidase mechanism.png|centre|600px]]

====Glycosyl-phosphate cleaving enzymes that lack a general acid====
A subset of family [[GH92]] α-mannosidases catalyze the hydrolysis of mannose-1-phosphate linkages found in the mannose-1-phosphate-6-mannose groups of yeast mannoproteins. In these enzymes the usual [[general acid]] glutamic acid found in other members of this family is replaced by a glutamine. It has been suggested that the phosphate aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst <cite>Tiels2012</cite>. This replacement may also reduce charge repulsion between the glutamic acid residue and the anionic phosphate aglycon. A related example may be found in the case of the family [[GH1]] myrosinases.

===Retaining glycoside hydrolases===
====Classical Koshland retaining mechanism====
Hydrolysis with net retention of configuration is most commonly achieved via a two step, double-displacement mechanism involving a covalent glycosyl-enzyme [[intermediate]], as is shown in the figure below. Each step passes through an [[oxocarbenium ion]]-like [[transition state]]. Reaction occurs with acid/base and nucleophilic assistance provided by two amino acid side chains, typically glutamate or aspartate, located 5.5 A apart. In the first step (often called the glycosylation step), one residue plays the role of a nucleophile, attacking the anomeric centre to displace the aglycon and form a glycosyl enzyme [[intermediate]]. At the same time the other residue functions as an acid catalyst and protonates the glycosidic oxygen as the bond cleaves. In the second step (known as the deglycosylation step), the glycosyl enzyme is hydrolyzed by water, with the other residue now acting as a base catalyst deprotonating the water molecule as it attacks. The pKa value of the acid/base group cycles between high and low values during catalysis to optimize it for its role at each step of catalysis <cite>McIntosh1996</cite>. In the case of sialidases, the catalytic nucleophile is a tyrosine residue (see below). This mechanism was originally proposed by Dan Koshland, although at the time the identities of the residues was unclear <cite>Koshland1953</cite>.

[[Image:Retaining_glycosidase_mechanism.png|centre|600px]]

====Neighboring group participation====
Enzymes of glycoside hydrolase families [[Glycoside Hydrolase Family 18|18]], [[Glycoside Hydrolase Family 20|20]], [[Glycoside Hydrolase Family 25|25]], [[Glycoside Hydrolase Family 56|56]], [[Glycoside Hydrolase Family 84|84]], and [[Glycoside Hydrolase Family 85|85]] hydrolyse substrates containing an ''N''-acetyl (acetamido) or ''N''-glycolyl group at the 2-position. These enzymes have no catalytic nucleophile: rather they utilize a mechanism in which the 2-acetamido group acts as an intramolecular nucleophile. Neighboring group participation by the 2-acetamido group leads to formation of an oxazoline (or more strictly an [[oxazolinium ion]]) [[intermediate]]. This mechanism was deduced from X-ray structures of complexes of chitinases with natural inhibitors <cite>Terwisscha1995</cite>, from the potent inhibition afforded by a stable thiazoline analogue of the oxazoline <cite>Knapp1996 Mark2001</cite>, and from detailed mechanistic analyses using substrates of modified reactivity <cite>Vocadlo2005</cite>. Typically, a stabilizing residue (a carboxylate) stabilizes the charge development in the [[transition state]]. Not all enzymes that cleave substrates possessing a 2-acetamido group utilize a neighboring groups participation mechanism; enzyme of glycoside hydrolase families [[Glycoside Hydrolase Family 3|3]] and [[Glycoside Hydrolase Family 22|22]] utilize a classical retaining mechanism with an enzymic nucleophile. Other hexosaminidases such as those of [[Glycoside Hydrolase Family 19]] utilize an inverting mechanism.

[[Image:Hex_neighboring_mechanism.png|centre|700px]]

====Myrosinases: Retaining glycoside hydrolases that lack a general acid and utilize an exogenous base====
Glycoside hydrolases termed myrosinases catalyze the hydrolysis of anionic thioglycosides (glucosinolates) found in plants. They are found in [[Glycoside Hydrolase Family 1]]. In these enzymes the usual acid/base glutamic acid found in other members of this family is replaced by a glutamine. This likely reduces charge repulsion between the anionic aglycon sulfate. The unusual aglycon is a sufficiently good leaving group to be able to cleave in the first glycosylation step to form the glycosyl enzyme [[intermediate]] without the requirement of an acid catalyst. However, since a base catalyst is required for the second step (hydrolysis or deglycosylation) these enzymes require an alternative basic group. This is provided by the co-enzyme L-ascorbate <cite>Burmeister2000</cite>.

[[Image:myrosinase.png|centre|550px]]

====Alternative nucleophiles====
Several groups of retaining glycosidases use atypical nucleophiles. These include the sialidases and trans-sialidases of glycoside hydrolase families [[Glycoside Hydrolase Family 33|33]] and [[Glycoside Hydrolase Family 34|34]], and 2-keto-3-deoxy-d-lyxo-heptulosaric acid hydrolases of [[glycoside hydrolase family 143|143]] <cite>Ndeh2017</cite>. Glycoside hydrolases of these families utilize a tyrosine as a catalytic nucleophile, which is believed to be activated by an adjacent base residue. A rationale for this unusual difference is that the use of a negatively charged carboxylate as a nucelophile will be disfavoured as the anomeric centre is itself negatively charged, and thus charge repulsion interferes. A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the [[intermediate]] with fluorosugars followed by peptide mapping and then crystallography <cite>Amaya2004 Watts2003</cite>, as well as via mechanistic studies on mutants <cite>Watson2003</cite>.

[[Image:sialidase_mechanism.png|center|700px]]

====NAD-dependent hydrolysis====
The glycoside hydrolases of family [[Glycoside Hydrolase Family 4|4]], [[Glycoside Hydrolase Family 109|109]], [[Glycoside Hydrolase Family 177|177]] and [[Glycoside Hydrolase Family 179|179]] use a mechanism that requires an NAD cofactor, which remains tightly bound throughout catalysis. The mechanism proceeds via anionic [[transition state]]s with elimination and redox steps rather than the classical mechanisms proceeding through[[oxocarbenium ion]]-like transition states. As shown below for a 6-phospho-β-glucosidase, the mechanism involves an initial oxidation of the 3-hydroxyl of the substrate by the enzyme-bound NAD cofactor. This increases the acidity of the C2 proton such that an E1cb elimination can occur with assistance from an enzymatic base. The α,β-unsaturated [[intermediate]] formed then undergoes addition of water at the anomeric centre and finally the ketone at C3 is reduced to generate the free sugar product. Thus, even though glycosidic bond cleavage occurred via an elimination mechanism, the overall reaction is hydrolysis. This mechanism was elucidated through a combination of stereochemical studies by NMR, kinetic isotope effects, linear free energy relationships, X-ray crystallography and UV/Vis spectrophotometry <cite>Yip2004 Rajan2004</cite>.

[[Image:Family_4_mechanism.png|center|700px]]

== References ==
<biblio>
#McCarter1994 pmid=7712292
#Koshland1953 Koshland, D. (1953) ''Biol. Rev.'' 28, 416.
#McIntosh1996 pmid=8756457
#Gebler1992 pmid=1618761
#Henrissat1995 pmid=7624375
#Terwisscha1995 pmid=7495789
#Mark2001 pmid=11124970
#Knapp1996 Knapp, S., Vocadlo, D., Gao, Z. N., Kirk, B., Lou, J. P., and Withers, S. G. (1996) ''Journal of the American Chemical Society 118'', 6804-6805.
#Vocadlo2005 pmid=16171396
#Burmeister2000 pmid=10978344
#Amaya2004 pmid=15130470
#Watts2003 pmid=12812490
#Watson2003 pmid=14580216
#Yip2004 pmid=15237973
#Rajan2004 pmid=15341727
#CAZyURL Carbohydrate Active Enzymes database; URL http://www.cazy.org/
#Henrissat1991 pmid=1747104
#Henrissat1996 pmid=8687420
#Gloster2008 pmid=18848471
#Yip2007 pmid=17676871
#Tiels2012 pmid=23159880
#DaviesHenrissat1995 pmid=8535779
#Ndeh2017 pmid=28329766
</biblio>
[[Category:Definitions and explanations]]

Glycoside Hydrolase Family 99

2022-09-17T07:27:22Z

Spencer Williams: /* Kinetics and Mechanism */ fixed figure legend

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''cat/''K''M for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Catalytic mechanism for GH99 endo-α-mannosidase/endo-α-mannanase. (A) [[Classical Koshland double-displacement mechanism]] of a typical retaining glycosidase. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate for family GH99.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the enzyme lacks a nucleophilic residue used for catalysis as the 2OH of the substrate acts as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012 Sobala2020A</cite>. According to this mechanism, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for human MANEA <cite>Sobala2020P</cite>, and bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, there is not nucleophilic residue within potential bonding distance to the anomeric centre, consistent with the neighboring group participation mechanism via an epoxide intermediate.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with and without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020P</cite>.

