CAZypedia - User contributions [en-ca]

Glycoside Hydrolase Family 20

2011-02-24T20:51:11Z

Ian Greig:

{{CuratorApproved}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH20'''
|-
|'''Clan'''
|GH-K
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/fam/GH20.html
|}
</div>


== Substrate specificities ==
GH20 members comprise enzymes from both eukaryotes and prokaryotes. In addition to exo-acting ''β''-''N''-acetylglucosaminidases, ''β''-''N''-acetylgalactosamindase and ''β''-6-SO3-''N''-acetylglucosaminidases, GH20 also contains ''exo''-acting lacto-''N''-biosidases that cleave ''β''-D-Gal-(1→3)-D-GlcNAc disaccharides from the non-reducing end of oligosaccharides. The best known members of this family are the human isoenzymes hexosaminidase A (a heterodimer of ''α'' and ''β'' subunits) and B (a homodimer of ''β'' subunits), which are responsible for the hydrolysis of the terminal GalNAc residue from the GM2 ganglioside (GalNAcβ(1–4)-[NANAα(2–3)-]-Galβ(1–4)-Glc-ceramide) within the lysosome. Mutations to these enzymes are responsible for the lysosomal storage disorders Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). Inhibitors of these enzymes are being developed as chemical chaperones to promote the partial restoration of enzyme activity ''in vivo'' and treat these genetic disorders.<cite>Mahuran04</cite> [[Image:GM2.jpg|thumb|300px|GM2 ganglioside is the natural target of human hexasaminidase A and B activity.]]

== Kinetics and Mechanism ==
Neighbouring group participation has long been established as a reasonable mechanism for glycoside hydrolysis in solution<cite>Sinnott76 Bruice67 Bruice68_1 Bruice68_2</cite> and originally outlined as a possible, though subsequently refuted, mechanism for the hen egg-white lysozyme-catalyzed cleavage of ''β''-aryl di-''N''-acetylchitobiosides<cite>Lowe67</cite>. The earliest kinetic evidence supporting a mechanism involving neighbouring group participation in an enzyme-catalyzed hydrolysis<cite>Yamamoto73 Yamamoto74</cite> can be found for an ''N''-acetyl-''β''-D-glucosaminidase isolated from ''Aspergillus oryzae''<cite>Mega70</cite>, likely a GH20 enzyme. This work used free energy relationships to infer neighbouring group participation although complete Michaelis-Menten kinetic parameters were not determined. Such kinetic parameters were determined for a ''β''-''N''-acetylglucosaminidase from ''Aspergillus niger'' and a similar free energy relationship-based analysis carried out to infer neighbouring group participation for this enzyme which, though unknown, is likely from GH20.<cite>Kosman80</cite> The potency of "NAG
-thiazoline" as a competitive inhibitor of the jack bean ''N''-acetyl-''β''-D-hexosaminidase (''K''i = 280 nM) has also been used to infer a mechanism of neighbouring group participation although, interestingly, the only retaining hexosaminidases reported currently (November 2010) reported in the CAZy database for the genus ''Canavalia'' are found in GH18.<cite>Knapp96</cite> Deacetylation of the non-reducing end of a series of chito-oligosaccharides results in a loss of activity of ''Serratia marscescens'' chitobiase, an established GH20 enzyme, towards these compounds, which instead act as competitive inhibitors.<cite>Armand1997</cite> Moreover the structure of ''Serratia marcescens'' chitobiase in complex with a substrate provides structural support for a substrate-assisted mechanism.[[Image:GH20inhibitors.jpg|thumb|450px|'''Competitive Inhibitors of hexosaminidases.''' "NAG-thiazoline" (upper panel) and non-reducing end deacetylated chito-oligosaccharides are competitive inhibitors of hexosamindase employing neighbouring group participation]] A comparative analysis of the activity of ''Streptomyces plicatus'' ''β''-hexosaminidase (SpHex, GH20) and ''Vibrio furnisii'' ''β''-hexosaminidase (ExoII, GH3) towards ''p''-nitrophenyl ''N''-acyl glucosaminides highlights contrasting reactivity trends expected for families of ''β''-glucosaminidase utilizing a mechanism of substrate-assisted catalysis (GH20) and those which do not (GH3): sharp decreases in activity with increasing ''N''-acyl fluorination are observed in the case of the SpHex enzyme whereas negligible changes in activity are observed for ExoII.<cite>SGW05</cite>

== Catalytic Residues ==

The key catalytic residues of GH20 enzymes are found in conserved D-E amino acid pair. This catalytic diad is preceded in the primary sequence by the consensus H-x-G-G motif. The glutamate residue functions as the catalytic general acid/base. As these enzymes employ neighbouring group participation the preceding aspartate is not a nucleophile. Rather kinetic and crystallographic studies have shown that this residue orients and polarizes the catalytic ''N''-acetyl residue.<cite>SJW2002</cite> It may function either as a general base by deprotonating the ''N''-acetyl group in the intermediate and forming a neutral oxazoline intermediate, or alternatively it may electrostatically stabilize a positively charge oxazolinium ion intermediate. The catalytic ''N''-acetyl group of the substrate is bound in a hydrophobic pocket defined by three conserved tryptophan residues. These three tryptophan residues define a compact pocket which does not accommodate (non-native) extended ''N''-acyl side-chains as readily as the elongated hydrophobic pocket found in GH84 enzymes.<cite>DJV2005</cite>

== Three-dimensional structures ==
The first GH20 enzyme to have its structure determined was the ''Serratia marscescens'' chitobiase.<cite>Tews1996</cite> This enzyme's active site is located at the C-terminal end of the third of four protein domains, a (''βα'')8-barrel. On the basis of a structure of this enzyme in complex with the substrate chitobiose, the invariant Glu540 was identified as the likely catalytic general acid/base. Furthermore the ''N''-acetyl group of the non-reducing ''N''-acetylglucosamine residue was found to have its carbonyl oxygen atom suitably positioned to act as the nucleophile.<cite>SJW2002</cite> [[Image:Sphex.jpg|thumb|300px|'''Ribbon diagram of ''SpHex'' with "NAG-thiazoline" bound in active site.''' The catalytic domain II is a (''βα'')8-barrel.]]

== Family Firsts ==
;First sterochemistry determination: The first stereochemical determination for a known member of GH20 was on the ''Serratia marscescens'' enzyme<cite>Armand1997</cite>. The stereochemistry of hydrolysis of three different hexosaminidases (human placenta, jack bean, and bovine kidney) was shown by the Withers group in 1994 <cite>Lai</cite> and it is now generally assumed that some of these are GH20 enzymes.
;First catalytic nucleophile identification: These enzymes employ neighbouring group participation. Prior to the advent of the CAZy system of classification, kinetic studies of the (likely GH20) ''β''-''N''-hexosaminidases from ''Aspergillus oryzae''<cite>Mega70</cite> and ''Aspergillus niger''<cite>Kosman80</cite> supported such a mechanism. This mechanism was further suggested by both the 3-D structure of ''Serratia marcescens'' chitobiase <cite>Tews1996</cite> (by analogy with GH18 enzymes), through work in which the non-reducing end sugar was de-acetylated resulting in total loss in activity <cite>Armand1997</cite>, and by potent inhibition of Jack Bean ''β''-hexosaminidase by NAG-thiazoline<cite>Knapp96</cite>.
;First general acid/base residue identification: Inferred from the 3-D structure <cite>Tews1996</cite> and by analogy with structurally related GH18 chitinases.
;First 3-D structure: The 3-D structure of the ''Serratia marscescens'' chitobiase <cite>Tews1996</cite>.