A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012 Sobala2020A</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
#Sobala2020A pmid=32490192
#Sobala2020P pmid=33154157
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 99

2022-09-17T07:26:01Z

Spencer Williams: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''cat/''K''M for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the enzyme lacks a nucleophilic residue used for catalysis as the 2OH of the substrate acts as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012 Sobala2020A</cite>. According to this mechanism, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for human MANEA <cite>Sobala2020P</cite>, and bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, there is not nucleophilic residue within potential bonding distance to the anomeric centre, consistent with the neighboring group participation mechanism via an epoxide intermediate.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with and without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020P</cite>.

A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012 Sobala2020A</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
#Sobala2020A pmid=32490192
#Sobala2020P pmid=33154157
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 99

2022-09-17T07:22:10Z

Spencer Williams: /* Catalytic Residues */

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''cat/''K''M for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the enzyme lacks a nucleophilic residue used for catalysis as the 2OH of the substrate acts as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012 Sobala2020A</cite>. According to this mechanism, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for human MANEA <cite>Sobala2020P</cite>, and bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, there is not nucleophilic residue within potential bonding distance to the anomeric centre, consistent with the neighboring group participation mechanism via an epoxide intermediate.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with and without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020P</cite>.
which was observed
A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012 Sobala2020A</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
#Sobala2020A pmid=32490192
#Sobala2020P pmid=33154157
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 99

2022-09-17T07:21:03Z

Spencer Williams: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''cat/''K''M for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the identity of the nucleophilic residue used for catalysis is more obscure and the 2OH of the substrate has been proposed to act as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012</cite>. According to this proposal, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for human MANEA <cite>Sobala2020P</cite>, and bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, there is not nucleophilic residue within potential bonding distance to the anomeric centre, consistent with the neighboring group participation mechanism via an epoxide intermediate.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with and without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020P</cite>.
which was observed
A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012 Sobala2020A</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
#Sobala2020A pmid=32490192
#Sobala2020P pmid=33154157
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 99

2022-09-17T07:20:23Z

Spencer Williams: /* Family Firsts */

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''cat/''K''M for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the identity of the nucleophilic residue used for catalysis is more obscure and the 2OH of the substrate has been proposed to act as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012</cite>. According to this proposal, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for human MANEA <cite>Sobala2020</cite>, and bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, there is not nucleophilic residue within potential bonding distance to the anomeric centre, consistent with the neighboring group participation mechanism via an epoxide intermediate.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with ad without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020</cite>.
which was observed
A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012 Sobala2020A</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
#Sobala2020A pmid=32490192
#Sobala2020P pmid=33154157
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 99

2022-09-17T07:20:09Z

Spencer Williams: /* References */

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''cat/''K''M for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the identity of the nucleophilic residue used for catalysis is more obscure and the 2OH of the substrate has been proposed to act as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012</cite>. According to this proposal, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for human MANEA <cite>Sobala2020</cite>, and bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, there is not nucleophilic residue within potential bonding distance to the anomeric centre, consistent with the neighboring group participation mechanism via an epoxide intermediate.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with ad without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020</cite>.
which was observed
A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012 Sobala2021</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
#Sobala2020A pmid=32490192
#Sobala2020P pmid=33154157
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 99

2022-09-17T07:19:55Z

Spencer Williams: /* References */

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''cat/''K''M for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the identity of the nucleophilic residue used for catalysis is more obscure and the 2OH of the substrate has been proposed to act as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012</cite>. According to this proposal, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for human MANEA <cite>Sobala2020</cite>, and bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, there is not nucleophilic residue within potential bonding distance to the anomeric centre, consistent with the neighboring group participation mechanism via an epoxide intermediate.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with ad without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020</cite>.
which was observed
A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012 Sobala2021</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
#Sobala2020A pmid=32490192
#Sobala2021P pmid=33154157
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 99

2022-09-17T07:19:29Z

Spencer Williams: /* Family Firsts */

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''cat/''K''M for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the identity of the nucleophilic residue used for catalysis is more obscure and the 2OH of the substrate has been proposed to act as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012</cite>. According to this proposal, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for human MANEA <cite>Sobala2020</cite>, and bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, there is not nucleophilic residue within potential bonding distance to the anomeric centre, consistent with the neighboring group participation mechanism via an epoxide intermediate.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with ad without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020</cite>.
which was observed
A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012 Sobala2021</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
#Sobala2020 pmid=32490192
#Sobala2021 pmid=33154157
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 99

2022-09-17T07:17:57Z

Spencer Williams: /* Three-dimensional structures */ added detail on MANEA structure

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''cat/''K''M for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the identity of the nucleophilic residue used for catalysis is more obscure and the 2OH of the substrate has been proposed to act as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012</cite>. According to this proposal, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for human MANEA <cite>Sobala2020</cite>, and bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, there is not nucleophilic residue within potential bonding distance to the anomeric centre, consistent with the neighboring group participation mechanism via an epoxide intermediate.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with ad without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020</cite>.
which was observed
A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: It has been proposed that GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
#Sobala2020 pmid=32490192
#Sobala2021 pmid=33154157
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 99

2022-09-17T06:59:19Z

Spencer Williams: /* References */

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''cat/''K''M for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the identity of the nucleophilic residue used for catalysis is more obscure and the 2OH of the substrate has been proposed to act as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012</cite>. According to this proposal, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, the precise identity of the nucleophilic residue is unclear, as in all GH99-inhibitor complexes with an occupied -1 subsite there is no candidate nucleophile within a reasonable distance to the "anomeric" carbon: in ''Bx''GH99 Glu333 is approximately 3.5 Å distant and the OH of Tyr46 and Tyr252 are 4.0 Å distant.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: It has been proposed that GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
#Sobala2020 pmid=32490192
#Sobala2021 pmid=33154157
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 99

2022-09-17T06:58:08Z

Spencer Williams: /* Kinetics and Mechanism */ added reference to Epoxide intermediate.

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''cat/''K''M for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the identity of the nucleophilic residue used for catalysis is more obscure and the 2OH of the substrate has been proposed to act as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012</cite>. According to this proposal, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, the precise identity of the nucleophilic residue is unclear, as in all GH99-inhibitor complexes with an occupied -1 subsite there is no candidate nucleophile within a reasonable distance to the "anomeric" carbon: in ''Bx''GH99 Glu333 is approximately 3.5 Å distant and the OH of Tyr46 and Tyr252 are 4.0 Å distant.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: It has been proposed that GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 99

2022-09-17T06:50:01Z

Spencer Williams: /* Substrate specificities */ added 'MANEA'

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]: [[User:Gideon Davies|Gideon Davies]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH99'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH99.html
|}
</div>


== Substrate specificities ==
[[Image:GH99_substrates.png|thumb|right|300px|'''Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.''']]
[[Glycoside hydrolases]] of family GH99 are [[endo]]-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers <cite>Spiro1997</cite>. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc1-3Man9GlcNAc2 (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose <cite>#Hakki2014</cite>.

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms <cite>Roth2003</cite>. Mammalian endo-α-mannosidases are localized to the Golgi apparatus <cite>Zuber2000</cite> and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum ''exo''-glucosidases I and II <cite>Dale1986</cite>. Mammalian [[endo]]-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches <cite>Spiro1997</cite>. There is evidence that mammalian [[endo]]-α-mannosidases act on dolichol-bound N-glycan precursors <cite>Spiro2000</cite>, as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway <cite>Kukushkin2011</cite>. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase <cite>Iwamoto2014</cite>. Studies of bacterial GH99 enzymes from ''Shewanella amazonensis'' <cite>Matsuda2011</cite>, ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' <cite>Thompson2012</cite> have shown that these enzymes can also process Glc1/3Man9/7GlcNAc2 structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of ''Bacteroides thetaiotaomicron'' GH99 on a fluorescently-labelled Glc3Man9GlcNAc2 structure yielded kinetic parameters that were similar to that found for the mammalian enzyme <cite>Thompson2012</cite>. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>Vogel2000</cite> or α-glucosyl-1,3-α-mannopyranosyl fluoride <cite>Thompson2012</cite>, but are inactive on aryl mannosides.

Bacterial GH99 enzymes from ''B. thetaiotaomicron'' and ''B. xylanisolvens'' have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases <cite>#Hakki2014</cite>. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside <cite>#Hakki2014</cite>.

== Kinetics and Mechanism ==
Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]

== Catalytic Residues ==
The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc3Man7GlcNAc2, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the identity of the nucleophilic residue used for catalysis is more obscure and the 2OH of the substrate has been proposed to act as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012</cite>. According to this proposal, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

== Three-dimensional structures ==
Three-dimensional structures are available for bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (β/α)8 TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate. Binary complexes with two inhibitors, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, the precise identity of the nucleophilic residue is unclear, as in all GH99-inhibitor complexes with an occupied -1 subsite there is no candidate nucleophile within a reasonable distance to the "anomeric" carbon: in ''Bx''GH99 Glu333 is approximately 3.5 Å distant and the OH of Tyr46 and Tyr252 are 4.0 Å distant.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.

A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.



== Family Firsts ==
;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by 1H NMR <cite>Thompson2012</cite>

;First [[catalytic nucleophile]] identification: It has been proposed that GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012</cite>

;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

;First 3-D structure of a GH99 enzyme: ''Bacteroides thetaiotaomicron'' and ''Bacteroides xylanisolvens'' ''endo''-α-mannosidases <cite>Thompson2012</cite>

== References ==
<biblio>
#Roth2003 pmid=12770767
#Spiro1997 pmid=9361017
#Zuber2000 pmid=11102520
#Dale1986 pmid=3087421
#Kukushkin2011 pmid=21585340
#Spiro2000 pmid=10764841
#Thompson2012 pmid=22219371
#Matsuda2011 pmid=21512220
#Vogel2000 Vogel C, Pohlentz G. ''Synthesis of α-D-glucopyranosyl-(1,3)-α-D-mannopyranosyl-(1,7)-4-methylumbelliferone, a fluorogenic substrate for endo-α-1,2-mannosidase.'' J. Carbohydr. Chem. 2000, 19: 1247-1258.
#Iwamoto2014 pmid=25036115
#Hakki2014 pmid=25487964
</biblio>

[[Category:Glycoside Hydrolase Families|GH099]]

Glycoside Hydrolase Family 76

2022-05-05T11:00:47Z

Spencer Williams: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]s: [[User:Gideon Davies|Gideon Davies]] and [[User:Harry Gilbert|Harry Gilbert]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH76'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH76.html
|}
</div>


== Substrate specificities ==
[[Glycoside hydrolases]] of family GH76 are [[endo]]-acting α-mannanases. GH76 genes are found within bacteria and fungi. Bacterial GH76 enzymes cleave α-1,6-mannans, such as those found within the α-1,6-linked backbone of fungal mannoproteins and mycobacterial cell wall lipomannan, lipoarabinomannan and phosphatidylinositol mannosides. This family was originally created from the cloning and characterization of Aman6 from ''Bacillus circulans'' TN-31 <cite>Maruyama2000</cite>, which appears to be the same enzyme as that characterized much earlier by Ballou and co-workers <cite>Nakajima1976</cite>. A related protein, Emn, has been cloned from ''Bacillus circulans'' TN-31 genomic DNA <cite>Angala2019</cite>. Aman6 degrades α-1,6-mannan to a mixture of the mannobiose and mannotriose <cite>Maruyama2000</cite>; mannotriose is the minimum substrate for the enzyme <cite> Nakajima1976</cite>. A possible GH76 enzyme has been detected within ''Mycobacterium smegmatis'', which has the capacity to degrade α-1,6-mannooligosaccharides <cite>Yokoyama1989</cite>.

Additional characterized GH76 enzymes include several from the gut bacterium ''Bacteroides thetaiotaomicron'' <cite>Cuskin2015</cite> and ShGH76 from the marine bacterium ''Salegentibacter'' sp Hel_I_6 <cite>Solanki2022</cite>. ''B. thetaiotaomicron'' expresses numerous GH76 enzymes. Several of these are found within polysaccharide utilization loci that are specifically up-regulated upon exposure to yeast α-mannan. Likewise the ''Salegentibacter'' genes reside in PUL-like clusters. These enzymes have the capacity to utilize unadorned linear α-1,6-mannan, but have little activity on highly branched wildtype α-mannan. Certain ''B. thetaiotaomicron'' GH76 enzymes are lipoenzymes that are associated with the cell surface, where they appear to act on large yeast mannan molecules that have undergone partial trimming to expose sections of the core α-1,6-mannan. Other periplasmic located GH76 enzymes have activity on shorter α-1,6-mannan fragments, which are obtained by importation of partially-digested fragments arising from the action of cell surface enzymes.

Fungal GH76 enzymes have been speculated to be involved in cross-linking of GPI-anchored proteins into the cell wall, where they are proposed to act as transglycosylases <cite>Kitagaki2002</cite>. DFG-5 from ''Neurospora crassa'' has an enzymatic activity and processes the α-1,6-mannose backbone of fungal N-linked galactomannan <cite>Patel2022</cite>. Cell wall glycoproteins co-purify with DFG-5 indicating a specific association between DFG-5 and cell wall glycoproteins. DFG-5 can discriminate between cell wall and secreted glycoproteins, and does not bind to the N-linked galactomannans on secreted glycoproteins.

== Kinetics and Mechanism ==
Family GH76 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of 4-nitrophenyl α-mannosyl-1,6-α-mannopyranoside by a ''Bacteroides thetaiotaomicron'' α-mannanase <cite>Cuskin2015</cite>. GH76 enzymes are believed to proceed through a [[classical Koshland double-displacement mechanism]]. Crystallographic evidence from a binary complexes of the catalytic domain of ''Bacillus circulans'' Aman6 with substrate and inhibitors, complemented by quantum mechanics/molecular mechanics calculations of preferred conformations on-enzyme supports a 1''S''5→''B''2,5‡→O''S''2 conformational reaction coordinate <cite>Thompson2015</cite>.

== Catalytic Residues ==
Inspection of the X-ray structure of ''Bacteroides thetaiotaomicron'' BT3792 revealed two consecutive asparate residues, D258 and D259, that were predicted to be catalytic residues <cite>Cuskin2015</cite>. The equivalent pair of conserved aspartic acid residues (D124 and D125 in the catalytic domain of ''Bacillus circulans'' Aman6) were identified as [[catalytic nucleophile]] and [[general acid/base]], respectively, based on X-ray analysis of substrate and inhibitor complexes, dovetailed with kinetic analysis of mutants <cite>Thompson2015</cite>.