== References ==
<biblio>
#Yamamoto73 Yamamoto, K, (1973) ''N''-Acyl Specificity of Taka-N-acetyl-''β''-D-glucosaminidase Studied by Synthetic Substrate Analogs II. Preparation of Some ''p''-Nitrophenyl 2-Halogenoacetylamino-2-deoxy-''β''-D-glucopyranoside and Their Susceptibility to Enzymic Hydrolysis. J. Biochem. 73, 749-753.
#Yamamoto74 Yamamoto, K, (1974) A Quantitative Approach to the Evaluation of ''β''-Acetamide Substituent Effects on the Hydrolysis by Taka-N-acetyl-''β''-D-glucosaminidase. Role of the Substrate 2-Acetamide Group in the ''N''-Acyl Specificity of the Enzyme J. Biochem. 76, 385-390.
#Mega70 Mega, T, Ikenaka, T, Matsushima, Y, (1970) Studies on ''N''-Acetyl-''β''-D-glucosaminidase of ''Aspergillus oryzae''. J. Biochem. 68, 109-117.
#Lowe67 Lowe, G, Sheppard, G, Sinnott, ML, Williams, A, (1967) Lysozyme-Catalysed Hydrolysis of some 'β''-Aryl Di-''N''-acetylchitobiosides. Biochem J. 104(3), 893-899.
#Sinnott76 Cocker, D, Sinnott, ML (1976) Acetolysis of 2,4-Dinitrophenyl Glycopyranosides. J. C. S. Perkin II 90, 618-620.
#Bruice67 Piszkiewicz, D, Bruice, T (1967) Glycoside Hydrolysis. I. Intramolecular Acetamido and Hydroxyl Group Catalysis in Glycoside Hydrolysis. J. Am. Chem. Soc. 89, 6237-6243.
#Bruice68_1 Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. II. Intramolecular Carboxyl and Acetamido Group Catalysis in β-Glycoside Hydrolysis. J. Am. Chem. Soc. 90, 2156-2163.
#Bruice68_2 Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. III. Intramolecular Acetamido Group Participation in the Specific Acid Catalyzed Hydrolysis of Methyl-2-Acetamido-2-deoxy-''β''-D-glucopyranoside. J. Am. Chem. Soc. 90, 5844-5848.
#Kosman80 Jones, CS, Kosman, DJ (1980) Purification, Properties, Kinetics, and Mechanism of ''β''-''N''-Acetylglucosaminidase from ''Aspergillus niger''. J. Biol. Chem. 255(24), 11861-11869.
#Knapp96 Knapp, S, Vocadlo, DJ, Gao, Z, Kirk, B, Lou, J, Withers, SG (1996) NAG-thiazoline, An ''N''-Acetyl-''β''-hexosaminidase Inhibitor That Implicates Acetamido Participation. J. Am. Chem. Soc. 118, 6804-6805.
#Mahuran04 pmid=14724290
#Comfort2007 pmid=17323919
#He1999 pmid=9312086
#DJV2005 pmid=15795231
#SJW2002 pmid=12171933
#SGW05 pmid=16171396
#3 isbn=978-0-240-52118-3
#MikesClassic 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]
#Armand1997 pmid=9396742
#Tews1996 pmid=8673609
#Lai pmid=7993902
</biblio>

[[Category:Glycoside Hydrolase Families|GH020]]

Glycoside Hydrolase Family 84

2011-02-24T20:50:38Z

Ian Greig:

{{CuratorApproved}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to ''β''-''N''-acetylhyaluronidases.<cite>Heckel98</cite> Biochemical analysis of a GH84 enzyme annotated in this way, however, revealed that it had no such activity<cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process hyaluronan. Humans possess only one enzyme belonging to GH84 first defined by its unique biochemical properties, including activity at neutral pH and selectivity for ''N''-acetylglucosamine residues. The human GH84 enzyme was labelled as HexC to distinguish it from the human GH20 lysosomal enzymes HexA and HexB. HexC was first cloned from a meningoma library using autologous serum and defined as meningoma expressed antigen 5 (MGEA5).<cite>Heckel98</cite> It was later cloned and biochemically characterized as ''O''-GlcNAcase, a nuclear and cytoplasmic enzyme targeting glycoprotein substrates modified by ''β''-linked GlcNAc residues linked to serine and threonine residues.<cite>Gao01 Dong94</cite> A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking a putative C-terminal histone acetyl transferase domain retains similar kinetic properties (aside from a much lower activity) and inhibition patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain, which shares similarity with other GH84 enzymes.<cite>DJV2009Trunc</cite> In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.

== Kinetics and Mechanism ==
Members of GH84 use a catalytic mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN)<cite>Guthrie89</cite> mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN)<cite>Guthrie89</cite> mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''a values (consistent with prior studies showing hydrolysis of 1-''S''-glucosaminides<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of the ''N''-acyl binding pockets of GH84 enzymes for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DJV2005 DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results clearly identified Asp175 as the general acid catalyst.

The mutant Asp174Ala showed significantly decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing either good or poor leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ (''Cp''OGA)<cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-glucosaminidase (''Bt''OGA)<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but both bacterial homologues are good models of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess good sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain, which separates the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OGA-bound thiazoline show that this intermediate is found in a 4C1conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound in this conformation to ''Bt''OGA active site mutants<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution <cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies <cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base <cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase <cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ <cite>DvA2006</cite>.

== References ==
<biblio>

#Gao01 pmid=11148210
#Guthrie89 Guthrie RD, Jencks WP. ''IUPAC Recommendations for the Representation of Reaction Mechanisms'' Acc. Chem. Res. 1989; 22(10): 343-349.
#Heckel98 pmid=9811929
#Dong94 pmid=8034696
#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2011-02-24T20:40:01Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to ''β''-''N''-acetylhyaluronidases.<cite>Heckel98</cite> Biochemical analysis of a GH84 enzyme annotated in this way, however, revealed that it had no such activity<cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process hyaluronan. Humans possess only one enzyme belonging to GH84 first defined by its unique biochemical properties, including activity at neutral pH and selectivity for ''N''-acetylglucosamine residues. The human GH84 enzyme was labelled as HexC to distinguish it from the human GH20 lysosomal enzymes HexA and HexB. HexC was first cloned from a meningoma library using autologous serum and defined as meningoma expressed antigen 5 (MGEA5).<cite>Heckel98</cite> It was later cloned and biochemically characterized as ''O''-GlcNAcase, a nuclear and cytoplasmic enzyme targeting glycoprotein substrates modified by ''β''-linked GlcNAc residues linked to serine and threonine residues.<cite>Gao01 Dong94</cite> A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking a putative C-terminal histone acetyl transferase domain retains similar kinetic properties (aside from a much lower activity) and inhibition patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain, which shares similarity with other GH84 enzymes.<cite>DJV2009Trunc</cite> In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.

== Kinetics and Mechanism ==
Members of GH84 use a catalytic mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN)<cite>Guthrie89</cite> mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN)<cite>Guthrie89</cite> mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''a values (consistent with prior studies showing hydrolysis of 1-''S''-glucosaminides<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of the ''N''-acyl binding pockets of GH84 enzymes for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DJV2005 DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results clearly identified Asp175 as the general acid catalyst.

The mutant Asp174Ala showed significantly decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing either good or poor leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ (''Cp''OGA)<cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-glucosaminidase (''Bt''OGA)<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but both bacterial homologues are good models of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess good sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain, which separates the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OGA-bound thiazoline show that this intermediate is found in a 4C1conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound in this conformation to ''Bt''OGA active site mutants<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution <cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies <cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base <cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase <cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ <cite>DvA2006</cite>.

== References ==
<biblio>

#Gao01 pmid=11148210
#Guthrie89 Guthrie RD, Jencks WP. ''IUPAC Recommendations for the Representation of Reaction Mechanisms'' Acc. Chem. Res. 1989; 22(10): 343-349.
#Heckel98 pmid=9811929
#Dong94 pmid=8034696
#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2011-02-24T20:01:35Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to ''β''-''N''-acetylhyaluronidases.<cite>Heckel98</cite> Biochemical analysis of a GH84 enzyme annotated in this way, however, revealed that it had no such activity<cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process hyaluronan. Humans possess only one enzyme belonging to GH84 first defined by its unique biochemical properties, including activity at neutral pH and selectivity for ''N''-acetylglucosamine residues. The human GH84 enzyme was labelled as HexC to distinguish it from the human GH20 lysosomal enzymes HexA and HexB. HexC was first cloned from a meningoma library using autologous serum and defined as meningoma expressed antigen 5 (MGEA5).<cite>Heckel98</cite> It was later cloned and biochemically characterized as ''O''-GlcNAcase, a nuclear and cytoplasmic enzyme targeting glycoprotein substrates modified by ''β''-linked GlcNAc residues linked to serine and threonine residues.<cite>Wells02</cite> A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking a putative C-terminal histone acetyl transferase domain retains similar kinetic properties (aside from a much lower activity) and inhibition patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain, which shares similarity with other GH84 enzymes.<cite>DJV2009Trunc</cite> In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.

== Kinetics and Mechanism ==
Members of GH84 use a catalytic mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN)<cite>Guthrie89</cite> mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN)<cite>Guthrie89</cite> mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''a values (consistent with prior studies showing hydrolysis of 1-''S''-glucosaminides<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of the ''N''-acyl binding pockets of GH84 enzymes for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DJV2005 DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results clearly identified Asp175 as the general acid catalyst.

The mutant Asp174Ala showed significantly decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing either good or poor leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ (''Cp''OGA)<cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-glucosaminidase (''Bt''OGA)<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but both bacterial homologues are good models of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess good sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain, which separates the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OGA-bound thiazoline show that this intermediate is found in a 4C1conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound in this conformation to ''Bt''OGA active site mutants<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution <cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies <cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base <cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase <cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ <cite>DvA2006</cite>.

== References ==
<biblio>

#Wells02 pmid=11788610

#Guthrie89 Guthrie RD, Jencks WP. ''IUPAC Recommendations for the Representation of Reaction Mechanisms'' Acc. Chem. Res. 1989; 22(10): 343-349.
#Heckel98 pmid=9811929
#Dong94 pmid=8034696
#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2011-02-24T19:38:05Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to ''β''-''N''-acetylhyaluronidases.<cite>Heckel98</cite> Biochemical analysis of a GH84 enzyme annotated in this way, however, revealed that it had no such activity<cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process hyaluronan. Humans possess only one enzyme belonging to GH84 first defined by its unique biochemical properties, including activity at neutral pH and selectivity for ''N''-acetylglucosamine residues. The human GH84 enzyme was labelled as HexC to distinguish it from the human GH20 lysosomal enzymes HexA and HexB. HexC was first cloned from a meningoma library using autologous serum and defined as meningoma expressed antigen 5 (MGEA5).<cite>Heckel98</cite> It was later cloned and biochemically characterized as ''O''-GlcNAcase, a nuclear and cytoplasmic enzyme targeting glycoprotein substrates modified by ''β''-linked GlcNAc residues linked to serine and threonine residues.<cite>Dong94</cite> A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking a putative C-terminal histone acetyl transferase domain retains similar kinetic properties (aside from a much lower activity) and inhibition patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain, which shares similarity with other GH84 enzymes.<cite>DJV2009Trunc</cite> In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.

== Kinetics and Mechanism ==
Members of GH84 use a catalytic mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN)<cite>Guthrie89</cite> mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN)<cite>Guthrie89</cite> mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''a values (consistent with prior studies showing hydrolysis of 1-''S''-glucosaminides<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of the ''N''-acyl binding pockets of GH84 enzymes for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DJV2005 DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results clearly identified Asp175 as the general acid catalyst.

The mutant Asp174Ala showed significantly decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing either good or poor leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ (''Cp''OGA)<cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-glucosaminidase (''Bt''OGA)<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but both bacterial homologues are good models of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess good sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain, which separates the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OGA-bound thiazoline show that this intermediate is found in a 4C1conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound in this conformation to ''Bt''OGA active site mutants<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution <cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies <cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base <cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase <cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ <cite>DvA2006</cite>.

== References ==
<biblio>

#Guthrie89 Guthrie RD, Jencks WP. ''IUPAC Recommendations for the Representation of Reaction Mechanisms'' Acc. Chem. Res. 1989; 22(10): 343-349.
#Heckel98 pmid=9811929
#Dong94 pmid=8034696
#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2011-02-24T19:10:28Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to ''β''-''N''-acetylhyaluronidases.<cite>Heckel98</cite> Biochemical analysis of a GH84 enzyme annotated in this way, however, revealed that it had no such activity<cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process hyaluronan. Humans possess only one enzyme belonging to GH84 first defined by its unique biochemical properties, including activity at neutral pH and selectivity for ''N''-acetylglucosamine residues. The human GH84 enzyme was labelled as HexC to distinguish it from the human GH20 lysosomal enzymes HexA and HexB. HexC was first cloned from a meningoma library using autologous serum and defined as meningoma expressed antigen 5 (MGEA5).<cite>Heckel98</cite> It was later cloned and biochemically characterized as ''O''-GlcNAcase, a nuclear and cytoplasmic enzyme targeting glycoprotein substrates modified by ''β''-linked GlcNAc residues linked to serine and threonine residues.<cite>Dong94</cite> A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking a putative C-terminal histone acetyl transferase domain retains similar kinetic properties (aside from a much lower activity) and inhibition patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain, which shares similarity with other GH84 enzymes.<cite>DJV2009Trunc</cite> In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.

== Kinetics and Mechanism ==
Members of GH84 use a catalytic mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN)<cite>Guthrie89</cite> mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN)<cite>Guthrie89</cite> mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''a values (consistent with prior studies showing hydrolysis of 1-''S''-glucosaminides<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in an ''N''-terminal domain <cite>DJV2009Trunc</cite>.

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme <cite>DJV2006</cite>.

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ <cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase <cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OG-bound thiazoline show that this intermediate is found in a 4C1conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound to (mutant) enzymes in this conformation <cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution <cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies <cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base <cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase <cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ <cite>DvA2006</cite>.

== References ==
<biblio>

#Guthrie89 Guthrie RD, Jencks WP. ''IUPAC Recommendations for the Representation of Reaction Mechanisms'' Acc. Chem. Res. 1989; 22(10): 343-349.

#Heckel98 pmid=9811929
#Dong94 pmid=8034696
#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2011-02-24T18:59:53Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to ''β''-''N''-acetylhyaluronidases.<cite>Heckel98</cite> Biochemical analysis of a GH84 enzyme annotated in this way, however, revealed that it had no such activity<cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process hyaluronan. Humans possess only one enzyme belonging to GH84 first defined by its unique biochemical properties, including activity at neutral pH and selectivity for ''N''-acetylglucosamine residues. The human GH84 enzyme was labelled as HexC to distinguish it from the human GH20 lysosomal enzymes HexA and HexB. HexC was first cloned from a meningoma library using autologous serum and defined as meningoma expressed antigen 5 (MGEA5).<cite>Heckel98</cite> It was later cloned and biochemically characterized as ''O''-GlcNAcase, a nuclear and cytoplasmic enzyme targeting glycoprotein substrates modified by ''β''-linked GlcNAc residues linked to serine and threonine residues.<cite>Dong94</cite> A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking a putative C-terminal histone acetyl transferase domain retains similar kinetic properties (aside from a much lower activity) and inhibition patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain, which shares similarity with other GH84 enzymes.<cite>DJV2009Trunc</cite> In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis <cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in an ''N''-terminal domain <cite>DJV2009Trunc</cite>.

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme <cite>DJV2006</cite>.

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ <cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase <cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OG-bound thiazoline show that this intermediate is found in a 4C1conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound to (mutant) enzymes in this conformation <cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution <cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies <cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base <cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase <cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ <cite>DvA2006</cite>.