== Three-dimensional structures ==
Three-dimensional structures are available for several bacterial members of GH76, including the catalytic domain of ''Bacillus circulans'' Aman6, ''Bacteroides thetaiotaomicron'' BT2949 and BT3792, ''Listeria innocua'' Clip11262 and ''Salegentibacter'' sp. Hel_I_6 ShGH76 (''see the [http://www.cazy.org/GH76_structure.html GH76 structure page in the CAZy Database]''). They have an (α/α)6 fold. A complex of mannopentaose bound in the active site of ''Bacillus circulans'' GH76 defined the -4 to +1 subsites, and showed the sugar binding in the -1 subsite in a 1''S''5 conformation. A complex of the same enzyme with α-1,6-mannobiose showed the disaccharide binding in the -3/-2 subsites (unpublished, PDB ID [{{PDBlink}}4boj 4boj]). Several inhibitor complexes have been reported. A complex with the inhibitor α-mannosyl-1,6-isofagomine revealed the inhibtior to bind in the -2/-1 subsites and displayed the isofagomine ring in a ''B''2,5 conformation, with the nitrogen of the inhibitor hydrogen-bonded to the nucleophile (D124) <cite>Thompson2015</cite>. The S-linked analogue α-mannosyl-1,6-''S''-isofagomine (ManSIFG) bound with similar affinity to the enzyme and also in the -2/-1 subsites, but instead the inhibitor bound in a relaxed 4''C''1 conformation with the inhibitor nitrogen hydrogen bonded to the acid/base (D125) <cite>Belz2017</cite>.

== Family Firsts ==
;First stereochemistry determination: ''Bacteroides thetaiotaomicron'' α-1,6-mannanase by 1H NMR <cite>Cuskin2015</cite>

;First [[catalytic nucleophile]] identification: D124 for ''Bacillus circulans'' catalytic domain <cite>Thompson2015</cite>.

;First [[general acid/base]] residue identification: D125 for ''Bacillus circulans'' catalytic domain <cite>Thompson2015</cite>.

;First 3-D structure of a GH76 enzyme: ''Listeria innocua'' Lin0763 (unpublished, PDB ID [{{PDBlink}}3k7x 3k7x])

== References ==
<biblio>
#Maruyama2000 pmid=11055417
#Nakajima1976 pmid=811665
#Yokoyama1989 pmid=2480954
#Kitagaki2002 pmid=12421307
#Cuskin2015 pmid=25567280
#Thompson2015 pmid=25772148
#Belz2017 pmid=28766587
#Angala2019 pmid=31835712
#Solanki2022 pmid=35414716
#Patel2022 pmid=35306147

</biblio>

[[Category:Glycoside Hydrolase Families|GH076]]

Glycoside Hydrolase Family 76

2022-05-05T11:00:20Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]s: [[User:Gideon Davies|Gideon Davies]] and [[User:Harry Gilbert|Harry Gilbert]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH76'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH76.html
|}
</div>


== Substrate specificities ==
[[Glycoside hydrolases]] of family GH76 are [[endo]]-acting α-mannanases. GH76 genes are found within bacteria and fungi. Bacterial GH76 enzymes cleave α-1,6-mannans, such as those found within the α-1,6-linked backbone of fungal mannoproteins and mycobacterial cell wall lipomannan, lipoarabinomannan and phosphatidylinositol mannosides. This family was originally created from the cloning and characterization of Aman6 from ''Bacillus circulans'' TN-31 <cite>Maruyama2000</cite>, which appears to be the same enzyme as that characterized much earlier by Ballou and co-workers <cite>Nakajima1976</cite>. A related protein, Emn, has been cloned from ''Bacillus circulans'' TN-31 genomic DNA <cite>Angala2019</cite>. Aman6 degrades α-1,6-mannan to a mixture of the mannobiose and mannotriose <cite>Maruyama2000</cite>; mannotriose is the minimum substrate for the enzyme <cite> Nakajima1976</cite>. A possible GH76 enzyme has been detected within ''Mycobacterium smegmatis'', which has the capacity to degrade α-1,6-mannooligosaccharides <cite>Yokoyama1989</cite>.

Additional characterized GH76 enzymes include several from the gut bacterium ''Bacteroides thetaiotaomicron'' <cite>Cuskin2015</cite> and ShGH76 from the marine bacterium ''Salegentibacter'' sp Hel_I_6 <cite>Solanki2022</cite>. ''B. thetaiotaomicron'' expresses numerous GH76 enzymes. Several of these are found within polysaccharide utilization loci that are specifically up-regulated upon exposure to yeast α-mannan. Likewise the ''Salegentibacter'' genes reside in PUL-like clusters. These enzymes have the capacity to utilize unadorned linear α-1,6-mannan, but have little activity on highly branched wildtype α-mannan. Certain ''B. thetaiotaomicron'' GH76 enzymes are lipoenzymes that are associated with the cell surface, where they appear to act on large yeast mannan molecules that have undergone partial trimming to expose sections of the core α-1,6-mannan. Other periplasmic located GH76 enzymes have activity on shorter α-1,6-mannan fragments, which are obtained by importation of partially-digested fragments arising from the action of cell surface enzymes.

Fungal GH76 enzymes have been speculated to be involved in cross-linking of GPI-anchored proteins into the cell wall, where they are proposed to act as transglycosylases <cite>Kitagaki2002</cite>. DFG-5 from ''Neurospora crassa'' has an enzymatic activity and processes the α-1,6-mannose backbone of fungal N-linked galactomannan <cite>Patel2022</cite>. Cell wall glycoproteins co-purify with DFG-5 indicating a specific association between DFG-5 and cell wall glycoproteins. DFG-5 can discriminate between cell wall and secreted glycoproteins, and does not bind to the N-linked galactomannans on secreted glycoproteins.

== Kinetics and Mechanism ==
Family GH76 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of 4-nitrophenyl α-mannosyl-1,6-α-mannopyranoside by a ''Bacteroides thetaiotaomicron'' α-mannanase <cite>Cuskin2015</cite>. GH76 enzymes are believed to proceed through a [[classical Koshland double-displacement mechanism]]. Crystallographic evidence from a binary complexes of the catalytic domain of ''Bacillus circulans'' Aman6 with substrate and inhibitors, complemented by quantum mechanics/molecular mechanics calculations of preferred conformations on-enzyme supports a 1''S''5→''B''2,5‡→O''S''2 conformational reaction coordinate <cite>Thompson2015</cite>.

== Catalytic Residues ==
Inspection of the X-ray structure of ''Bacteroides thetaiotaomicron'' BT3792 revealed two consecutive asparate residues, D258 and D259, that were predicted to be catalytic residues <cite>Cuskin2015</cite>. The equivalent pair of conserved aspartic acid residues (D124 and D125 in the catalytic domain of ''Bacillus circulans'' Aman6) were identified as [[catalytic nucleophile]] and [[general acid/base]], respectively, based on X-ray analysis of substrate and inhibitor complexes, dovetailed with kinetic analysis of mutants <cite>Thompson2015</cite>.

== Three-dimensional structures ==
Three-dimensional structures are available for several bacterial members of GH76, including the catalytic domain of ''Bacillus circulans'' Aman6, ''Bacteroides thetaiotaomicron'' BT2949 and BT3792, ''Listeria innocua'' Clip11262 and ''Salegentibacter'' sp. Hel_I_6 ShGH76 (''see the [http://www.cazy.org/GH76_structure.html GH76 structure page in the CAZy Database]''). They have an (α/α)6 fold. A complex of mannopentaose bound in the active site of ''Bacillus circulans'' GH76 defined the -4 to +1 subsites, and showed the sugar binding in the -1 subsite in a 1''S''5 conformation. A complex of the same enzyme with α-1,6-mannobiose showed the disaccharide binding in the -3/-2 subsites (unpublished, PDB ID [{{PDBlink}}4boj 4boj]). Several inhibtior complezes have been reported. A complex with the inhibitor α-mannosyl-1,6-isofagomine revealed the inhibtior to bind in the -2/-1 subsites and displayed the isofagomine ring in a ''B''2,5 conformation, with the nitrogen of the inhibitor hydrogen-bonded to the nucleophile (D124) <cite>Thompson2015</cite>. The S-linked analogue α-mannosyl-1,6-''S''-isofagomine (ManSIFG) bound with similar affinity to the enzyme and also in the -2/-1 subsites, but instead the inhibitor bound in a relaxed 4''C''1 conformation with the inhibitor nitrogen hydrogen bonded to the acid/base (D125) <cite>Belz2017</cite>.