== References ==
<biblio>

#Heckel98 pmid=9811929
#Dong94 pmid=8034696
#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2011-02-24T18:49:34Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to ''β''-''N''-acetylhyaluronidases.<cite>Heckel98</cite> Biochemical analysis of a GH84 enzyme annotated in this way, however, revealed that it had no such activity<cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process hyaluronan. Humans possess only one enzyme belonging to GH84 first defined by its unique biochemical properties, including activity at neutral pH and selectivity for ''N''-acetylglucosamine residues. The human GH84 enzyme was labelled as HexC to distinguish it from the human GH20 lysosomal enzymes HexA and HexB. HexC was first cloned from a meningoma library using autologous serum and defined as meningoma expressed antigen 5 (MGEA5).<cite>Heckel98</cite> It was later cloned and biochemically characterized as ''O''-GlcNAcase, a nuclear and cytoplasmic enzyme targeting glycoprotein substrates modified by ''β''-linked GlcNAc residues linked to serine and threonine residues.<cite>Dong94</cite> ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis <cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in an ''N''-terminal domain <cite>DJV2009Trunc</cite>.

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme <cite>DJV2006</cite>.

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ <cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase <cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OG-bound thiazoline show that this intermediate is found in a 4C1conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound to (mutant) enzymes in this conformation <cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution <cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies <cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base <cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase <cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ <cite>DvA2006</cite>.

== References ==
<biblio>

#Heckel98 pmid=9811929
#Dong94 pmid=8034696
#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2011-02-24T18:44:41Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to ''β''-''N''-acetylhyaluronidases.<cite>Heckel98</cite> Biochemical analysis of a GH84 enzyme annotated in this way, however, revealed that it had no such activity<cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process hyaluronan. Humans possess only one enzyme belonging to GH84 first defined by its unique biochemical properties, including activity at neutral pH and selectivity for ''N''-acetylglucosamine residues. The human GH84 enzyme was labelled as HexC to distinguish it from the human GH20 lysosomal enzymes HexA and HexB. HexC was first cloned from a meningoma library using autologous serum and defined as meningoma expressed antigen 5 (MGEA5).<cite>Heckel98</cite> It was later cloned and biochemically characterized as ''O''-GlcNAcase, a nuclear and cytoplasmic enzyme targeting glycoprotein substrates modified by 'β''-linked GlcNAc residues linked to serine and threonine residues. ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis <cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in an ''N''-terminal domain <cite>DJV2009Trunc</cite>.

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme <cite>DJV2006</cite>.

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ <cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase <cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OG-bound thiazoline show that this intermediate is found in a 4C1conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound to (mutant) enzymes in this conformation <cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution <cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies <cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base <cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase <cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ <cite>DvA2006</cite>.

== References ==
<biblio>

#Heckel98 pmid=9811929

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2011-02-24T18:30:39Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to ''β''-''N''-acetylhyaluronidases.<cite>Heckel98</cite> Biochemical analysis of a GH84 enzyme annotated in this way, however, revealed that it had no such activity<cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process hyaluronan. Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis <cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in an ''N''-terminal domain <cite>DJV2009Trunc</cite>.

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme <cite>DJV2006</cite>.

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ <cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase <cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OG-bound thiazoline show that this intermediate is found in a 4C1conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound to (mutant) enzymes in this conformation <cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution <cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies <cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base <cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase <cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ <cite>DvA2006</cite>.

== References ==
<biblio>

#Heckel98 pmid=9811929

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2011-02-23T23:35:03Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to 'β''-''N''-acetylhyaluronidases.<cite>Heckel98</cite> Biochemical analysis of a GH84 enzyme annotated in this way, however, revealed that it had no such activity<cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process hyaluronan. Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis <cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in an ''N''-terminal domain <cite>DJV2009Trunc</cite>.

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme <cite>DJV2006</cite>.

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ <cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase <cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OG-bound thiazoline show that this intermediate is found in a 4C1conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound to (mutant) enzymes in this conformation <cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution <cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies <cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base <cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase <cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ <cite>DvA2006</cite>.

== References ==
<biblio>

#Heckel98 pmid=9811929

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 20

2011-02-23T23:15:13Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH20'''
|-
|'''Clan'''
|GH-K
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/fam/GH20.html
|}
</div>


== Substrate specificities ==
GH20 members comprise enzymes from both eukaryotes and prokaryotes. In addition to exo-acting ''β''-''N''-acetylglucosaminidases, ''β''-''N''-acetylgalactosamindase and ''β''-6-SO3-''N''-acetylglucosaminidases, GH20 also contains ''exo''-acting lacto-''N''-biosidases that cleave ''β''-D-Gal-(1→3)-D-GlcNAc disaccharides from the non-reducing end of oligosaccharides. The best known members of this family are the human isoenzymes hexosaminidase A (a heterodimer of ''α'' and ''β'' subunits) and B (a homodimer of ''β'' subunits), which are responsible for the hydrolysis of the terminal GalNAc residue from the GM2 ganglioside (GalNAcβ(1–4)-[NANAα(2–3)-]-Galβ(1–4)-Glc-ceramide) within the lysosome. Mutations to these enzymes are responsible for the lysosomal storage disorders Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). Inhibitors of these enzymes are being developed as chemical chaperones to promote the partial restoration of enzyme activity ''in vivo'' and treat these genetic disorders.<cite>Mahuran04</cite> [[Image:GM2.jpg|thumb|300px|GM2 ganglioside is the natural target of human hexasaminidase A and B activity.]]

== Kinetics and Mechanism ==
Neighbouring group participation has long been established as a reasonable mechanism for glycoside hydrolysis in solution<cite>Sinnott76 Bruice67 Bruice68_1 Bruice68_2</cite> and originally outlined as a possible, though subsequently refuted, mechanism for the hen egg-white lysozyme-catalyzed cleavage of ''β''-aryl di-''N''-acetylchitobiosides<cite>Lowe67</cite>. The earliest kinetic evidence supporting a mechanism involving neighbouring group participation in an enzyme-catalyzed hydrolysis<cite>Yamamoto73 Yamamoto74</cite> can be found for an ''N''-acetyl-''β''-D-glucosaminidase isolated from ''Aspergillus oryzae''<cite>Mega70</cite>, likely a GH20 enzyme. This work used free energy relationships to infer neighbouring group participation although complete Michaelis-Menten kinetic parameters were not determined. Such kinetic parameters were determined for a ''β''-''N''-acetylglucosaminidase from ''Aspergillus niger'' and a similar free energy relationship-based analysis carried out to infer neighbouring group participation for this enzyme which, though unknown, is likely from GH20.<cite>Kosman80</cite> The potency of "NAG
-thiazoline" as a competitive inhibitor of the jack bean ''N''-acetyl-''β''-D-hexosaminidase (''K''i = 280 nM) has also been used to infer a mechanism of neighbouring group participation although, interestingly, the only retaining hexosaminidases reported currently (November 2010) reported in the CAZy database for the genus ''Canavalia'' are found in GH18.<cite>Knapp96</cite> Deacetylation of the non-reducing end of a series of chito-oligosaccharides results in a loss of activity of ''Serratia marscescens'' chitobiase, an established GH20 enzyme, towards these compounds, which instead act as competitive inhibitors.<cite>Armand1997</cite> Moreover the structure of ''Serratia marcescens'' chitobiase in complex with a substrate provides structural support for a substrate-assisted mechanism.[[Image:GH20inhibitors.jpg|thumb|450px|'''Competitive Inhibitors of hexosaminidases.''' "NAG-thiazoline" (upper panel) and non-reducing end deacetylated chito-oligosaccharides are competitive inhibitors of hexosamindase employing neighbouring group participation]] A comparative analysis of the activity of ''Streptomyces plicatus'' ''β''-hexosaminidase (SpHex, GH20) and ''Vibrio furnisii'' ''β''-hexosaminidase (ExoII, GH3) towards ''p''-nitrophenyl ''N''-acyl glucosaminides highlights contrasting reactivity trends expected for families of ''β''-glucosaminidase utilizing a mechanism of substrate-assisted catalysis (GH20) and those which do not (GH3): sharp decreases in activity with increasing ''N''-acyl fluorination are observed in the case of the SpHex enzyme whereas negligible changes in activity are observed for ExoII.<cite>SGW05</cite>

== Catalytic Residues ==

The key catalytic residues of GH20 enzymes are found in conserved D-E amino acid pair. This catalytic diad is preceded in the primary sequence by the consensus H-x-G-G motif. The glutamate residue functions as the catalytic general acid/base. As these enzymes employ neighbouring group participation the preceding aspartate is not a nucleophile. Rather kinetic and crystallographic studies have shown that this residue orients and polarizes the catalytic ''N''-acetyl residue.<cite>SJW2002</cite> It may function either as a general base by deprotonating the ''N''-acetyl group in the intermediate and forming a neutral oxazoline intermediate, or alternatively it may electrostatically stabilize a positively charge oxazolinium ion intermediate. The catalytic ''N''-acetyl group of the substrate is bound in a hydrophobic pocket defined by three conserved tryptophan residues. These three tryptophan residues define a compact pocket which does not accommodate (non-native) extended ''N''-acyl side-chains as readily as the elongated hydrophobic pocket found in GH84 enzymes.<cite>DJV2005</cite>

== Three-dimensional structures ==
The first GH20 enzyme to have its structure determined was the ''Serratia marscescens'' chitobiase.<cite>Tews1996</cite> This enzyme's active site is located at the C-terminal end of the third of four protein domains, a (''βα'')8-barrel. On the basis of a structure of this enzyme in complex with the substrate chitobiose, the invariant Glu540 was identified as the likely catalytic general acid/base. Furthermore the ''N''-acetyl group of the non-reducing ''N''-acetylglucosamine residue was found to have its carbonyl oxygen atom suitably positioned to act as the nucleophile.<cite>SJW2002</cite> [[Image:Sphex.jpg|thumb|300px|'''Ribbon diagram of ''SpHex'' with "NAG-thiazoline" bound in active site.''' The catalytic domain II is a (''βα'')8-barrel.]]

== Family Firsts ==
;First sterochemistry determination: The first stereochemical determination for a known member of GH20 was on the ''Serratia marscescens'' enzyme<cite>Armand1997</cite>. The stereochemistry of hydrolysis of three different hexosaminidases (human placenta, jack bean, and bovine kidney) was shown by the Withers group in 1994 <cite>Lai</cite> and it is now generally assumed that some of these are GH20 enzymes.
;First catalytic nucleophile identification: These enzymes employ neighbouring group participation. Prior to the advent of the CAZy system of classification, kinetic studies of the (likely GH20) ''β''-''N''-hexosaminidases from ''Aspergillus oryzae''<cite>Mega70</cite> and ''Aspergillus niger''<cite>Kosman80</cite> supported such a mechanism. This mechanism was further suggested by both the 3-D structure of ''Serratia marcescens'' chitobiase <cite>Tews1996</cite> (by analogy with GH18 enzymes), through work in which the non-reducing end sugar was de-acetylated resulting in total loss in activity <cite>Armand1997</cite>, and by potent inhibition of Jack Bean ''β''-hexosaminidase by NAG-thiazoline<cite>Knapp96</cite>.
;First general acid/base residue identification: Inferred from the 3-D structure <cite>Tews1996</cite> and by analogy with structurally related GH18 chitinases.
;First 3-D structure: The 3-D structure of the ''Serratia marscescens'' chitobiase <cite>Tews1996</cite>.

== References ==
<biblio>
#Yamamoto73 Yamamoto, K, (1973) ''N''-Acyl Specificity of Taka-N-acetyl-''β''-D-glucosaminidase Studied by Synthetic Substrate Analogs II. Preparation of Some ''p''-Nitrophenyl 2-Halogenoacetylamino-2-deoxy-''β''-D-glucopyranoside and Their Susceptibility to Enzymic Hydrolysis. J. Biochem. 73, 749-753.
#Yamamoto74 Yamamoto, K, (1974) A Quantitative Approach to the Evaluation of ''β''-Acetamide Substituent Effects on the Hydrolysis by Taka-N-acetyl-''β''-D-glucosaminidase. Role of the Substrate 2-Acetamide Group in the ''N''-Acyl Specificity of the Enzyme J. Biochem. 76, 385-390.
#Mega70 Mega, T, Ikenaka, T, Matsushima, Y, (1970) Studies on ''N''-Acetyl-''β''-D-glucosaminidase of ''Aspergillus oryzae''. J. Biochem. 68, 109-117.
#Lowe67 Lowe, G, Sheppard, G, Sinnott, ML, Williams, A, (1967) Lysozyme-Catalysed Hydrolysis of some 'β''-Aryl Di-''N''-acetylchitobiosides. Biochem J. 104(3), 893-899.
#Sinnott76 Cocker, D, Sinnott, ML (1976) Acetolysis of 2,4-Dinitrophenyl Glycopyranosides. J. C. S. Perkin II 90, 618-620.
#Bruice67 Piszkiewicz, D, Bruice, T (1967) Glycoside Hydrolysis. I. Intramolecular Acetamido and Hydroxyl Group Catalysis in Glycoside Hydrolysis. J. Am. Chem. Soc. 89, 6237-6243.
#Bruice68_1 Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. II. Intramolecular Carboxyl and Acetamido Group Catalysis in β-Glycoside Hydrolysis. J. Am. Chem. Soc. 90, 2156-2163.
#Bruice68_2 Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. III. Intramolecular Acetamido Group Participation in the Specific Acid Catalyzed Hydrolysis of Methyl-2-Acetamido-2-deoxy-''β''-D-glucopyranoside. J. Am. Chem. Soc. 90, 5844-5848.
#Kosman80 Jones, CS, Kosman, DJ (1980) Purification, Properties, Kinetics, and Mechanism of ''β''-''N''-Acetylglucosaminidase from ''Aspergillus niger''. J. Biol. Chem. 255(24), 11861-11869.
#Knapp96 Knapp, S, Vocadlo, DJ, Gao, Z, Kirk, B, Lou, J, Withers, SG (1996) NAG-thiazoline, An ''N''-Acetyl-''β''-hexosaminidase Inhibitor That Implicates Acetamido Participation. J. Am. Chem. Soc. 118, 6804-6805.
#Mahuran04 pmid=14724290
#Comfort2007 pmid=17323919
#He1999 pmid=9312086
#DJV2005 pmid=15795231
#SJW2002 pmid=12171933
#SGW05 pmid=16171396
#3 isbn=978-0-240-52118-3
#MikesClassic 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]
#Armand1997 pmid=9396742
#Tews1996 pmid=8673609
#Lai pmid=7993902
</biblio>

[[Category:Glycoside Hydrolase Families|GH020]]

Glycoside Hydrolase Family 20

2011-02-23T22:58:58Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH20'''
|-
|'''Clan'''
|GH-K
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/fam/GH20.html
|}
</div>


== Substrate specificities ==
GH20 members comprise enzymes from both eukaryotes and prokaryotes. In addition to exo-acting ''β''-''N''-acetylglucosaminidases, ''β''-''N''-acetylgalactosamindase and ''β''-6-SO3-''N''-acetylglucosaminidases, GH20 also contains ''exo''-acting lacto-''N''-biosidases that cleave ''β''-D-Gal-(1→3)-D-GlcNAc disaccharides from the non-reducing end of oligosaccharides. The best known members of this family are the human isoenzymes hexosaminidase A (a heterodimer of ''α'' and ''β'' subunits) and B (a homodimer of ''β'' subunits), which are responsible for the hydrolysis of the terminal GalNAc residue from the GM2 ganglioside (GalNAcβ(1–4)-[NANAα(2–3)-]-Galβ(1–4)-Glc-ceramide) within the lysosome. Mutations to these enzymes are responsible for the lysosomal storage disorders Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). Inhibitors of these enzymes are being developed as chemical chaperones to promote the partial restoration of enzyme activity ''in vivo'' and treat these genetic disorders.<cite>Mahuran04</cite> [[Image:GM2.jpg|thumb|300px|GM2 ganglioside is the natural target of human hexasaminidase A and B activity.]]