== Family Firsts ==
;First stereochemistry determination: ''Bacteroides thetaiotaomicron'' α-1,6-mannanase by 1H NMR <cite>Cuskin2015</cite>

;First [[catalytic nucleophile]] identification: D124 for ''Bacillus circulans'' catalytic domain <cite>Thompson2015</cite>.

;First [[general acid/base]] residue identification: D125 for ''Bacillus circulans'' catalytic domain <cite>Thompson2015</cite>.

;First 3-D structure of a GH76 enzyme: ''Listeria innocua'' Lin0763 (unpublished, PDB ID [{{PDBlink}}3k7x 3k7x])

== References ==
<biblio>
#Maruyama2000 pmid=11055417
#Nakajima1976 pmid=811665
#Yokoyama1989 pmid=2480954
#Kitagaki2002 pmid=12421307
#Cuskin2015 pmid=25567280
#Thompson2015 pmid=25772148
#Belz2017 pmid=28766587
#Angala2019 pmid=31835712
#Solanki2022 pmid=35414716
#Patel2022 pmid=35306147

</biblio>

[[Category:Glycoside Hydrolase Families|GH076]]

Glycoside Hydrolase Family 76

2022-05-05T10:59:08Z

Spencer Williams:

{{CuratorApproved}}
* [[Author]]: [[User:Spencer Williams|Spencer Williams]]
* [[Responsible Curator]]s: [[User:Gideon Davies|Gideon Davies]] and [[User:Harry Gilbert|Harry Gilbert]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH76'''
|-
|'''Clan'''
|not assigned
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH76.html
|}
</div>


== Substrate specificities ==
[[Glycoside hydrolases]] of family GH76 are [[endo]]-acting α-mannanases. GH76 genes are found within bacteria and fungi. Bacterial GH76 enzymes cleave α-1,6-mannans, such as those found within the α-1,6-linked backbone of fungal mannoproteins and mycobacterial cell wall lipomannan, lipoarabinomannan and phosphatidylinositol mannosides. This family was originally created from the cloning and characterization of Aman6 from ''Bacillus circulans'' TN-31 <cite>Maruyama2000</cite>, which appears to be the same enzyme as that characterized much earlier by Ballou and co-workers <cite> Nakajima1976</cite>. A related protein, Emn, has been cloned from ''Bacillus circulans'' TN-31 genomic DNA <cite Angala2019>. Aman6 degrades α-1,6-mannan to a mixture of the mannobiose and mannotriose <cite>Maruyama2000</cite>; mannotriose is the minimum substrate for the enzyme <cite> Nakajima1976</cite>. A possible GH76 enzyme has been detected within ''Mycobacterium smegmatis'', which has the capacity to degrade α-1,6-mannooligosaccharides <cite>Yokoyama1989</cite>.

Additional characterized GH76 enzymes include several from the gut bacterium ''Bacteroides thetaiotaomicron'' <cite>Cuskin2015</cite> and ShGH76 from the marine bacterium ''Salegentibacter'' sp Hel_I_6 <cite Solanki2022>. ''B. thetaiotaomicron'' expresses numerous GH76 enzymes. Several of these are found within polysaccharide utilization loci that are specifically up-regulated upon exposure to yeast α-mannan. Likewise the ''Salegentibacter'' genes reside in PUL-like clusters. These enzymes have the capacity to utilize unadorned linear α-1,6-mannan, but have little activity on highly branched wildtype α-mannan. Certain ''B. thetaiotaomicron'' GH76 enzymes are lipoenzymes that are associated with the cell surface, where they appear to act on large yeast mannan molecules that have undergone partial trimming to expose sections of the core α-1,6-mannan. Other periplasmic located GH76 enzymes have activity on shorter α-1,6-mannan fragments, which are obtained by importation of partially-digested fragments arising from the action of cell surface enzymes.

Fungal GH76 enzymes have been speculated to be involved in cross-linking of GPI-anchored proteins into the cell wall, where they are proposed to act as transglycosylases <cite>Kitagaki2002</cite>. DFG-5 from ''Neurospora crassa'' has an enzymatic activity and processes the α-1,6-mannose backbone of fungal N-linked galactomannan <cite Patel2022>. Cell wall glycoproteins co-purify with DFG-5 indicating a specific association between DFG-5 and cell wall glycoproteins. DFG-5 can discriminate between cell wall and secreted glycoproteins, and does not bind to the N-linked galactomannans on secreted glycoproteins.

== Kinetics and Mechanism ==
Family GH76 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by 1H NMR analysis of the hydrolysis of 4-nitrophenyl α-mannosyl-1,6-α-mannopyranoside by a ''Bacteroides thetaiotaomicron'' α-mannanase <cite>Cuskin2015</cite>. GH76 enzymes are believed to proceed through a [[classical Koshland double-displacement mechanism]]. Crystallographic evidence from a binary complexes of the catalytic domain of ''Bacillus circulans'' Aman6 with substrate and inhibitors, complemented by quantum mechanics/molecular mechanics calculations of preferred conformations on-enzyme supports a 1''S''5→''B''2,5‡→O''S''2 conformational reaction coordinate <cite>Thompson2015</cite>.

== Catalytic Residues ==
Inspection of the X-ray structure of ''Bacteroides thetaiotaomicron'' BT3792 revealed two consecutive asparate residues, D258 and D259, that were predicted to be catalytic residues <cite>Cuskin2015</cite>. The equivalent pair of conserved aspartic acid residues (D124 and D125 in the catalytic domain of ''Bacillus circulans'' Aman6) were identified as [[catalytic nucleophile]] and [[general acid/base]], respectively, based on X-ray analysis of substrate and inhibitor complexes, dovetailed with kinetic analysis of mutants <cite>Thompson2015</cite>.

== Three-dimensional structures ==
Three-dimensional structures are available for several bacterial members of GH76, including the catalytic domain of ''Bacillus circulans'' Aman6, ''Bacteroides thetaiotaomicron'' BT2949 and BT3792, ''Listeria innocua'' Clip11262 and ''Salegentibacter'' sp. Hel_I_6 ShGH76 (''see the [http://www.cazy.org/GH76_structure.html GH76 structure page in the CAZy Database]''). They have an (α/α)6 fold. A complex of mannopentaose bound in the active site of ''Bacillus circulans'' GH76 defined the -4 to +1 subsites, and showed the sugar binding in the -1 subsite in a 1''S''5 conformation. A complex of the same enzyme with α-1,6-mannobiose showed the disaccharide binding in the -3/-2 subsites (unpublished, PDB ID [{{PDBlink}}4boj 4boj]). Several inhibtior complezes have been reported. A complex with the inhibitor α-mannosyl-1,6-isofagomine revealed the inhibtior to bind in the -2/-1 subsites and displayed the isofagomine ring in a ''B''2,5 conformation, with the nitrogen of the inhibitor hydrogen-bonded to the nucleophile (D124) <cite>Thompson2015</cite>. The S-linked analogue α-mannosyl-1,6-''S''-isofagomine (ManSIFG) bound with similar affinity to the enzyme and also in the -2/-1 subsites, but instead the inhibitor bound in a relaxed 4''C''1 conformation with the inhibitor nitrogen hydrogen bonded to the acid/base (D125) <cite>Belz2017</cite>.

== Family Firsts ==
;First stereochemistry determination: ''Bacteroides thetaiotaomicron'' α-1,6-mannanase by 1H NMR <cite>Cuskin2015</cite>

;First [[catalytic nucleophile]] identification: D124 for ''Bacillus circulans'' catalytic domain <cite>Thompson2015</cite>.

;First [[general acid/base]] residue identification: D125 for ''Bacillus circulans'' catalytic domain <cite>Thompson2015</cite>.