== Kinetics and Mechanism ==
Neighbouring group participation has long been established as a reasonable mechanism for glycoside hydrolysis in solution<cite>Sinnott76 Bruice67 Bruice68_1 Bruice68_2</cite> and originally outlined as a possible, though subsequently refuted, mechanism for the hen egg-white lysozyme-catalyzed cleavage of ''β''-aryl di-''N''-acetylchitobiosides<cite>Lowe67</cite>. The earliest kinetic evidence supporting a mechanism involving neighbouring group participation in an enzyme-catalyzed hydrolysis<cite>Yamamoto73 Yamamoto74</cite> can be found for an ''N''-acetyl-''β''-D-glucosaminidase isolated from ''Aspergillus oryzae''<cite>Mega70</cite>, likely a GH20 enzyme. This work used free energy relationships to infer neighbouring group participation although complete Michaelis-Menten kinetic parameters were not determined. Such kinetic parameters were determined for a ''β''-''N''-acetylglucosaminidase from ''Aspergillus niger'' and a similar free energy relationship-based analysis carried out to infer neighbouring group participation for this enzyme which, though unknown, is likely from GH20.<cite>Kosman80</cite> The potency of "NAG
-thiazoline" as a competitive inhibitor of the jack bean ''N''-acetyl-''β''-D-hexosaminidase (''K''i = 280 nM) has also been used to infer a mechanism of neighbouring group participation although, interestingly, the only retaining hexosaminidases reported currently (November 2010) reported in the CAZy database for the genus ''Canavalia'' are found in GH18.<cite>Knapp96</cite> Deacetylation of the non-reducing end of a series of chito-oligosaccharides results in a loss of activity of ''Serratia marscescens'' chitobiase, an established GH20 enzyme, towards these compounds, which instead act as competitive inhibitors.<cite>Armand1997</cite> Moreover the structure of ''Serratia marcescens'' chitobiase in complex with a substrate provides structural support for a substrate-assisted mechanism.[[Image:GH20inhibitors.jpg|thumb|450px|'''Competitive Inhibitors of hexosaminidases.''' "NAG-thiazoline" (upper panel) and non-reducing end deacetylated chito-oligosaccharides are competitive inhibitors of hexosamindase employing neighbouring group participation]] A comparative analysis of the activity of ''Streptomyces plicatus'' ''β''-hexosaminidase (SpHex, GH20) and ''Vibrio furnisii'' ''β''-hexosaminidase (ExoII, GH3) towards ''p''-nitrophenyl ''N''-acyl glucosaminides highlights contrasting reactivity trends expected for families of ''β''-glucosaminidase utilizing a mechanism of substrate-assisted catalysis (GH20) and those which do not (GH3): sharp decreases in activity with increasing ''N''-acyl fluorination are observed in the case of the SpHex enzyme whereas negligible changes in activity are observed for ExoII.<cite>SGW05</cite>

== Catalytic Residues ==

The key catalytic residues of GH20 enzymes are found in conserved D-E amino acid pair. This catalytic diad is preceded in the primary sequence by the consensus H-x-G-G motif. The glutamate residue functions as the catalytic general acid/base. As these enzymes employ neighbouring group participation the preceding aspartate is not a nucleophile. Rather kinetic and crystallographic studies have shown that this residue orients and polarizes the catalytic ''N''-acetyl residue.<cite>SJW2002</cite> It may function either as a general base by deprotonating the ''N''-acetyl group in the intermediate and forming a neutral oxazoline intermediate, or alternatively it may electrostatically stabilize a positively charge oxazolinium ion intermediate. The catalytic ''N''-acetyl group of the substrate is bound in a hydrophobic pocket defined by three conserved tryptophan residues. These three tryptophan residues define a compact pocket which does not accommodate (non-native) extended ''N''-acyl side-chains as readily as the elongated hydrophobic pocket found in GH84 enzymes.<cite>DJV2005</cite>

== Three-dimensional structures ==
The first GH20 enzyme to have its structure determined was the ''Serratia marscescens'' chitobiase.<cite>Tews1996</cite> This enzyme's active site is located at the C-terminal end of the third of four protein domains, a (''βα'')8-barrel. On the basis of a structure of this enzyme in complex with the substrate chitobiose, the invariant Glu540 was identified as the likely catalytic general acid/base. Furthermore the ''N''-acetyl group of the non-reducing ''N''-acetylglucosamine residue was found to have its carbonyl oxygen atom suitably positioned to act as the nucleophile.<cite>SJW2002</cite> [[Image:Sphex.jpg|thumb|300px|'''Ribbon diagram of ''SpHex'' with "NAG-thiazoline" bound in active site.''' The catalytic domain II is a (''βα'')8-barrel.]]

== Family Firsts ==
;First sterochemistry determination: The stereochemistry of hydrolysis of three different hexosaminidases (human placenta, jack bean, and bovine kidney) was shown by the Withers group in 1994 <cite>Lai</cite> and it is (now) assumed that (some of) these are GH20 enzymes. The first stereochemical determination for a fully sequenced GH20 was on the ''Serratia marscescens'' enzyme <cite>Armand1997</cite>.
;First catalytic nucleophile identification: These enzymes employ neighbouring group participation. Prior to the advent of the CAZy system of classification, kinetic studies of the (likely GH20) ''β''-''N''-hexosaminidases from ''Aspergillus oryzae''<cite>Mega70</cite> and ''Aspergillus niger''<cite>Kosman80</cite> supported such a mechanism. This mechanism is further suggested by both the 3-D structure of ''Serratia marcescens'' chitobiase <cite>Tews1996</cite> (by analogy with GH18 enzymes) and through work in which the non-reducing end sugar was de-acetylated resulting in total loss in activity <cite>Armand1997</cite>.
;First general acid/base residue identification: Inferred from the 3-D structure <cite>Tews1996</cite> and by analogy with closely related GH18 chitinases.
;First 3-D structure: The 3-D structure of the ''Serratia marscescens'' chitobiase <cite>Tews1996</cite>.

== References ==
<biblio>
#Yamamoto73 Yamamoto, K, (1973) ''N''-Acyl Specificity of Taka-N-acetyl-''β''-D-glucosaminidase Studied by Synthetic Substrate Analogs II. Preparation of Some ''p''-Nitrophenyl 2-Halogenoacetylamino-2-deoxy-''β''-D-glucopyranoside and Their Susceptibility to Enzymic Hydrolysis. J. Biochem. 73, 749-753.
#Yamamoto74 Yamamoto, K, (1974) A Quantitative Approach to the Evaluation of ''β''-Acetamide Substituent Effects on the Hydrolysis by Taka-N-acetyl-''β''-D-glucosaminidase. Role of the Substrate 2-Acetamide Group in the ''N''-Acyl Specificity of the Enzyme J. Biochem. 76, 385-390.
#Mega70 Mega, T, Ikenaka, T, Matsushima, Y, (1970) Studies on ''N''-Acetyl-''β''-D-glucosaminidase of ''Aspergillus oryzae''. J. Biochem. 68, 109-117.
#Lowe67 Lowe, G, Sheppard, G, Sinnott, ML, Williams, A, (1967) Lysozyme-Catalysed Hydrolysis of some 'β''-Aryl Di-''N''-acetylchitobiosides. Biochem J. 104(3), 893-899.
#Sinnott76 Cocker, D, Sinnott, ML (1976) Acetolysis of 2,4-Dinitrophenyl Glycopyranosides. J. C. S. Perkin II 90, 618-620.
#Bruice67 Piszkiewicz, D, Bruice, T (1967) Glycoside Hydrolysis. I. Intramolecular Acetamido and Hydroxyl Group Catalysis in Glycoside Hydrolysis. J. Am. Chem. Soc. 89, 6237-6243.
#Bruice68_1 Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. II. Intramolecular Carboxyl and Acetamido Group Catalysis in β-Glycoside Hydrolysis. J. Am. Chem. Soc. 90, 2156-2163.
#Bruice68_2 Piszkiewicz, D, Bruice, T (1968) Glycoside Hydrolysis. III. Intramolecular Acetamido Group Participation in the Specific Acid Catalyzed Hydrolysis of Methyl-2-Acetamido-2-deoxy-''β''-D-glucopyranoside. J. Am. Chem. Soc. 90, 5844-5848.
#Kosman80 Jones, CS, Kosman, DJ (1980) Purification, Properties, Kinetics, and Mechanism of ''β''-''N''-Acetylglucosaminidase from ''Aspergillus niger''. J. Biol. Chem. 255(24), 11861-11869.
#Knapp96 Knapp, S, Vocadlo, DJ, Gao, Z, Kirk, B, Lou, J, Withers, SG (1996) NAG-thiazoline, An ''N''-Acetyl-''β''-hexosaminidase Inhibitor That Implicates Acetamido Participation. J. Am. Chem. Soc. 118, 6804-6805.
#Mahuran04 pmid=14724290
#Comfort2007 pmid=17323919
#He1999 pmid=9312086
#DJV2005 pmid=15795231
#SJW2002 pmid=12171933
#SGW05 pmid=16171396
#3 isbn=978-0-240-52118-3
#MikesClassic 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]
#Armand1997 pmid=9396742
#Tews1996 pmid=8673609
#Lai pmid=7993902
</biblio>