;First 3-D structure of a GH76 enzyme: ''Listeria innocua'' Lin0763 (unpublished, PDB ID [{{PDBlink}}3k7x 3k7x])

== References ==
<biblio>
#Maruyama2000 pmid=11055417
#Nakajima1976 pmid=811665
#Yokoyama1989 pmid=2480954
#Kitagaki2002 pmid=12421307
#Cuskin2015 pmid=25567280
#Thompson2015 pmid=25772148
#Belz2017 pmid=28766587
#Angala2019 pmid=31835712
#Solanki2022 pmid=35414716
#Patel2022 pmid=35306147

</biblio>

[[Category:Glycoside Hydrolase Families|GH076]]

Reaction intermediate

2021-10-17T22:31:16Z

Spencer Williams:

{{CuratorApproved}}
* Author: [[User:Withers|Stephen Withers]]
* Responsible Curator: [[User:SpencerWilliams|Spencer Williams]]

----

An '''intermediate''' in a reaction is a species formed that has a real lifetime, best defined as being greater than the time for a bond vibration (approx 10-14 sec, ie a few femtoseconds). This will correspond to a minimum in the reaction coordinate diagram located between the reactant and product. Depending on their lifetimes and the tools available, intermediates can sometimes be trapped and observed by physical and chemical techniques.

[[Image:ReactionCoordinate.png|center|400px]]

[[Category:Definitions and explanations]]

Glycosyltransferases

2021-01-07T11:47:01Z

Spencer Williams: /* References */

{{CuratorApproved}}
* Author: [[User:SpencerWilliams|Spencer Williams]]
* Responsible Curator: [[User:SpencerWilliams|Spencer Williams]]
----
__TOC__
== Overview ==
Glycosyltransferases are enzymes that catalyze the formation of the glycosidic linkage to form a glycoside. These enzymes utilize 'activated' sugar phosphates as glycosyl donors, and catalyze glycosyl group transfer to a nucleophilic group, usually an alcohol. The product of glycosyl transfer may be an O-, N-, S-, or C-glycoside; the glycoside may be part of a monosaccharide, oligosaccharide, or polysaccharide <cite>StickWilliams Lairson2008 CoutinhoJMB2003 CampbellBJ1997 Coutinho2009</cite>.

== Glycosyl donor substrates ==
Glycosyltransferases can utilize a range of donor substrates. Sugar mono- or diphosphonucleotides are sometimes termed Leloir donors (after Nobel prize winner, Luis Leloir); the corresponding enzymes are termed Leloir glycosyltransferases.

[[Image:Leloir_donors.png|center|500px]]

Glycosyltransferases that utilize non-nucleotide donors, which may be polyprenol pyrophosphates, polyprenol phosphates, sugar-1-phosphates, or sugar-1-pyrophosphates, are termed non-Leloir glycosyltransferases.

[[Image:non-Leloir_donors.png|center|500px]]

In the last two cases, the enzymes that catalyze the transfer of a glycosyl group from a glycosyl phosphate or pyrophosphate are more commonly referred to as [[phosphorylases]] and pyrophosphorylases. Some of these enzymes are classified into [[glycoside hydrolase]] (GH) families (eg sucrose phosphorylase, [[GH13]]) and others are classified into GT familes (eg glycogen phosphorylase [[GT35]]).

''For specific examples of glycosyl donor substrates, please see the [[#Common sugar donors|Common sugar donors]] section below.''

== Mechanism ==
Glycosyltransferases catalyze the transfer of glycosyl groups to a nucleophilic acceptor with either retention or inversion of configuration at the anomeric centre. This allows the classification of glycosyltransferases as either retaining or inverting enzymes.

=== Inverting glycosyltransferases ===
Structural and kinetic data for inverting glycosyltransferases support a mechanism that proceeds through a single nucleophilic substitution step, facilitated by an enzymic general base catalyst. The [[transition state]] is believed to possess substantial [[oxocarbenium ion]] character. Most inverting glycosyltransferases require a divalent cation (typically Mg2+ or Mn2+) although metal-independent enzymes are known <cite>Lairson2008</cite>.
[[Image:Inverting_glycosyltransferase_mechanism.png|center|700px]]

=== Retaining glycosyltransferases ===
Mechanistic evidence for the catalytic process that results in retention of configuration of glycosyltransferases is scant. One mechanism that seems reasonable by comparison with the [[classical Koshland retaining mechanism]] for [[glycoside hydrolases]], involves an enzymic nucleophile. In this mechanism, the enzymic nucleophile reacts with the glycosyl donor to generate a glycosyl enzyme with inversion of stereochemistry, followed by reaction with the glycosyl acceptor with a second inversion of stereochemistry, to yield a product glycoside with a net retention of anomeric stereochemistry. However, attempts to trap a glycosyl enzyme on a wildtype glycosyltransferase have met with universal failure.

Further, crystal structures of retaining glycosyltransferases are mostly ambiguous with respect to identifying possible candidate nucleophiles, with some lacking any suitable nucleophile situated close enough to the glycosyl donor to be able to form a covalent intermediate. In other cases structural analysis has identified possible candidate nucleophiles, which mutagenesis studies have supported. In some cases mutagenesis of the candidate residue to a non-nucleophilic amino acid has not resulted in the expected, dramatic loss in catalytic activity (of the order of 106), as seen for retaining glycoside hydrolases.

The most common alternative mechanism that is invoked is one that involves an SNi process, also termed 'internal return'. In this process, the leaving group on the donor departs, and the nucleophile attacks from the same face, with the other face of the donor being blocked by the enzyme. An open question is whether this process is concerted (''i.e.'' involving a single [[transition state]] in which bonds are formed and broken at similar times) or stepwise (''i.e.'' involving two [[transition state]]s, and therefore an oxocarbenium ion intermediate). A detailed kinetic study of the retaining glycosyltransferase OtsA (which catalyzes the synthesis of trehalose-6-phosphate from UDP-glucose and glucose-6-phosphate) yielded data that was consistent with a frontside, SNi-type reaction <cite>Lee2011</cite>.

[[Image:Retaining glycosyltransferase mechanism.png|center|700px]]

== Classification ==
=== Sequence based classification ===
[[Sequence-based classification]] uses algorithmic methods to assign protein sequences to various families. The glycosyltransferases have been classified into more than 90 families <cite>Campbell1997 Countinho2003</cite>. Each family (GT family) contains proteins that are related by sequence, and by corollary, fold. This allows several useful predictions to be made since the catalytic machinery is conserved within each family. Usually, the mechanism used (''i.e.'' retaining or inverting) is also conserved within a GT family. An actively curated list of GT families and members is available through the Carbohydrate Active enZyme database <cite>CAZyURL</cite>.

== 3-D folds ==
In striking contrast to [[glycoside hydrolases]], which exhibit a wide variety of folds, GTs exhibit a much narrower range of folds.

=== Leloir GTs ===
Sugar nucleotide-dependent (Leloir) glycosyltransferases have been found to possess a range of different folds. The main folds are termed the GT-A and GT-B folds <cite>Unligil2000</cite>. The GT-A fold is typified by the first member to have its X-ray structure determined, SpsA from''Bacillus subtilus'' <cite>Charnock1999</cite>. The GT-A fold consists of two dissimilar domains, one involved in nucleotide binding and the other binding the acceptor. The GT-B fold was exemplified by its first member, the beta-glucosyltransferase from bacteriophage T4 <cite>Vrielink1994</cite>. The GT-B fold consists of two similar Rossmann fold subdomains. The mannosyltransferases of family [[GT108]] possess a 5-bladed beta-propeller fold, similar to family [[GH130]] glycoside phosphorylases <cite>Sernee2019</cite>.