[[Category:Glycoside Hydrolase Families|GH020]]

User:Ian Greig

2010-12-15T17:38:49Z

Ian Greig:

I completed my PhD in pysical organic chemistry under the guidance of Professor Tony Kirby (University of Cambridge, UK). Post-doctoral periods with Professors Steven Withers (University of British Columbia, Canada) and Ian Williams (Univeristy of Bath, UK) followed taking up a Leverhulme Trust Early Career Fellowship at the University of Bath. I am currently back on Canada's west coast, working part-time in the Vocadlo lab and following up on some earlier mechanistic studies of GH84 enzymes. My interests lie in understanding a wide variety of biologically and synthetically interesting reaction mechanisms through combinations of experimental and theoretical approaches.<cite>Greig2010</cite>

<biblio>
#Greig2010 pmid=20419174
</biblio>

[[Category:Contributors|Greig, Ian]]

User:Ian Greig

2010-12-15T17:38:23Z

Ian Greig:

I completed my PhD in pysical organic chemistry under the guidance of Tony Kirby (University of Cambridge, UK). Post-doctoral periods with Professors Steven Withers (University of British Columbia, Canada) and Ian Williams (Univeristy of Bath, UK) followed taking up a Leverhulme Trust Early Career Fellowship at the University of Bath. I am currently back on Canada's west coast, working part-time in the Vocadlo lab and following up on some earlier mechanistic studies of GH84 enzymes. My interests lie in understanding a wide variety of biologically and synthetically interesting reaction mechanisms through combinations of experimental and theoretical approaches.<cite>Greig2010</cite>

<biblio>
#Greig2010 pmid=20419174
</biblio>

[[Category:Contributors|Greig, Ian]]

Glycoside Hydrolase Family 84

2010-12-13T23:32:32Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines,<cite>Stick2007</cite> and 6-acetamido-6-deoxy-castanospermine<cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005 Whitworth2007 Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in an ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005</cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine<cite>Mac2010</cite> and the 7-membered ring-containing azepane<cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Early studies of the wild-type ''Bt''OG-bound thiazoline show that this intermediate is found in a 4C1conformation;<cite>DJV2005</cite> subsequent studies have shown that oxazoline intermediates are bound to (mutant) enzymes in this conformation.<cite>GJD2010</cite>

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-13T21:48:00Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines,<cite>Stick2007</cite> and 6-acetamido-6-deoxy-castanospermine<cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005 Whitworth2007 Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in an ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005</cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-Acetamido-6-deoxy-castanospermine<cite>Mac2010</cite> and the 7-membered ring-containing azepane<cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Early studies of the wild-type ''Bt''OG-bound thiazoline show that this intermediate is found in a 4C1conformation;<cite>DJV2005</cite> subsequent studies have shown that oxazoline intermediates are bound to (mutant) enzymes in this conformation.<cite>GJD2010</cite>

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-13T21:28:04Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines,<cite>Stick2007</cite> and 6-acetamido-6-deoxy-castanospermine<cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005 Whitworth2007 Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in an ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005</cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-Acetamido-6-deoxy-castanospermine<cite>Mac2010</cite> and the 7-membered ring-containing azepane<cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-13T21:24:50Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines,<cite>Stick2007</cite> and 6-acetamido-6-deoxy-castanospermine<cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005 Whitworth2007 Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in an ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005</cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-Acetamido-6-deoxy-castanospermine<cite>Mac2010</cite> and the 7-membered ring-containing azepane<cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-13T21:23:55Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines,<cite>Stick2007</cite> and 6-acetamido-6-deoxy-castanospermine<cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005 Whitworth2007 Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in an ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005</cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]

A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the stable 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-Acetamido-6-deoxy-castanospermine<cite>Mac2010</cite> and the 7-membered ring-containing azepane<cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-13T21:18:04Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines,<cite>Stick2007</cite> and 6-acetamido-6-deoxy-castanospermine<cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-Acetamido-6-deoxy-castanospermine<cite>Mac2010</cite> and the 7-membered ring-containing azepane<cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-13T21:17:20Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of [[neighbouring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines,<cite>Stick2007</cite> and 6-acetamido-6-deoxy-castanospermine<cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-Acetamido-6-deoxy-castanospermine<cite>Mac2010</cite> and the 7-membered ring-containing azepane<cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-13T21:12:08Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines,<cite>Stick2007</cite> and 6-acetamido-6-deoxy-castanospermine<cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-Acetamido-6-deoxy-castanospermine<cite>Mac2010</cite> and the 7-membered ring-containing azepane<cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-13T19:39:25Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines,<cite>Stick2007</cite> and 6-Acetamido-6-deoxy-castanospermine<cite>Mac2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-Acetamido-6-deoxy-castanospermine<cite>Mac2010</cite> and the 7-membered ring-containing azepane<cite>Ble2009</cite>. This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-13T19:27:41Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines,<cite>Stick2007</cite> and 6-Acetamido-6-deoxy-castanospermine<cite>Mac2010</cite> . The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327
#Mac2010 pmid=20851343

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T21:35:47Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite> and 6-''epi''-valeinamines<cite>Stick2007</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T21:35:14Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human ''O''-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite> and 6-''epi''-valeinamines<cite>Stick2007</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T21:33:44Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite> and 6-''epi''-valeinamines<cite>Stick2007</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particularly potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T21:29:51Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite> and 6-''epi''-valeinamines<cite>Stick2007</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes,<cite>DvA2008 GJD2009</cite> nor is a particular potent inhibitor of human ''O''-GlcNAcase<cite>DJV2005 Kudlow2001 Sato1999</cite>.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2009 pmid=19217614
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868
#Kudlow2001 pmid=11336633
#Sato1999 pmid=9917327

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T21:11:53Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite> and 6-epi-valeinamines<cite>Stick2007</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin <cite>DvA2008</cite>
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2010 pmid=20863279
#DvA2008 pmid=18721751
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T21:02:22Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite> and 6-epi-valeinamines<cite>Stick2007</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative ''C''-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#Stick2007 pmid=17728868

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T20:52:09Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T20:48:11Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and is often annotated as containing enzymes possessing ''β''-''N''-acetylhyaluronidase activity (though see reference <cite>Black2006</cite>). Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human O-GlcNAcase established that the ''β''-configured hemiacetal product is formed prior to anomerisation.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' O-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#Black2006 pmid=16822234
#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T20:42:26Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and ''β''-''N''-acetylhyaluronidase activities. Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human O-GlcNAcase established that the ''β''-configured hemiacetal product is formed prior to anomerisation.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' O-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T20:38:39Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and ''β''-''N''-acetylhyaluronidase activities. Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",<cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatins,<cite>Dorf2006 Vasella2006</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.<cite>DJV2005</cite><cite>Whitworth2007</cite><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human O-GlcNAcase established that the ''β''-configured hemiacetal product is formed prior to anomerisation.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' O-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#Bartlett1997 pmid=11851452
#Vasella2006 pmid=17057847
#He1999 pmid=9312086
#3 isbn=978-0-240-52118-3
#MikesClassic 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]