=== Non-Leloir GTs ===
Non-Leloir glycosyltransferases possess non-GT-A and -B folds. For example, the polymerizing glycosyltransferase transglycosylase, which catalyzes the condensation of oligosaccharyl polyprenolphosphate to generate the carbohydrate backbone of peptidoglycan has a bacteriophage-lysozyme-like fold <cite>Lovering2007</cite>. The ''Pyrococcus furiosius'' oligosaccharyltransferase STT3, which catalyzes the transfer of a preformed oligosaccharide from a dolichol phosphate glycosyl donor to form asparagine-linked glycoproteins, possesses a novel fold; however, this crystallized fragment is catalytically-inactive and may not represent the structure of the complete glycosyltransferase <cite>Igura2008</cite>. The same fold is also observed in the periplasmic domain of the PglB oligosaccharyltransferase of ''Camplyobacter lari''; this latter protein was been crystallized as a full length protein incorporating a transmembrane domain that retains transferase activity <cite>Lizak2011</cite>. A structure of PglB oligosaccharyltransferase in complex with a hexapeptide substrate showed that the peptide binds to both the periplasmic domain and the transmembrane domain. These data demonstrate that the transmembrane domain of this bacterial oligosaccharyltransferase is required for both substrate binding and catalysis.


== Role of metals ==
Many, but not all, glycosyltransferases utilize divalent metal ion cofactors such as Mn2+ and Mg2+. These metals are found mainly in glycosyltransferases that are diphosphonucleoside-dependent. X-ray crystallographic analysis reveals that the metal ion is coordinated to an oxygen of each of the two phosphate groups, as well as to side-chain carboxylates derived from the protein. Much has been made of the so-called ‘DXD’ amino acid sequence as an identifier of glycosyltransferases, where the aspartate residues of this sequence are presumed to comprise the metal-binding residues of the active site. However, it is to be cautioned that no part of the DXD motif is invariant among glycosyltransferases, with this motif being present in more than 50% of all protein sequences. Moreover, it is emphasized that many glycosyltransferases are metal-ion independent, and thus do not bind metals at the active site, and so do not require a metal-binding motif at their active site.

== Common sugar donors ==

<gallery widths=270px perrow=3 caption="Examples of sugar nucleotide donors">

File:UDPGlc.png|'''uridine diphospho-D-glucose''' (UDP-Glc)

File:UDPGal.png|'''uridine diphospho-D-galactose''' (UDP-Gal)

File:UDPXyl.png|'''uridine diphospho-D-xylose''' (UDP-Xyl)

File:UDPGlcNAc.png|'''uridine diphospho-''N''-acetyl-D-glucosamine''' (UDP-GlcNAc)

File:UDPGalNAc.png|'''uridine diphospho-''N''-acetyl-D-galactosamine''' (UDP-GalNAc)

File:UDPGlcA.png|'''uridine diphospho-D-glucuronic acid''' (UDP-GlcA)

File:UDPGalf.png|'''uridine diphospho-D-galactofuranose''' (UDP-Gal''f'')

File:GDPMan.png|'''guanosine diphospho-D-mannose''' (GDP-Man)

File:GDPFuc.png|'''guanosine diphospho-L-fucose''' (GDP-Fuc)

File:GDPRha.png|'''guanosine diphospho-L-rhamnose''' (GDP-Rha)

File:CMPNeu5Ac.png|'''cytidine monophospho-''N''-acetylneuraminic acid''' (CMP-Neu5Ac)

File:CMPKdo.png|'''cytidine monophospho-2-keto-3-deoxy-D-mannooctanoic acid''' (CMP-Kdo)

</gallery>

<gallery widths=350px perrow=2 caption="Examples of sugar phospholipid donors">
File:DPMan.png|'''dolichol phosphomannose''' (DP-Man)

File:PPAra.png|'''decaprenolphosphoarabinose''' (PP-Ara)
</gallery>

<gallery widths=740px perrow=1>
File:DolGlc3Man9GlcNAc2.png|'''dolichol-PP-Glc3Man9GlcNac2''' 
</gallery>

== References ==
<biblio>
#StickWilliams isbn=9780240521183
#Lairson2008 pmid=18518825
#CoutinhoJMB2003 pmid=12691742
#CampbellBJ1997 pmid=9334165
#Coutinho2009 isbn=9780470016671 //''Chapter 5:'' Coutinho PM, Rancurel C, Stam M, Bernard T, Couto FM, Danchin EGJ, Henrissat B. "Carbohydrate-active Enzymes Database: Principles and Classification of Glycosyltransferases."
#Charnock1999 pmid=10350455
#Vrielink1994 pmid=8062817
#Unligil2000 pmid=11042447
#Lovering2007 pmid=17347437
#Igura2008 pmid=18046457
#CAZyURL Carbohydrate Active Enzymes database; URL http://www.cazy.org/
#Lizak2011 pmid=21677752
#Lee2011 pmid=21822275
#Sernee2019 pmid=31513773
</biblio>

[[Category:Definitions and explanations]]

Glycosyltransferases

2021-01-07T11:45:52Z

Spencer Williams: /* Leloir GTs */

{{CuratorApproved}}
* Author: [[User:SpencerWilliams|Spencer Williams]]
* Responsible Curator: [[User:SpencerWilliams|Spencer Williams]]
----
__TOC__
== Overview ==
Glycosyltransferases are enzymes that catalyze the formation of the glycosidic linkage to form a glycoside. These enzymes utilize 'activated' sugar phosphates as glycosyl donors, and catalyze glycosyl group transfer to a nucleophilic group, usually an alcohol. The product of glycosyl transfer may be an O-, N-, S-, or C-glycoside; the glycoside may be part of a monosaccharide, oligosaccharide, or polysaccharide <cite>StickWilliams Lairson2008 CoutinhoJMB2003 CampbellBJ1997 Coutinho2009</cite>.

== Glycosyl donor substrates ==
Glycosyltransferases can utilize a range of donor substrates. Sugar mono- or diphosphonucleotides are sometimes termed Leloir donors (after Nobel prize winner, Luis Leloir); the corresponding enzymes are termed Leloir glycosyltransferases.

[[Image:Leloir_donors.png|center|500px]]

Glycosyltransferases that utilize non-nucleotide donors, which may be polyprenol pyrophosphates, polyprenol phosphates, sugar-1-phosphates, or sugar-1-pyrophosphates, are termed non-Leloir glycosyltransferases.

[[Image:non-Leloir_donors.png|center|500px]]

In the last two cases, the enzymes that catalyze the transfer of a glycosyl group from a glycosyl phosphate or pyrophosphate are more commonly referred to as [[phosphorylases]] and pyrophosphorylases. Some of these enzymes are classified into [[glycoside hydrolase]] (GH) families (eg sucrose phosphorylase, [[GH13]]) and others are classified into GT familes (eg glycogen phosphorylase [[GT35]]).

''For specific examples of glycosyl donor substrates, please see the [[#Common sugar donors|Common sugar donors]] section below.''

== Mechanism ==
Glycosyltransferases catalyze the transfer of glycosyl groups to a nucleophilic acceptor with either retention or inversion of configuration at the anomeric centre. This allows the classification of glycosyltransferases as either retaining or inverting enzymes.

=== Inverting glycosyltransferases ===
Structural and kinetic data for inverting glycosyltransferases support a mechanism that proceeds through a single nucleophilic substitution step, facilitated by an enzymic general base catalyst. The [[transition state]] is believed to possess substantial [[oxocarbenium ion]] character. Most inverting glycosyltransferases require a divalent cation (typically Mg2+ or Mn2+) although metal-independent enzymes are known <cite>Lairson2008</cite>.
[[Image:Inverting_glycosyltransferase_mechanism.png|center|700px]]

=== Retaining glycosyltransferases ===
Mechanistic evidence for the catalytic process that results in retention of configuration of glycosyltransferases is scant. One mechanism that seems reasonable by comparison with the [[classical Koshland retaining mechanism]] for [[glycoside hydrolases]], involves an enzymic nucleophile. In this mechanism, the enzymic nucleophile reacts with the glycosyl donor to generate a glycosyl enzyme with inversion of stereochemistry, followed by reaction with the glycosyl acceptor with a second inversion of stereochemistry, to yield a product glycoside with a net retention of anomeric stereochemistry. However, attempts to trap a glycosyl enzyme on a wildtype glycosyltransferase have met with universal failure.

Further, crystal structures of retaining glycosyltransferases are mostly ambiguous with respect to identifying possible candidate nucleophiles, with some lacking any suitable nucleophile situated close enough to the glycosyl donor to be able to form a covalent intermediate. In other cases structural analysis has identified possible candidate nucleophiles, which mutagenesis studies have supported. In some cases mutagenesis of the candidate residue to a non-nucleophilic amino acid has not resulted in the expected, dramatic loss in catalytic activity (of the order of 106), as seen for retaining glycoside hydrolases.

The most common alternative mechanism that is invoked is one that involves an SNi process, also termed 'internal return'. In this process, the leaving group on the donor departs, and the nucleophile attacks from the same face, with the other face of the donor being blocked by the enzyme. An open question is whether this process is concerted (''i.e.'' involving a single [[transition state]] in which bonds are formed and broken at similar times) or stepwise (''i.e.'' involving two [[transition state]]s, and therefore an oxocarbenium ion intermediate). A detailed kinetic study of the retaining glycosyltransferase OtsA (which catalyzes the synthesis of trehalose-6-phosphate from UDP-glucose and glucose-6-phosphate) yielded data that was consistent with a frontside, SNi-type reaction <cite>Lee2011</cite>.

[[Image:Retaining glycosyltransferase mechanism.png|center|700px]]

== Classification ==
=== Sequence based classification ===
[[Sequence-based classification]] uses algorithmic methods to assign protein sequences to various families. The glycosyltransferases have been classified into more than 90 families <cite>Campbell1997 Countinho2003</cite>. Each family (GT family) contains proteins that are related by sequence, and by corollary, fold. This allows several useful predictions to be made since the catalytic machinery is conserved within each family. Usually, the mechanism used (''i.e.'' retaining or inverting) is also conserved within a GT family. An actively curated list of GT families and members is available through the Carbohydrate Active enZyme database <cite>CAZyURL</cite>.

== 3-D folds ==
In striking contrast to [[glycoside hydrolases]], which exhibit a wide variety of folds, GTs exhibit a much narrower range of folds.

=== Leloir GTs ===
Sugar nucleotide-dependent (Leloir) glycosyltransferases have been found to possess a range of different folds. The main folds are termed the GT-A and GT-B folds <cite>Unligil2000</cite>. The GT-A fold is typified by the first member to have its X-ray structure determined, SpsA from''Bacillus subtilus'' <cite>Charnock1999</cite>. The GT-A fold consists of two dissimilar domains, one involved in nucleotide binding and the other binding the acceptor. The GT-B fold was exemplified by its first member, the beta-glucosyltransferase from bacteriophage T4 <cite>Vrielink1994</cite>. The GT-B fold consists of two similar Rossmann fold subdomains. The mannosyltransferases of family [[GT108]] possess a 5-bladed beta-propeller fold, similar to family [[GH130]] glycoside phosphorylases <cite>Sernee2019</cite>.

=== Non-Leloir GTs ===
Non-Leloir glycosyltransferases possess non-GT-A and -B folds. For example, the polymerizing glycosyltransferase transglycosylase, which catalyzes the condensation of oligosaccharyl polyprenolphosphate to generate the carbohydrate backbone of peptidoglycan has a bacteriophage-lysozyme-like fold <cite>Lovering2007</cite>. The ''Pyrococcus furiosius'' oligosaccharyltransferase STT3, which catalyzes the transfer of a preformed oligosaccharide from a dolichol phosphate glycosyl donor to form asparagine-linked glycoproteins, possesses a novel fold; however, this crystallized fragment is catalytically-inactive and may not represent the structure of the complete glycosyltransferase <cite>Igura2008</cite>. The same fold is also observed in the periplasmic domain of the PglB oligosaccharyltransferase of ''Camplyobacter lari''; this latter protein was been crystallized as a full length protein incorporating a transmembrane domain that retains transferase activity <cite>Lizak2011</cite>. A structure of PglB oligosaccharyltransferase in complex with a hexapeptide substrate showed that the peptide binds to both the periplasmic domain and the transmembrane domain. These data demonstrate that the transmembrane domain of this bacterial oligosaccharyltransferase is required for both substrate binding and catalysis.


== Role of metals ==
Many, but not all, glycosyltransferases utilize divalent metal ion cofactors such as Mn2+ and Mg2+. These metals are found mainly in glycosyltransferases that are diphosphonucleoside-dependent. X-ray crystallographic analysis reveals that the metal ion is coordinated to an oxygen of each of the two phosphate groups, as well as to side-chain carboxylates derived from the protein. Much has been made of the so-called ‘DXD’ amino acid sequence as an identifier of glycosyltransferases, where the aspartate residues of this sequence are presumed to comprise the metal-binding residues of the active site. However, it is to be cautioned that no part of the DXD motif is invariant among glycosyltransferases, with this motif being present in more than 50% of all protein sequences. Moreover, it is emphasized that many glycosyltransferases are metal-ion independent, and thus do not bind metals at the active site, and so do not require a metal-binding motif at their active site.

== Common sugar donors ==

<gallery widths=270px perrow=3 caption="Examples of sugar nucleotide donors">

File:UDPGlc.png|'''uridine diphospho-D-glucose''' (UDP-Glc)

File:UDPGal.png|'''uridine diphospho-D-galactose''' (UDP-Gal)

File:UDPXyl.png|'''uridine diphospho-D-xylose''' (UDP-Xyl)

File:UDPGlcNAc.png|'''uridine diphospho-''N''-acetyl-D-glucosamine''' (UDP-GlcNAc)

File:UDPGalNAc.png|'''uridine diphospho-''N''-acetyl-D-galactosamine''' (UDP-GalNAc)

File:UDPGlcA.png|'''uridine diphospho-D-glucuronic acid''' (UDP-GlcA)

File:UDPGalf.png|'''uridine diphospho-D-galactofuranose''' (UDP-Gal''f'')

File:GDPMan.png|'''guanosine diphospho-D-mannose''' (GDP-Man)

File:GDPFuc.png|'''guanosine diphospho-L-fucose''' (GDP-Fuc)

File:GDPRha.png|'''guanosine diphospho-L-rhamnose''' (GDP-Rha)

File:CMPNeu5Ac.png|'''cytidine monophospho-''N''-acetylneuraminic acid''' (CMP-Neu5Ac)

File:CMPKdo.png|'''cytidine monophospho-2-keto-3-deoxy-D-mannooctanoic acid''' (CMP-Kdo)

</gallery>

<gallery widths=350px perrow=2 caption="Examples of sugar phospholipid donors">
File:DPMan.png|'''dolichol phosphomannose''' (DP-Man)

File:PPAra.png|'''decaprenolphosphoarabinose''' (PP-Ara)
</gallery>

<gallery widths=740px perrow=1>
File:DolGlc3Man9GlcNAc2.png|'''dolichol-PP-Glc3Man9GlcNac2''' 
</gallery>

== References ==
<biblio>
#StickWilliams isbn=9780240521183
#Lairson2008 pmid=18518825
#CoutinhoJMB2003 pmid=12691742
#CampbellBJ1997 pmid=9334165
#Coutinho2009 isbn=9780470016671 //''Chapter 5:'' Coutinho PM, Rancurel C, Stam M, Bernard T, Couto FM, Danchin EGJ, Henrissat B. "Carbohydrate-active Enzymes Database: Principles and Classification of Glycosyltransferases."
#Charnock1999 pmid=10350455
#Vrielink1994 pmid=8062817
#Unligil2000 pmid=11042447
#Lovering2007 pmid=17347437
#Igura2008 pmid=18046457
#CAZyURL Carbohydrate Active Enzymes database; URL http://www.cazy.org/
#Lizak2011 pmid=21677752
#Lee2011 pmid=21822275
</biblio>

[[Category:Definitions and explanations]]