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T20:23:59Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and ''β''-''N''-acetylhyaluronidase activities. Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",REF, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatin, <cite>Dorf2006</cite. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases.REFS><cite>Dorf2010</cite> Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues.
Substrate distortion.<cite>DJV2009 DJV2010</cite>

A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human O-GlcNAcase established that the ''β''-configured hemiacetal product is formed prior to anomerisation.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' O-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#Bartlett1997 pmid=11851452
#He1999 pmid=9312086
#3 isbn=978-0-240-52118-3
#MikesClassic 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]

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T19:23:41Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and ''β''-''N''-acetylhyaluronidase activities. Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",REF, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatin, <cite>Dorf2006</cite><cite>Dorf2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) compared to human lysosomal ''β''-hexosaminidases.REFS Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite>Bartlett1997</cite> transition state analogues.
Substrate distortion.<cite>DJV2009 DJV2010</cite>
A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human O-GlcNAcase established that the ''β''-configured hemiacetal product is formed prior to anomerisation.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' O-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#Bartlett1997 pmid=11851452
#He1999 pmid=9312086
#3 isbn=978-0-240-52118-3
#MikesClassic 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]

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T19:20:23Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and ''β''-''N''-acetylhyaluronidase activities. Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",REF, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatin, <cite>Dorf2006</cite><cite>Dorf2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) compared to human lysosomal ''β''-hexosaminidases.REFS Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.<cite>Whitworth2007</cite> As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)<cite></cite> transition state analogues.
Substrate distortion.<cite>DJV2009 DJV2010</cite>
A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human O-GlcNAcase established that the ''β''-configured hemiacetal product is formed prior to anomerisation.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' O-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Whitworth2007 pmid=17227027
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#He1999 pmid=9312086
#3 isbn=978-0-240-52118-3
#MikesClassic 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]

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T19:14:35Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and ''β''-''N''-acetylhyaluronidase activities. Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",REF, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatin, <cite>Dorf2006</cite><cite>Dorf2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) compared to human lysosomal ''β''-hexosaminidases.REFS Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate. As such
Substrate distortion.<cite>DJV2009 DJV2010</cite>
A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human O-GlcNAcase established that the ''β''-configured hemiacetal product is formed prior to anomerisation.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' O-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#He1999 pmid=9312086
#3 isbn=978-0-240-52118-3
#MikesClassic 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]

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T19:04:31Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and ''β''-''N''-acetylhyaluronidase activities. Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",REF, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatin, <cite>Dorf2006</cite><cite>Dorf2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) compared to human lysosomal ''β''-hexosaminidases.REFS
Substrate distortion.<cite>DJV2009 DJV2010</cite>
A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human O-GlcNAcase established that the ''β''-configured hemiacetal product is formed prior to anomerisation.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' O-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#He1999 pmid=9312086
#3 isbn=978-0-240-52118-3
#MikesClassic 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]

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T19:00:15Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and ''β''-''N''-acetylhyaluronidase activities. Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).
Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",REF, PUGNAc (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite> GlcNAcstatin, <cite>Dorf2006</cite><cite>Dorf2010</cite>. The greater tolerance of GH84 enzymes' acyl binding pockets for bulky sidechains compared to those of GH20 enzymes has led to inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) compared to human lysosomal ''β''-hexosaminidases.REFS
Substrate distortion.<cite>DJV2009 DJV2010</cite>
A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human O-GlcNAcase established that the ''β''-configured hemiacetal product is formed prior to anomerisation.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' O-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Stubbs06 pmid=16493467
#Dorf2006 pmid=17177381
#Dorf2010 pmid=21095575
#Comfort2007 pmid=17323919
#He1999 pmid=9312086
#3 isbn=978-0-240-52118-3
#MikesClassic 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]

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T18:48:53Z

Ian Greig:

{{UnderConstruction}}
* [[Author]]: ^^^Ian Greig^^^
* [[Responsible Curator]]: ^^^David Vocadlo^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH84'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/GH84.html
|}
</div>


== Substrate specificities ==

GH84 contains ''β''-''N''-acetylglucosaminidases and ''β''-''N''-acetylhyaluronidase activities. Human O-GlcNAcase is a nucleocytoplasmic enzyme whose ''in vivo'' targets are glycoprotein serine and threonine residues modified by a single ''β''-linked GlcNAc residue. In contrast to the ''β''-hexosaminidases of GH20 a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated.<cite>DJV2005</cite>

== Kinetics and Mechanism ==
Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human ''O''-GlNAcase.<cite>DJV2005</cite> More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures.<cite>DJV2009</cite> For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''a below 11 (with the chemical step rate-determining for substrates with higher p''K''a values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''a greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''as (consistent with prior studies of ''S''-glucosaminide hydrolysis<cite>DJV2005Thio</cite>).
Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",REF, PUGNac (''O''-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-''N''-phenylcarbamate),<cite>Stubbs06</cite>
Substrate distortion.<cite>DJV2009 DJV2010</cite>
A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the N-terminal domain.<cite>DJV2009Trunc</cite>

== Catalytic Residues ==

Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.<cite>DJV2006</cite>

The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.

== Three-dimensional structures ==
The reported crystallization of ''Clostridium perfringens'' NagJ<cite>ABB2005 </cite> was followed by solved structures for that enzyme<cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the exo-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')8-barrel structure. The structure of human ''O''-GlcNAcase has not been solved but ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.<cite>DvA2010</cite> Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
[[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i) binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the 4C1 conformation.]]
A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. <cite>Ble2009</cite> This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside.<cite>GJD2010</cite> Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.<cite>DJV2005</cite> Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.<cite>GJD2010</cite>.

== Family Firsts ==
;First sterochemistry determination: 1H-NMR studies of human O-GlcNAcase established that the ''β''-configured hemiacetal product is formed prior to anomerisation.<cite>DJV2009</cite>.
;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.<cite>DJV2005</cite>.
;First general acid/base residue identification: Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.<cite>DJV2006</cite>.
;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' O-GlcNAcase<cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ<cite>DvA2006</cite>.

== References ==
<biblio>

#ABB2005 pmid=16511172
#Ble2009 pmid=19331390
#DJV2009Trunc pmid=19423084
#DJV2005 pmid=15795231
#DJV2005Thio pmid=16332065
#DJV2009 pmid=19715310
#DJV2010 pmid=20067256
#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067 PMID:16533067]
#GJD2006 pmid=16565725
#GJD2010 pmid=20067256
#DvA2006 pmid=16541109
#DvA2006 pmid=20863279
#Stubbs06 pmid=16493467
#Comfort2007 pmid=17323919
#He1999 pmid=9312086
#3 isbn=978-0-240-52118-3
#MikesClassic 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]

</biblio>

[[Category:Glycoside Hydrolase Families|GH084]]

Glycoside Hydrolase Family 84

2010-12-10T18:37:09Z

Ian Greig:

Glycoside Hydrolase Family 84

2010-12-09T17:06:58Z

Ian Greig:

Glycoside Hydrolase Family 84

2010-12-08T20:12:17Z

Ian Greig:

Glycoside Hydrolase Family 84

2010-12-08T18:58:59Z

Ian Greig:

Glycoside Hydrolase Family 84

2010-12-08T18:55:00Z

Ian Greig:

Glycoside Hydrolase Family 84

2010-12-08T18:52:46Z

Ian Greig:

File:GH84conf.jpg

2010-12-08T18:38:34Z

Ian Greig:

Glycoside Hydrolase Family 84

2010-12-07T19:14:26Z

Ian Greig: