https://www.cazypedia.org/api.php?action=feedcontributions&user=Steve+Withers&feedformat=atomCAZypedia - User contributions [en-ca]2024-03-28T17:33:00ZUser contributionsMediaWiki 1.35.10https://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15485Glycoside Hydrolase Family 332020-06-23T17:13:49Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^ ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acids (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acid <cite>Kim2011 Varki2007 Vimir2004</cite>. These α(2,8) linked sialic acids can occur in polymeric form as polysialic acids, in the brain, as well as in capsular polysaccharides of some bacteria. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
The GH33 family includes most bacterial and simple eukaryotic sialidases, trans-sialidases <cite> Amaya2004</cite> and intramolecular trans-sialidases <cite> Li1990 Tailford2015 </cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
GH33 sialidases and trans-sialidases hydrolyse, or transfer sialic acids to acceptor sugars, with retention of anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a covalent glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue and the labelled amino acid residue identified as Tyr342 by peptide mapping. Subsequent crystal structures provided further insight into the mechanism of the protozoan sialidase <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS confirmed the expected ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
<br />
The catalytic machinery is conserved throughout this family and, as shown by studies on TcTS, three key residues are involved: E230, Y342, D59. Since Watts first trapped the covalent 3F-sialyl enzyme intermediate on Tyr342 in 2003, many confirmatory structures have been solved. It is likely that tyrosine has evolved as the catalytic nucleophile, rather than the carboxylate moiety found in most retaining glycosidases, in order to minimize repulsive charge-charge interactions with the C-1 carboxylate of the sialic acids. The neutral tyrosine can more readily approach the anomeric centre, and is rendered more nucleophilic by an invariant glutamate, Glu230 for TcTS, that serves as a base catalyst for deprotonation/reprotonation of the tyrosine hydroxyl. See Chan et al <cite> ChanBennet2012 </cite> for a detailed analysis of the role of this residue in the GH33 sialidase from ''M. viridifaciens''. <br />
===Acid Base Catalyst===<br />
The third mechanistically relevant conserved residue in the active site is Asp59, which serves as the acid/base catalyst, protonating the glycosidic oxygen as bond cleavage occurs. Following formation of the intermediate, Asp59 then serves as a base catalyst, activating the incoming water molecule (for simple hydrolytic pathways) or glycosyl acceptor (in the case of trans-sialidases) towards trans-glycosylation of the glycosyl-enzyme intermediate. This second attack leads to a double inversion at the anomeric center and a net retention of stereochemistry observed in the released product, Neu5Ac <cite> Amaya2004 Newstead2008 Damager2008 </cite>.<br />
<br />
== Three-dimensional structures ==<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the ''T. cruzi'' trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al (1981) 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
#Tailford2015 pmid=26154892<br />
<br />
#Li1990 pmid=2254319<br />
<br />
#ChanBennet2012 pmid=22133027<br />
<br />
#WatsonBennet2004 pmid=15527797<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15471Glycoside Hydrolase Family 342020-06-23T00:07:38Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH34'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|Tyr/Glu and Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH34.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
All enzymes in GH34 are Neuraminidases (also known as sialidases; EC. 3.2.1.18) that are found, along with the sialic acid-binding protein Hemagglutinin, on the surface of influenza viruses that are pathogenic to mammalian or avian species. These are the H and N of H1N1. Viruses bind to the cell surface and enter via interaction of the hemagglutinin with cell surface sialic acids. The function of the neuraminidase is to cleave sialic acid from the cell surface after budding of progeny virus, to assist viral spread to other cells. GH34 neuraminidases have therefore been major drug design targets, and are very effectively inhibited by the drugs Tamiflu (Oseltamivir) and Relenza (Zanamivir).<br />
<br />
The cell surface sialic acids they cleave are linked α(2,3) or α(2,6) to galactose or N-acetyl galactosamine residues that terminate glycolipid or glycoprotein structures. Sialic acids are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them are found in families GH33, GH34, GH83 and, along with GH93 arabinanases constitute Clan GH-E.<br />
<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases of the GH34 family have been shown to cleave sialic acid residues with retention of anomeric stereochemistry <cite> vonItzstein1992 </cite>. The mechanism of this hydrolysis has been extensively studied, and for years was thought to occur through a sialosyl cation intermediate. However, trapping of the covalent enzyme intermediate of the viral neuraminidase using 3-fluoro-glycosylfluorides <cite> KimWithers2013 </cite> confirmed a covalent structure. Therefore, the mechanism of these neuraminidases is a classic Koshland type double-displacement involving a covalent sialyl-enzyme intermediate on a Tyrosine residue. This is followed by base-catalysed attack of water on the sialyl enzyme, releasing Neu5Ac with net retention of anomeric stereochemistry.<br />
<br />
== Catalytic Residues ==<br />
The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Y406 by use of 3-fluoro-sialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue E277 to be appropriately positioned to act as the general acid/base pair for activation of Y406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same paper Asp151 was considered as a candidate for the acid/base catalyst based upon its interaction with C2OH in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276, though subsequent structures showed that this residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to D151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for D151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base.<br />
<br />
<br />
== Three-dimensional structures ==<br />
These are members of Clan GH-E, along with GH33, 83 and 93. Three dimensional crystallographic structures have been described for neuraminidases of all subtypes of Influenza A (N1-N10) and B (see Structures section of CAZy for PDBs). These structures consist of cytoplasmic, transmembrane, ‘head’ and ‘stem’ domains. Homotetramers of these enzymes form mushroom-like structures on the surface of the virus <cite> Bovin2009 </cite>. Each of these enzymes displays the sialidase 6-blade beta-propeller fold in the catalytic ‘head’ domain, as well as a calcium-binding domain common to this class of glycoside hydrolase. The viral neuraminidases have eight highly conserved residues in the active site that form key stabilizing interactions (hydrogen bonding, hydrophobic interactions, charge-charge interactions) with the bound substrates, and an additional ten conserved residues that are thought to be key structural factors for these enzymes <cite> vonItzstein2007 </cite>. An arginine triad interacts with the carboxylate residue of active site-bound sialic acid. Nearby is the nucleophilic tyrosine residue Tyr406 and its partner glutamate Glu277, which serves as an acid/base for deprotonation/reprotonation of Tyr406 during turnover. Also nearby is the probable acid/base Asp151 for protonation of the glycosidic oxygen and deprotonation of incoming water. Structures of trapped 3-fluorosialosyl enzyme intermediates are available (<cite> KimWithers2013 Vavricka2013 </cite>.<br />
<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: Determined for influenza NA by NMR by the von Itzstein group <cite> vonItzstein1992 </cite>.<br />
;First catalytic nucleophile identification: Implied by X-ray structures, although intermediate was thought to be a carbocation. Determined definitively using a 2,3-difluorosialic acid in the Withers group <cite> KimWithers2013 </cite> and by Vavricka <cite> Vavricka2013 </cite>.<br />
;First general acid/base residue identification: Inferred from X-ray structure below <cite>Varghese1983</cite>.<br />
;First 3-D structure: Influenza neuraminidase determined by Colman group: <cite> Varghese1983 </cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Varghese1983 pmid=6843658<br />
#vonItzstein1992 pmid=1628657<br />
<br />
#KimWithers2013 pmid=23429702<br />
<br />
#Vavricka2013 pmid=23422659<br />
<br />
#Varghese1992 pmid=1438172<br />
<br />
#Lentz1987 pmid=3314986<br />
<br />
#GhateAir1998 pmid=9874196<br />
<br />
#ZhuWilson2012 pmid=23015718<br />
<br />
#Bovin2009 pmid=22649600<br />
#vonItzstein2007 pmid=18049471<br />
</biblio><br />
<br />
<br />
[[Category:Glycoside Hydrolase Families|GH034]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15470Glycoside Hydrolase Family 342020-06-23T00:06:19Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH34'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|Tyr/Glu and Asp<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH34.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
All enzymes in GH34 are Neuraminidases (also known as sialidases; EC. 3.2.1.18) that are found, along with the sialic acid-binding protein Hemagglutinin, on the surface of influenza viruses that are pathogenic to mammalian or avian species. These are the H and N of H1N1. Viruses bind to the cell surface and enter via interaction of the hemagglutinin with cell surface sialic acids. The function of the neuraminidase is to cleave sialic acid from the cell surface after budding of progeny virus, to assist viral spread to other cells. GH34 neuraminidases have therefore been major drug design targets, and are very effectively inhibited by the drugs Tamiflu (Oseltamivir) and Relenza (Zanamivir).<br />
<br />
The cell surface sialic acids they cleave are linked α(2,3) or α(2,6) to galactose or N-acetyl galactosamine residues that terminate glycolipid or glycoprotein structures. Sialic acids are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them are found in families GH33, GH34, GH83 and, along with GH93 arabinanases constitute Clan GH-E.<br />
<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases of the GH34 family have been shown to cleave sialic acid residues with retention of anomeric stereochemistry <cite> vonItzstein1992 </cite>. The mechanism of this hydrolysis has been extensively studied, and for years was thought to occur through a sialosyl cation intermediate. However, trapping of the covalent enzyme intermediate of the viral neuraminidase using 3-fluoro-glycosylfluorides <cite> KimWithers2013 </cite> confirmed a covalent structure. Therefore, the mechanism of these neuraminidases is a classic Koshland type double-displacement involving a covalent sialyl-enzyme intermediate on a Tyrosine residue. This is followed by base-catalysed attack of water on the sialyl enzyme, releasing Neu5Ac with net retention of anomeric stereochemistry.<br />
<br />
== Catalytic Residues ==<br />
The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Y406 by use of 3-fluoro-sialosyl fluorides to trap the covalent intermediate, followed by peptide mapping <cite> KimWithers2013 </cite>. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue E277 to be appropriately positioned to act as the general acid/base pair for activation of Y406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex <cite> KimWithers2013 Vavricka2013 </cite>. This role had been discussed previously <cite> Varghese1992 </cite> though not favoured. In the same paper Asp151 was considered as a candidate for the acid/base catalyst based upon its interaction with C2OH in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276, though subsequent structures showed that this residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to D151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants <cite> ZhuWilson2012 </cite>. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for D151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base.<br />
<br />
<br />
== Three-dimensional structures ==<br />
These are members of Clan GH-E, along with GH33, 83 and 93. Three dimensional crystallographic structures have been described for neuraminidases of all subtypes of Influenza A (N1-N10) and B (see Structures section of CAZy for PDBs). These structures consist of cytoplasmic, transmembrane, ‘head’ and ‘stem’ domains. Homotetramers of these enzymes form mushroom-like structures on the surface of the virus <cite> Bovin2009 </cite>. Each of these enzymes displays the sialidase 6-blade beta-propeller fold in the catalytic ‘head’ domain, as well as a calcium-binding domain common to this class of glycoside hydrolase. The viral neuraminidases have eight highly conserved residues in the active site that form key stabilizing interactions (hydrogen bonding, hydrophobic interactions, charge-charge interactions) with the bound substrates, and an additional ten conserved residues that are thought to be key structural factors for these enzymes <cite> vonItzstein2007 </cite>. An arginine triad interacts with the carboxylate residue of active site-bound sialic acid. Nearby is the nucleophilic tyrosine residue Tyr406 and its partner glutamate Glu277, which serves as an acid/base for deprotonation/reprotonation of Tyr406 during turnover. Also nearby is the probable acid/base Asp151 for protonation of the glycosidic oxygen and deprotonation of incoming water. Structures of trapped 3-fluorosialosyl enzyme intermediates are available (<cite> KimWithers2013 Vavricka2013 </cite>.<br />
<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: Determined for influenza NA by NMR by the von Itzstein group <cite> vonItzstein1992 </cite>.<br />
;First catalytic nucleophile identification: Implied by X-ray structures, although intermediate was thought to be a carbocation. Determined definitively using a 2,3-difluorosialic acid in the Withers group <cite> KimWithers2013 </cite> and by Vavricka <cite> Vavricka2013 </cite>.<br />
;First general acid/base residue identification: Inferred from X-ray structure below <cite>Varghese1983</cite>.<br />
;First 3-D structure: Influenza neuraminidase determined by Colman group: <cite> Varghese1983 </cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Varghese1983 pmid=6843658<br />
#vonItzstein1992 pmid=1628657<br />
<br />
#KimWithers2013 pmid=23429702<br />
<br />
#Vavricka2013 pmid=23422659<br />
<br />
#Varghese1992 pmid=1438172<br />
<br />
#Lentz1987 pmid=3314986<br />
<br />
#GhateAir1998 pmid=9874196<br />
<br />
#ZhuWilson2012 pmid=23015718<br />
<br />
#Bovin2009 pmid=22649600<br />
#vonItzstein2007<br />
</biblio><br />
<br />
<br />
[[Category:Glycoside Hydrolase Families|GH034]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15469Glycoside Hydrolase Family 342020-06-22T23:25:14Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH34'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|Tyr/Glu<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH34.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
All enzymes in GH34 are Neuraminidases (also known as sialidases; EC. 3.2.1.18) that are found, along with the sialic acid-binding protein Hemagglutinin, on the surface of influenza viruses that are pathogenic to mammalian or avian species. These are the H and N of H1N1. Viruses bind to the cell surface and enter via interaction of the hemagglutinin with cell surface sialic acids. The function of the neuraminidase is to cleave sialic acid from the cell surface after budding of progeny virus, to assist viral spread to other cells. GH34 neuraminidases have therefore been major drug design targets, and are very effectively inhibited by the drugs Tamiflu (Oseltamivir) and Relenza (Zanamivir).<br />
<br />
The cell surface sialic acids they cleave are linked α(2,3) or α(2,6) to galactose or N-acetyl galactosamine residues that terminate glycolipid or glycoprotein structures. Sialic acids are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them are found in families GH33, GH34, GH83 and, along with GH93 arabinanases constitute Clan GH-E.<br />
<br />
This is an example of how to make references to a journal article <cite>Comfort2007</cite>. (See the References section below). Multiple references can go in the same place like this <cite>Comfort2007 He1999</cite>. You can even cite books using just the ISBN <cite>StickWilliams</cite>. References that are not in PubMed can be typed in by hand <cite>Sinnott1990</cite>. <br />
<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases of the GH34 family have been shown to cleave sialic acid residues with retention of anomeric stereochemistry (REF). The mechanism of this hydrolysis has been extensively studied, and for years was thought to occur through a sialosyl cation intermediate (von Itzstein). However, trapping of the covalent enzyme intermediate of the viral neuraminidase using 3-fluoro-glycosylfluorides (Kim 2013) confirmed a covalent structure. Therefore, the mechanism of these neuraminidases is a classic Koshland type double-displacement involving a covalent sialyl-enzyme intermediate on a Tyrosine residue. This is followed by base-catalysed attack of water on the sialyl enzyme, releasing Neu5Ac with net retention of anomeric stereochemistry.<br />
<br />
== Catalytic Residues ==<br />
The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Y406 by use of 3-fluoro-sialosyl fluorides to trap the covalent intermediate, followed by peptide mapping (Withers 2013, REF). An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue E277 to be appropriately positioned to act as the general acid/base pair for activation of Y406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex. This role had been discussed previously (Varghese Proteins 1992) though not favoured. In the same paper Asp151 is considered as a candidate for the acid/base catalyst based upon its interaction with C2OH in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of mutants by Lentz et al 1987 Biochemistry, on which basis they suggested Glu276, though subsequent structures showed this residue interacting with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging Ghate and Air (Eur J Biochem 1998). More recently Zhu and Wilson investigated why mutations to D151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for D151 since this is the classical kinetic signature. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base..<br />
<br />
<br />
== Three-dimensional structures ==<br />
These are members of Clan GH-E along with GH33, 83 and 93. Three dimensional crystallographic structures have been described for neuraminidases of all subtypes of Influenza A (N1-N10) and B (see Structures section of CAZy for PDBs). These structures consist of cytoplasmic, transmembrane, ‘head’ and ‘stem’ domains, homotetramers of these enzymes form mushroom-like structures on the surface of the virus (Bovin ActaNature 2009). Each of these enzymes displays the sialidase 6-blade beta-propeller fold in the catalytic ‘head’ domain, as well as a calcium-binding domain common to this class of glycoside hydrolase. The viral neuraminidases have eight highly conserved residues in the active site that form key stabilizing interactions (hydrogen bonding, hydrophobic interactions, charge-charge interactions) with the bound substrates, and an additional ten conserved residues that are thought to be key structural factors for these enzymes (von Itzstein 2007). An arginine triad interacts with the carboxylate residue of active site-bound sialic acid. Nearby is the nucleophilic tyrosine residue Tyr406 and its partner glutamate Glu277, which serves as an acid/base for deprotonation/reprotonation of Tyr406 during turnover. Also nearby is the probable acid/base Asp151 for protonation of the glycosidic oxygen and deprotonation of incoming water. Structures of trapped 3-fluorosialosyl enzyme intermediates are available (Kim et al; Vavricka et al Gao 2013).<br />
<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: Determined for influenza NA by NMR by the von Itzstein group <cite> vonItzstein1992 </cite>.<br />
;First catalytic nucleophile identification: Implied by X-ray structures, although intermediate was thought to be a carbocation. Determined definitively using a 2,3-difluorosialic acid in the Withers group Kim et al Science (2013) 340, 71-75. <cite> KimWithers2013 </cite>.<br />
;First general acid/base residue identification: Inferred from X-ray structure below <cite>Varghese1983</cite>.<br />
;First 3-D structure: Influenza neuraminidase determined by Colman group: <cite> Varghese1983 </cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Varghese1983 pmid=6843658<br />
#vonItzstein1992 pmid=1628657<br />
<br />
#KimWithers2013pmid=23429702<br />
<br />
</biblio><br />
<br />
<br />
[[Category:Glycoside Hydrolase Families|GH034]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15468Glycoside Hydrolase Family 342020-06-22T23:15:55Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH34'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|Tyr/Glu<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH34.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
All enzymes in GH34 are Neuraminidases (also known as sialidases; EC. 3.2.1.18) that are found, along with the sialic acid-binding protein Hemagglutinin, on the surface of influenza viruses that are pathogenic to mammalian or avian species. These are the H and N of H1N1. Viruses bind to the cell surface and enter via interaction of the hemagglutinin with cell surface sialic acids. The function of the neuraminidase is to cleave sialic acid from the cell surface after budding of progeny virus, to assist viral spread to other cells. GH34 neuraminidases have therefore been major drug design targets, and are very effectively inhibited by the drugs Tamiflu (Oseltamivir) and Relenza (Zanamivir).<br />
<br />
The cell surface sialic acids they cleave are linked α(2,3) or α(2,6) to galactose or N-acetyl galactosamine residues that terminate glycolipid or glycoprotein structures. Sialic acids are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them are found in families GH33, GH34, GH83 and, along with GH93 arabinanases constitute Clan GH-E.<br />
<br />
This is an example of how to make references to a journal article <cite>Comfort2007</cite>. (See the References section below). Multiple references can go in the same place like this <cite>Comfort2007 He1999</cite>. You can even cite books using just the ISBN <cite>StickWilliams</cite>. References that are not in PubMed can be typed in by hand <cite>Sinnott1990</cite>. <br />
<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases of the GH34 family have been shown to cleave sialic acid residues with retention of anomeric stereochemistry (REF). The mechanism of this hydrolysis has been extensively studied, and for years was thought to occur through a sialosyl cation intermediate (von Itzstein). However, trapping of the covalent enzyme intermediate of the viral neuraminidase using 3-fluoro-glycosylfluorides (Kim 2013) confirmed a covalent structure. Therefore, the mechanism of these neuraminidases is a classic Koshland type double-displacement involving a covalent sialyl-enzyme intermediate on a Tyrosine residue. This is followed by base-catalysed attack of water on the sialyl enzyme, releasing Neu5Ac with net retention of anomeric stereochemistry.<br />
<br />
== Catalytic Residues ==<br />
The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Y406 by use of 3-fluoro-sialosyl fluorides to trap the covalent intermediate, followed by peptide mapping (Withers 2013, REF). An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue E277 to be appropriately positioned to act as the general acid/base pair for activation of Y406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex. This role had been discussed previously (Varghese Proteins 1992) though not favoured. In the same paper Asp151 is considered as a candidate for the acid/base catalyst based upon its interaction with C2OH in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of mutants by Lentz et al 1987 Biochemistry, on which basis they suggested Glu276, though subsequent structures showed this residue interacting with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging Ghate and Air (Eur J Biochem 1998). More recently Zhu and Wilson investigated why mutations to D151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for D151 since this is the classical kinetic signature. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base..<br />
<br />
<br />
== Three-dimensional structures ==<br />
These are members of Clan GH-E along with GH33, 83 and 93. Three dimensional crystallographic structures have been described for neuraminidases of all subtypes of Influenza A (N1-N10) and B (see Structures section of CAZy for PDBs). These structures consist of cytoplasmic, transmembrane, ‘head’ and ‘stem’ domains, homotetramers of these enzymes form mushroom-like structures on the surface of the virus (Bovin ActaNature 2009). Each of these enzymes displays the sialidase 6-blade beta-propeller fold in the catalytic ‘head’ domain, as well as a calcium-binding domain common to this class of glycoside hydrolase. The viral neuraminidases have eight highly conserved residues in the active site that form key stabilizing interactions (hydrogen bonding, hydrophobic interactions, charge-charge interactions) with the bound substrates, and an additional ten conserved residues that are thought to be key structural factors for these enzymes (von Itzstein 2007). An arginine triad interacts with the carboxylate residue of active site-bound sialic acid. Nearby is the nucleophilic tyrosine residue Tyr406 and its partner glutamate Glu277, which serves as an acid/base for deprotonation/reprotonation of Tyr406 during turnover. Also nearby is the probable acid/base Asp151 for protonation of the glycosidic oxygen and deprotonation of incoming water. Structures of trapped 3-fluorosialosyl enzyme intermediates are available (Kim et al; Vavricka et al Gao 2013).<br />
<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: Determined for influenza NA by NMR by the von Itzstein group Eur. J. Biochem. (1992) 207, 335-343. <cite>Comfort2007</cite>.<br />
;First catalytic nucleophile identification: Implied by X-ray structures, though intermediate thought to be a carbocation. Determined definitively using a 2,3-difluorosialic acid in the Withers group Kim et al Science (2013) 340, 71-75. <cite>Sinnott1990</cite>.<br />
;First general acid/base residue identification: Inferred from X-ray structure below <cite>He1999</cite>.<br />
;First 3-D structure: Influenza neuraminidase determined by Colman group: <cite> Varghese1983 </cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Varghese1983 pmid6843658<br />
<br />
<br />
#Comfort2007 pmid=17323919<br />
<br />
#He1999 pmid=9312086<br />
#StickWilliams isbn=9780240521183<br />
#Sinnott1990 Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. [http://dx.doi.org/10.1021/cr00105a006 DOI: 10.1021/cr00105a006]<br />
</biblio><br />
<br />
<br />
[[Category:Glycoside Hydrolase Families|GH034]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15467Glycoside Hydrolase Family 342020-06-22T23:09:23Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH34'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|Tyr/Glu<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH34.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
All enzymes in GH34 are Neuraminidases (also known as sialidases; EC. 3.2.1.18) that are found, along with the sialic acid-binding protein Hemagglutinin, on the surface of influenza viruses that are pathogenic to mammalian or avian species. These are the H and N of H1N1. Viruses bind to the cell surface and enter via interaction of the hemagglutinin with cell surface sialic acids. The function of the neuraminidase is to cleave sialic acid from the cell surface after budding of progeny virus, to assist viral spread to other cells. GH34 neuraminidases have therefore been major drug design targets, and are very effectively inhibited by the drugs Tamiflu (Oseltamivir) and Relenza (Zanamivir).<br />
<br />
The cell surface sialic acids they cleave are linked a-2,3 or a-2,6 to galactose or N-acetyl galactosamine residues that terminate glycolipid or glycoprotein structures. Sialic acids are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them are found in families GH33, GH34, GH83 and, along with GH93 arabinanases constitute Clan GH-E.<br />
<br />
This is an example of how to make references to a journal article <cite>Comfort2007</cite>. (See the References section below). Multiple references can go in the same place like this <cite>Comfort2007 He1999</cite>. You can even cite books using just the ISBN <cite>StickWilliams</cite>. References that are not in PubMed can be typed in by hand <cite>Sinnott1990</cite>. <br />
<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases of the GH34 family have been shown to cleave sialic acid residues from host polysaccharides with retention of anomeric stereochemistry (REF). The mechanism of this hydrolysis has been extensively studied, and for years was thought to occur through a sialosyl cation intermediate (von Itzstein). However, trapping of the covalent enzyme intermediate of the viral neuraminidase using 3-fluoro-glycosylfluorides (Kim 2013) confirmed a covalent structure. Therefore, the mechanism of these neuraminidases is a classic Koshland type double-displacement involving a covalent sialyl-enzyme intermediate on a Tyrosine residue. This is followed by base-catalysed attack of water on the sialyl enzyme, releasing Neu5Ac with net retention of anomeric stereochemistry.<br />
<br />
== Catalytic Residues ==<br />
The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Y406 by use of 3-fluoro-sialosyl fluorides to trap the covalent intermediate, followed by peptide mapping (Withers 2013, REF). An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue E277 to be appropriately positioned to act as the general acid/base pair for activation of Y406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex. This role had been discussed previously (Varghese Proteins 1992) though not favoured. In the same paper Asp151 is considered as a candidate for the acid/base catalyst based upon its interaction with C2OH in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of mutants by Lentz et al 1987 Biochemistry, on which basis they suggested Glu276, though subsequent structures showed this residue interacting with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging Ghate and Air (Eur J Biochem 1998). More recently Zhu and Wilson investigated why mutations to D151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for D151 since this is the classical kinetic signature. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base..<br />
<br />
<br />
== Three-dimensional structures ==<br />
These are members of Clan GH-E along with GH33, 83 and 93. Three dimensional crystallographic structures have been described for neuraminidases of all subtypes of Influenza A (N1-N10) and B (see Structures section of CAZy for PDBs). These structures consist of cytoplasmic, transmembrane, ‘head’ and ‘stem’ domains, homotetramers of these enzymes form mushroom-like structures on the surface of the virus (Bovin ActaNature 2009). Each of these enzymes displays the sialidase 6-blade beta-propeller fold in the catalytic ‘head’ domain, as well as a calcium-binding domain common to this class of glycoside hydrolase. The viral neuraminidases have eight highly conserved residues in the active site that form key stabilizing interactions (hydrogen bonding, hydrophobic interactions, charge-charge interactions) with the bound substrates, and an additional ten conserved residues that are thought to be key structural factors for these enzymes (von Itzstein 2007). An arginine triad interacts with the carboxylate residue of active site-bound sialic acid. Nearby is the nucleophilic tyrosine residue Tyr406 and its partner glutamate Glu277, which serves as an acid/base for deprotonation/reprotonation of Tyr406 during turnover. Also nearby is the probable acid/base Asp151 for protonation of the glycosidic oxygen and deprotonation of incoming water. Structures of trapped 3-fluorosialosyl enzyme intermediates are available (Kim et al; Vavricka et al Gao 2013).<br />
<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: Determined for influenza NA by NMR by the von Itzstein group Eur. J. Biochem. (1992) 207, 335-343. <cite>Comfort2007</cite>.<br />
;First catalytic nucleophile identification: Implied by X-ray structures, though intermediate thought to be a carbocation. Determined definitively using a 2,3-difluorosialic acid in the Withers group Kim et al Science (2013) 340, 71-75. <cite>Sinnott1990</cite>.<br />
;First general acid/base residue identification: Inferred from X-ray structure below <cite>He1999</cite>.<br />
;First 3-D structure: Influenza neuraminidase determined by Colman group: Varghese et al (1983) Nature 303, 35-40. <cite>StickWilliams</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Comfort2007 pmid=17323919<br />
#He1999 pmid=9312086<br />
#StickWilliams isbn=9780240521183<br />
#Sinnott1990 Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. [http://dx.doi.org/10.1021/cr00105a006 DOI: 10.1021/cr00105a006]<br />
</biblio><br />
<br />
<br />
[[Category:Glycoside Hydrolase Families|GH034]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15466Glycoside Hydrolase Family 332020-06-22T22:58:54Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^ ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acids (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acid <cite>Kim2011 Varki2007 Vimir2004</cite>. These α(2,8) linked sialic acids can occur in polymeric form as polysialic acids, in the brain, as well as in capsular polysaccharides of some bacteria. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
The GH33 family includes most bacterial and simple eukaryotic sialidases, trans-sialidases <cite> Amaya2004</cite> and intramolecular trans-sialidases <cite> Li1990 Tailford2015 </cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
GH33 sialidases and trans-sialidases hydrolyse, or transfer sialic acids to acceptor sugars, with retention of anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a covalent glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue and the labelled amino acid residue identified as Tyr342by peptide mapping. Subsequent crystal structures provided further insight into the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS confirmed the expected ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
<br />
The catalytic machinery is conserved throughout this family and, as shown by studies on TcTS, three key residues are involved: E167, Y342, D59. Since Watts first trapped the covalent 3F-sialyl enzyme intermediate on Tyr342 in 2003, many confirmatory structures have been solved. It is likely that tyrosine has evolved as the catalytic nucleophile, rather than the carboxylate moiety found in most retaining glycosidases, in order to minimize repulsive charge-charge interactions with the C-1 carboxylate of the sialic acids. The neutral tyrosine can more readily approach the anomeric centre, and is rendered more nucleophilic by an invariant glutamate, Glu167 for TcTS, that serves as a base catalyst for deprotonation/reprotonation of the tyrosine hydroxyl. See Chan et al <cite> ChanBennet2012 </cite> for a detailed analysis of the role of this residue in the GH33 sialidase from M. viridifaciens. The third mechanistically relevant conserved residue in the active site is Asp59, which serves as the acid/base catalyst, protonating the glycosidic oxygen as bond cleavage occurs. Following formation of the intermediate, Asp59 then serves as a base catalyst, activating the incoming water molecule (for simple hydrolytic pathways) or glycosyl acceptor (in the case of trans-sialidases) towards trans-glycosylation of the glycosyl-enzyme intermediate. This second attack leads to a double inversion at the anomeric center and a net retention of stereochemistry observed in the released product, Neu5Ac <cite> Amaya2004 Newstead2008 Damager2008 </cite>.<br />
===Acid Base Catalyst===<br />
The third mechanistically relevant conserved residue in the active site is Asp59, which serves as the acid/base catalyst, protonating the glycosidic oxygen as bond cleavage occurs. Following formation of the intermediate, Asp59 then serves as a base catalyst, activating the incoming water molecule (for simple hydrolytic pathways) or glycosyl acceptor (in the case of trans-sialidases) towards trans-glycosylation of the glycosyl-enzyme intermediate. The acid/base catalyst has been identified primarily from X-ray crystal structures, but also from the kinetic behaviour of mutants modified at that position <cite> Crennell1993 Amaya2004 Damager2008 </cite>. A nice attempt to further probe the potential role of that Asp in the M viridifaciens sialidase by kinetic analysis of mutants modified at that position was sidelined by use of carboxylate buffers, which likely effected chemical rescue, leading to underestimates of the effects of mutation on activity <cite> WatsonBennet2004 </cite><br />
<br />
== Three-dimensional structures ==<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al (1981) 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
#Tailford2015 pmid=26154892<br />
<br />
#Li1990 pmid=2254319<br />
<br />
#ChanBennet2012 pmid=22133027<br />
<br />
#WatsonBennet2004 pmid15527797<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15465Glycoside Hydrolase Family 332020-06-22T22:57:43Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^ ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acids (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acid <cite>Kim2011 Varki2007 Vimir2004</cite>. These α(2,8) linked sialic acids can occur in polymeric form as polysialic acids, in the brain, as well as in capsular polysaccharides of some bacteria. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
The GH33 family includes most bacterial and simple eukaryotic sialidases, trans-sialidases <cite> Amaya2004</cite> and intramolecular trans-sialidases <cite> Li1990 Tailford2015 </cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
GH33 sialidases and trans-sialidases hydrolyse, or transfer sialic acids to acceptor sugars, with retention of anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a covalent glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue and the labelled amino acid residue identified as Tyr342by peptide mapping. Subsequent crystal structures provided further insight into the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS confirmed the expected ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
'''===Nucleophile==='''<br />
<br />
The catalytic machinery is conserved throughout this family and, as shown by studies on TcTS, three key residues are involved: E167, Y342, D59. Since Watts first trapped the covalent 3F-sialyl enzyme intermediate on Tyr342 in 2003, many confirmatory structures have been solved. It is likely that tyrosine has evolved as the catalytic nucleophile, rather than the carboxylate moiety found in most retaining glycosidases, in order to minimize repulsive charge-charge interactions with the C-1 carboxylate of the sialic acids. The neutral tyrosine can more readily approach the anomeric centre, and is rendered more nucleophilic by an invariant glutamate, Glu167 for TcTS, that serves as a base catalyst for deprotonation/reprotonation of the tyrosine hydroxyl. See Chan et al <cite> ChanBennet2012 </cite> for a detailed analysis of the role of this residue in the GH33 sialidase from M. viridifaciens. The third mechanistically relevant conserved residue in the active site is Asp59, which serves as the acid/base catalyst, protonating the glycosidic oxygen as bond cleavage occurs. Following formation of the intermediate, Asp59 then serves as a base catalyst, activating the incoming water molecule (for simple hydrolytic pathways) or glycosyl acceptor (in the case of trans-sialidases) towards trans-glycosylation of the glycosyl-enzyme intermediate. This second attack leads to a double inversion at the anomeric center and a net retention of stereochemistry observed in the released product, Neu5Ac <cite> Amaya2004 Newstead2008 Damager2008 </cite>.<br />
===Acid Base Catalyst===<br />
The third mechanistically relevant conserved residue in the active site is Asp59, which serves as the acid/base catalyst, protonating the glycosidic oxygen as bond cleavage occurs. Following formation of the intermediate, Asp59 then serves as a base catalyst, activating the incoming water molecule (for simple hydrolytic pathways) or glycosyl acceptor (in the case of trans-sialidases) towards trans-glycosylation of the glycosyl-enzyme intermediate. The acid/base catalyst has been identified primarily from X-ray crystal structures, but also from the kinetic behaviour of mutants modified at that position <cite> Crennell1993 Amaya2004 Damager2008 </cite>. A nice attempt to further probe the potential role of that Asp in the M viridifaciens sialidase by kinetic analysis of mutants modified at that position was sidelined by use of carboxylate buffers, which likely effected chemical rescue, leading to underestimates of the effects of mutation on activity <cite> WatsonBennet2004 </cite><br />
<br />
== Three-dimensional structures ==<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al (1981) 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
#Tailford2015 pmid=26154892<br />
<br />
#Li1990 pmid=2254319<br />
<br />
#ChanBennet2012 pmid=22133027<br />
<br />
#WatsonBennet2004 pmid15527797<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15464Glycoside Hydrolase Family 332020-06-22T22:48:02Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^ ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acids (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acid <cite>Kim2011 Varki2007 Vimir2004</cite>. These α(2,8) linked sialic acids can occur in polymeric form as polysialic acids, in the brain, as well as in capsular polysaccharides of some bacteria. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
The GH33 family includes most bacterial and simple eukaryotic sialidases, trans-sialidases <cite> Amaya2004</cite> and intramolecular trans-sialidases <cite> Li1990 Tailford2015 </cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
GH33 sialidases and trans-sialidases hydrolyse, or transfer sialic acids to acceptor sugars, with retention of anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a covalent glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue and the labelled amino acid residue identified as Tyr342by peptide mapping. Subsequent crystal structures provided further insight into the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS confirmed the expected ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===The catalytic machinery is conserved throughout this family and, as shown by studies on TcTS, three key residues are involved: E167, Y342, D59. Since Watts first trapped the covalent 3F-sialyl enzyme intermediate on Tyr342 in 2003, many confirmatory structures have been solved. It is likely that tyrosine has evolved as the catalytic nucleophile, rather than the carboxylate moiety found in most retaining glycosidases, in order to minimize repulsive charge-charge interactions with the C-1 carboxylate of the sialic acids. The neutral tyrosine can more readily approach the anomeric centre, and is rendered more nucleophilic by an invariant glutamate, Glu167 for TcTS, that serves as a base catalyst for deprotonation/reprotonation of the tyrosine hydroxyl. See Chan et al <cite> ChanBennet2012 </cite> for a detailed analysis of the role of this residue in the GH33 sialidase from M. viridifaciens. The third mechanistically relevant conserved residue in the active site is Asp59, which serves as the acid/base catalyst, protonating the glycosidic oxygen as bond cleavage occurs. Following formation of the intermediate, Asp59 then serves as a base catalyst, activating the incoming water molecule (for simple hydrolytic pathways) or glycosyl acceptor (in the case of trans-sialidases) towards trans-glycosylation of the glycosyl-enzyme intermediate. This second attack leads to a double inversion at the anomeric center and a net retention of stereochemistry observed in the released product, Neu5Ac <cite> Amaya2004 Newstead2008 Damager2008 </cite>.<br />
<br />
<br />
===Acid Base Catalyst===<br />
The third mechanistically relevant conserved residue in the active site is Asp59, which serves as the acid/base catalyst, protonating the glycosidic oxygen as bond cleavage occurs. Following formation of the intermediate, Asp59 then serves as a base catalyst, activating the incoming water molecule (for simple hydrolytic pathways) or glycosyl acceptor (in the case of trans-sialidases) towards trans-glycosylation of the glycosyl-enzyme intermediate. The acid/base catalyst has been identified primarily from X-ray crystal structures, but also from the kinetic behaviour of mutants modified at that position <cite> Crennell1993 Amaya2004 Damager2008 </cite>:<br />
<br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al (1981) 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
#Tailford2015 pmid=26154892<br />
<br />
#Li1990 pmid=2254319<br />
<br />
#ChanBennet2012 pmid=22133027<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15463Glycoside Hydrolase Family 332020-06-22T22:36:30Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^ ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acids (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acid <cite>Kim2011 Varki2007 Vimir2004</cite>. These α(2,8) linked sialic acids can occur in polymeric form as polysialic acids, in the brain, as well as in capsular polysaccharides of some bacteria. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
The GH33 family includes most bacterial and simple eukaryotic sialidases, trans-sialidases <cite> Amaya2004</cite> and intramolecular trans-sialidases <cite> Li1990 Tailford2015 </cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
GH33 sialidases and trans-sialidases hydrolyse, or transfer sialic acids to acceptor sugars, with retention of anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a covalent glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue and the labelled amino acid residue identified as Tyr342by peptide mapping. Subsequent crystal structures provided further insight into the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS confirmed the expected ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===The catalytic machinery is conserved throughout this family and, as shown by studies on TcTS, three key residues are involved: E167, Y342, D59. Since Watts first trapped the covalent 3F-sialyl enzyme intermediate on Tyr342 in 2003, many confirmatory structures have been solved. It is likely that tyrosine has evolved as the catalytic nucleophile, rather than the carboxylate moiety found in most retaining glycosidases, in order to minimize repulsive charge-charge interactions with the C-1 carboxylate of the sialic acids. The neutral tyrosine can more readily approach the anomeric centre, and is rendered more nucleophilic by an invariant glutamate, Glu167 for TcTS, that serves as a base catalyst for deprotonation/reprotonation of the tyrosine hydroxyl. See Chan et al <cite> ChanBennet2012 </cite> for a detailed analysis of the role of this residue in the GH33 sialidase from M. viridifaciens. The third mechanistically relevant conserved residue in the active site is Asp59, which serves as the acid/base catalyst, protonating the glycosidic oxygen as bond cleavage occurs. Following formation of the intermediate, Asp59 then serves as a base catalyst, activating the incoming water molecule (for simple hydrolytic pathways) or glycosyl acceptor (in the case of trans-sialidases) towards trans-glycosylation of the glycosyl-enzyme intermediate. This second attack leads to a double inversion at the anomeric center and a net retention of stereochemistry observed in the released product, Neu5Ac.<br />
<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al (1981) 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
#Tailford2015 pmid=26154892<br />
<br />
#Li1990 pmid=2254319<br />
<br />
#ChanBennet2012 pmid=22133027<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15462Glycoside Hydrolase Family 332020-06-22T18:15:37Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^ ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acids (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acid <cite>Kim2011 Varki2007 Vimir2004</cite>. These α(2,8) linked sialic acids can occur in polymeric form as polysialic acids, in the brain, as well as in capsular polysaccharides of some bacteria. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
The GH33 family includes most bacterial and simple eukaryotic sialidases, trans-sialidases <cite> Amaya2004</cite> and intramolecular trans-sialidases <cite> Li1990 Tailford2015 </cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
GH33 sialidases and trans-sialidases hydrolyse, or transfer sialic acids to acceptor sugars, with retention of anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a covalent glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue and the labelled amino acid residue identified as Tyr342by peptide mapping. Subsequent crystal structures provided further insight into the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS confirmed the expected ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al (1981) 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
#Tailford2015 pmid=26154892<br />
<br />
#Li1990 pmid=2254319<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15461Glycoside Hydrolase Family 332020-06-22T17:46:50Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^ ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al (1981) 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
#Tailford2015 pmid=26154892<br />
<br />
#Li1990 pmid=2254319<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15460Glycoside Hydrolase Family 332020-06-22T17:36:47Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^ ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al (1981) 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15459Glycoside Hydrolase Family 332020-06-22T17:36:17Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson, Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al (1981) 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15458Glycoside Hydrolase Family 332020-06-22T17:35:44Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson and Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al (1981) 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15457Glycoside Hydrolase Family 332020-06-22T17:32:28Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. (1981) 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15456Glycoside Hydrolase Family 332020-06-22T17:31:40Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. (1981) 3, 321-326.<br />
<br />
#Crennell1993 pmid=8234325<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15455Glycoside Hydrolase Family 332020-06-22T17:27:06Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. (1981) 3, 321-326.<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15454Glycoside Hydrolase Family 332020-06-22T17:24:56Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al <cite> Friebolin1981 </cite>.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through paptide mapping after labelling with 2,3-difluorosialic acid. Watts et al J. Am. Chem. Soc. (2003) 125, 7532-7533.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. (1981) 3, 321-326.<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15453Glycoside Hydrolase Family 332020-06-22T17:23:07Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al Biochem. Int. (1981) 3, 321-326.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through paptide mapping after labelling with 2,3-difluorosialic acid. Watts et al J. Am. Chem. Soc. (2003) 125, 7532-7533.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. (1981) 3, 321-326.<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15452Glycoside Hydrolase Family 332020-06-22T17:19:41Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration <cite> Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al Biochem. Int. (1981) 3, 321-326.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through paptide mapping after labelling with 2,3-difluorosialic acid. Watts et al J. Am. Chem. Soc. (2003) 125, 7532-7533.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al Biochem. Int. (1981) 3, 321-326.<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15451Glycoside Hydrolase Family 332020-06-22T17:11:52Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration<cite/cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al Biochem. Int. (1981) 3, 321-326.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through paptide mapping after labelling with 2,3-difluorosialic acid. Watts et al J. Am. Chem. Soc. (2003) 125, 7532-7533.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin1981 Friebolin, H. et al Biochem. Int. (1981) 3, 321-326.<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15450Glycoside Hydrolase Family 332020-06-22T17:10:01Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al Biochem. Int. (1981) 3, 321-326.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through paptide mapping after labelling with 2,3-difluorosialic acid. Watts et al J. Am. Chem. Soc. (2003) 125, 7532-7533.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
<br />
#Friebolin 1981 Friebolin, H. et al Biochem. Int. (1981) 3, 321-326.<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15449Glycoside Hydrolase Family 332020-06-22T17:09:22Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al Biochem. Int. (1981) 3, 321-326.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through paptide mapping after labelling with 2,3-difluorosialic acid. Watts et al J. Am. Chem. Soc. (2003) 125, 7532-7533.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
</biblio><br />
<br />
#Friebolin 1981 Friebolin, H. et al Biochem. Int. (1981) 3, 321-326.<br />
<br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15442Glycoside Hydrolase Family 342020-06-22T04:47:33Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH34'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|Tyr/Glu<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH34.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
All enzymes in GH34 are Neuraminidases (also known as sialidases; EC. 3.2.1.18) that are found, along with the sialic acid-binding protein Hemagglutinin, on the surface of influenza viruses that are pathogenic to mammalian or avian species. These are the H and N of H1N1. Viruses bind to the cell surface and enter via interaction of the hemagglutinin with cell surface sialic acids. The function of the neuraminidase is to cleave sialic acid from the cell surface after budding of progeny virus, to assist viral spread to other cells. GH34 neurmainidases have therefore been major drug design targets, and are very effectively inhibited by the drugs Tamiflu and Zanamivir (Relenza).<br />
<br />
The cell surface sialic acids they cleave are linked a-2,3 or a-2,6 to galactose or N-acetyl galactosamine residues that terminate glycolipid or glycoprotein structures. Sialic acids are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them are found in families GH33, GH34, GH83 and, along with GH93 arabinanases constitute Clan GH-E.<br />
<br />
This is an example of how to make references to a journal article <cite>Comfort2007</cite>. (See the References section below). Multiple references can go in the same place like this <cite>Comfort2007 He1999</cite>. You can even cite books using just the ISBN <cite>StickWilliams</cite>. References that are not in PubMed can be typed in by hand <cite>Sinnott1990</cite>. <br />
<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases of the GH34 family have been shown to cleave sialic acid residues from host polysaccharides with retention of anomeric stereochemistry (REF). The mechanism of this hydrolysis has been extensively studied, and for years was thought to occur through a sialosyl cation intermediate (von Itzstein). However, trapping of the covalent enzyme intermediate of the viral neuraminidase using 3-fluoro-glycosylfluorides (Kim 2013) confirmed a covalent structure. Therefore, the mechanism of these neuraminidases is a classic Koshland type double-displacement involving a covalent sialyl-enzyme intermediate on a Tyrosine residue. This is followed by base-catalysed attack of water on the sialyl enzyme, releasing Neu5Ac with net retention of anomeric stereochemistry.<br />
<br />
== Catalytic Residues ==<br />
The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Y406 by use of 3-fluoro-sialosyl fluorides to trap the covalent intermediate, followed by peptide mapping (Withers 2013, REF). An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue E277 to be appropriately positioned to act as the general acid/base pair for activation of Y406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex. This role had been discussed previously (Varghese Proteins 1992) though not favoured. In the same paper Asp151 is considered as a candidate for the acid/base catalyst based upon its interaction with C2OH in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of mutants by Lentz et al 1987 Biochemistry, on which basis they suggested Glu276, though subsequent structures showed this residue interacting with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging Ghate and Air (Eur J Biochem 1998). More recently Zhu and Wilson investigated why mutations to D151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for D151 since this is the classical kinetic signature. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base..<br />
<br />
<br />
== Three-dimensional structures ==<br />
These are members of Clan GH-E along with GH33, 83 and 93. Three dimensional crystallographic structures have been described for neuraminidases of all subtypes of Influenza A (N1-N10) and B (see Structures section of CAZy for PDBs). These structures consist of cytoplasmic, transmembrane, ‘head’ and ‘stem’ domains, homotetramers of these enzymes form mushroom-like structures on the surface of the virus (Bovin ActaNature 2009). Each of these enzymes displays the sialidase 6-blade beta-propeller fold in the catalytic ‘head’ domain, as well as a calcium-binding domain common to this class of glycoside hydrolase. The viral neuraminidases have eight highly conserved residues in the active site that form key stabilizing interactions (hydrogen bonding, hydrophobic interactions, charge-charge interactions) with the bound substrates, and an additional ten conserved residues that are thought to be key structural factors for these enzymes (von Itzstein 2007). An arginine triad interacts with the carboxylate residue of active site-bound sialic acid. Nearby is the nucleophilic tyrosine residue Tyr406 and its partner glutamate Glu277, which serves as an acid/base for deprotonation/reprotonation of Tyr406 during turnover. Also nearby is the probable acid/base Asp151 for protonation of the glycosidic oxygen and deprotonation of incoming water. Structures of trapped 3-fluorosialosyl enzyme intermediates are available (Kim et al; Vavricka et al Gao 2013).<br />
<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: Determined for influenza NA by NMR by the von Itzstein group Eur. J. Biochem. (1992) 207, 335-343. <cite>Comfort2007</cite>.<br />
;First catalytic nucleophile identification: Implied by X-ray structures, though intermediate thought to be a carbocation. Determined definitively using a 2,3-difluorosialic acid in the Withers group Kim et al Science (2013) 340, 71-75. <cite>Sinnott1990</cite>.<br />
;First general acid/base residue identification: Inferred from X-ray structure below <cite>He1999</cite>.<br />
;First 3-D structure: Influenza neuraminidase determined by Colman group: Varghese et al (1983) Nature 303, 35-40. <cite>StickWilliams</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Comfort2007 pmid=17323919<br />
#He1999 pmid=9312086<br />
#StickWilliams isbn=9780240521183<br />
#Sinnott1990 Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. [http://dx.doi.org/10.1021/cr00105a006 DOI: 10.1021/cr00105a006]<br />
</biblio><br />
<br />
<br />
[[Category:Glycoside Hydrolase Families|GH034]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15355Glycoside Hydrolase Family 342020-06-16T00:55:27Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Kyle Robinson^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH34'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|Tyr/Glu<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH34.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Content is to be added here.<br />
<br />
This is an example of how to make references to a journal article <cite>Comfort2007</cite>. (See the References section below). Multiple references can go in the same place like this <cite>Comfort2007 He1999</cite>. You can even cite books using just the ISBN <cite>StickWilliams</cite>. References that are not in PubMed can be typed in by hand <cite>Sinnott1990</cite>. <br />
<br />
<br />
== Kinetics and Mechanism ==<br />
Content is to be added here.<br />
<br />
<br />
== Catalytic Residues ==<br />
Content is to be added here.<br />
<br />
<br />
== Three-dimensional structures ==<br />
Content is to be added here.<br />
<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: Determined for influenza NA by NMR by the von Itzstein group Eur. J. Biochem. (1992) 207, 335-343. <cite>Comfort2007</cite>.<br />
;First catalytic nucleophile identification: Implied by X-ray structures, though intermediate thought to be a carbocation. Determined definitively using a 2,3-difluorosialic acid in the Withers group Kim et al Science (2013) 340, 71-75. <cite>Sinnott1990</cite>.<br />
;First general acid/base residue identification: Inferred from X-ray structure below <cite>He1999</cite>.<br />
;First 3-D structure: Influenza neuraminidase determined by Colman group: Varghese et al (1983) Nature 303, 35-40. <cite>StickWilliams</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Comfort2007 pmid=17323919<br />
#He1999 pmid=9312086<br />
#StickWilliams isbn=9780240521183<br />
#Sinnott1990 Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. [http://dx.doi.org/10.1021/cr00105a006 DOI: 10.1021/cr00105a006]<br />
</biblio><br />
<br />
<br />
[[Category:Glycoside Hydrolase Families|GH034]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15354Glycoside Hydrolase Family 332020-06-16T00:40:53Z<p>Steve Withers: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Junho Lee^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''<br />
|-<br />
|'''Clan''' <br />
|GH-E<br />
|-<br />
|'''Mechanism'''<br />
|Retaining<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}GH33.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.<br />
<br />
GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.<br />
<br />
== Catalytic Residues ==<br />
===Nucleophile===<br />
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. <br />
<br />
===Acid Base Catalyst===<br />
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.<br />
<br />
From <cite>Amaya2004</cite>:<br />
<blockquote><br />
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."<br />
</blockquote><br />
<br />
== Three-dimensional structures ==<br />
<br />
All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.<br />
<br />
The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.<br />
<br />
Bacterial sialidases may also contain a membrane binding domain, signal domain and a lectin-like domain. Although not all bacterial sialidases have a lectin domain, the lectin domain can be used to recognize the sialic acid in certain species, such as ''V. cholerae'' <cite> Moustafa2004</cite>. Also it is not uncommon for a bacterial sialidase to have a carbohydrate binding module (CBM) as one of its domains, such as in ''M. viridifaciens'' sialidase <cite> Gaskell1995 Watson2005 </cite>.<br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: First determined by proton NMR by Friebolin et al Biochem. Int. (1981) 3, 321-326.<br />
;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through paptide mapping after labelling with 2,3-difluorosialic acid. Watts et al J. Am. Chem. Soc. (2003) 125, 7532-7533.<br />
;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor Proc. Natl. Acad. Sci. USA (1993) 90, 9852.<br />
== References ==<br />
<biblio><br />
#Varki1997 pmid=9068613<br />
#Kim2011 pmid=21544654<br />
#Varki2007 pmid=17460663<br />
#Vimir2004 pmid=15007099<br />
#Buschiazzo2008 pmid=18625334<br />
#Amaya2004 pmid=15130470<br />
#Mizan2000 pmid=11092845<br />
#Watts2003 pmid=12812490<br />
#Damager2008 pmid=18284211<br />
#Watts2006 pmid=16298994<br />
#Newstead2008 pmid=18218621<br />
#Chong1992 pmid=1628657<br />
#Burmeister1993 pmid=8069621<br />
#Moustafa2004 pmid=15226294<br />
#Watson2005 pmid=16206228<br />
<br />
#Gaskell1995 pmid=8591030<br />
</biblio><br />
<br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_46&diff=3672Glycoside Hydrolase Family 462010-02-05T17:08:52Z<p>Steve Withers: /* Family Firsts */</p>
<hr />
<div><!-- CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Ryszard Brzezinski^^^<br />
* [[Responsible Curator]]: ^^^Ryszard Brzezinski^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GHnn'''<br />
|-<br />
|'''Clan''' <br />
|GH-I<br />
|-<br />
|'''Mechanism'''<br />
|inverting<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH46.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
Glycoside hydrolases of family 46 are essentially ''endo''-beta-1,4-chitosanases (EC [{{EClink}}3.2.1.132 3.2.1.132]) that hydrolyze various links in chitosan, a polymer of beta-1,4-linked D-glucosamine (GlcN) units with a variable content (mostly 0 - 35%) of N-acetyl-D-glucosamine (GlcNAc) <cite>Yabuki1988 Boucher1992</cite>. Among the four types of links occurring between these two kinds of subunits in chitosan, all the enzymes examined for their cleavage specificity recognized productively the GlcN-GlcN links. In addition, the chitosanase from ''Bacillus circulans'' MH-K1 recognized also GlcN-GlcNAc links <cite>Mitsutomi1996</cite>, while the chitosanase from ''Streptomyces'' sp. N174 recognized the GlcNAc-GlcN links <cite>Fukamizo1995</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
Family GH46 enzymes utilize an inverting mechanism, as shown by NMR <cite>Fukamizo1995</cite>.<br />
<br />
== Catalytic Residues ==<br />
The catalytic residues have been identified by site-directed mutagenesis and crystallography in the chitosanase from Streptomyces sp. N174. The general acid residue is Glu22, while Asp40 is the general base residue <cite>Boucher1995 Marcotte1996</cite>. The latter could activate the nucleophilic water molecule with assistance from residue Thr45 <cite>Lacombe-Harvey2009</cite>. Analysis of sequence alignments as well as crystallographic evidence showed that the same function is played by residues Glu37, Asp55 and Thr60 in the chitosanase from ''Bacillus circulans'' MH-K1 <cite>Saito1999</cite>.<br />
<br />
== Three-dimensional structures ==<br />
Two structures have been solved using X-ray crystallography, for the chitosanases from Streptomyces sp. N174 <cite>Marcotte1996</cite> and from Bacillus circulans MH-K1 (wild-type enzyme <cite>Saito1999</cite> and mutant K218P <cite>Fukamizo2005</cite>. These enzymes have essentially an alpha-helical fold, with two globular domains separated by the active site cleft for substrate binding. The cleft is bordered on the upper face by a three-stranded beta-sheet. The structure is similar to the 3D fold of the well studied lysozyme of bacteriophage T4 of ''Escherichia coli'' belonging to family GH24 <cite>Marcotte1996</cite> and, to some extent, to the structures of lysozymes from families GH22, GH23 as well the chitinases from family GH19 <cite>Monzingo1996</cite>. These five families are sometimes grouped in the "lysozyme superfamily" <cite>Holm1994 Lacombe-Harvey2009</cite>. <br />
The crystal structures, completed by site-directed mutagenesis have also revealed several residues involved in substrate binding <cite>Marcotte1996 Fukamizo2005 Tremblay2001 Katsumi2005</cite>. While a 4+2 model of substrate binding has been initially proposed for a GlcN hexasaccharide <cite>Marcotte1996</cite>, the mode of binding was later established as being in conformity with a 3+3 model, based on the analysis of products of hydrolysis <cite>Tremblay2001</cite>. <br />
<br />
== Family Firsts ==<br />
;First primary sequence determination: Chitosanase from ''Bacillus circulans'' MH-K1 <cite>Ando1992</cite>.<br />
;First sterochemistry determination: Chitosanase from ''Streptomyces'' sp. N174 by NMR <cite>Fukamizo1995</cite>.<br />
;First general base residue identification: Chitosanase from ''Streptomyces'' sp. N174 by sequence conservation and mutagenesis <cite>Boucher1995</cite> and by X-ray crystallography <cite>Marcotte1996</cite>.<br />
;First general acid residue identification: Chitosanase from ''Streptomyces'' sp. N174 by sequence conservation and mutagenesis <cite>Boucher1995</cite> and by X-ray crystallography <cite>Marcotte1996</cite>.<br />
;First 3-D structure: Chitosanase from ''Streptomyces'' sp. N174 by X-ray crystallography <cite>Marcotte1996</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Yabuki1988 Yabuki, M., Uchiyama, A., Suzuki, K., Ando, A., Fujii, T. (1988) Purification and properties of chitosanase from ''Bacillus circulans'' MH-K1. Journal of General and Applied Microbiology 34:255-270.<br />
#Boucher1992 Boucher, I., Dupuy, A., Vidal, P., Neugebauer, WA., Brzezinski, R. (1992) Purification and characterization of a chitosanase from ''Streptomyces'' N174. Applied Microbiology and Biotechnology 38:188-193.<br />
#Mitsutomi1996 Mitsutomi, M., Ueda, M., Arai, M., Ando, A., Watanabe, T. (1996) Action patterns of microbial chitinases and chitosanases on partially ''N''-acetylated chitosan. Chitin Enzymology, vol. 2, pp 273-284.<br />
#Fukamizo1995 pmid=7487871<br />
#Boucher1995 pmid=8537367<br />
#Lacombe-Harvey2009 pmid=19143844<br />
#Saito1999 pmid=10521473<br />
#Marcotte1996 pmid=8564542<br />
#Fukamizo2005 pmid=16272568<br />
#Monzingo1996 pmid=8564539<br />
#Holm1994 pmid=8119396<br />
#Tremblay2001 pmid=11686931<br />
#Katsumi2005 pmid=16288718<br />
#Ando1992 Ando, A., Noguchi, K., Yanagi, M., Shinoyama, H., Kagawa, Y., Hirata, H., Yabuki, M., Fujii, T. (1992) Primary structure of chitosanase produced by ''Bacillus circulans'' MH-K1. Journal of General and Applied Microbiology 38:135-144.<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH046]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_29&diff=3499Glycoside Hydrolase Family 292010-01-15T22:25:58Z<p>Steve Withers: </p>
<hr />
<div><!-- CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Gerlind Sulzenbacher^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH 29'''<br />
|-<br />
|'''Clan''' <br />
|none<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH29.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The [[glycoside hydrolases]] of this family are exo-acting α-fucosidases from archaeal, bacterial and eukaryotic origin. No other activities have been observed for GH29 family members. So far the only other CAZY family containing α-fucosidases is family [[GH95]]. The human enzyme FucA1 is of medical interest because its deficiency leads to fucosidosis, an autosomal recessive lysosomal storage disease <cite>1</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
GH29 α-fucosidases are [[retaining]] enzymes following a [[classical Koshland double-displacement mechanism]], as first proposed in 1987 for human liver α-fucosidase ''via'' burst kinetics experiments and using methanol as an alternative glycone acceptor to produce methyl-α-L-fucoside <cite>2</cite>. This has been further confirmed by <sup>1</sup>H NMR monitoring of the reaction catalyzed by an α-L-fucosidase from ''Thermus sp.'' <cite>3</cite>, and a α-L-fucosidase from the marine mollusc ''Pecten maximus'' <cite>4</cite>, as well as by COSY and <sup>1</sup>H-<sup>13</sup>C NMR spectroscopy analysis of the interglycosidic linkage of disaccharides formed by the transglycosylation action of ''Sulfolobus solfataricus'' α-L-fucosidase, Ssα-fuc <cite>5</cite>. [[GH95]] α-fucosidases, in contrast, operate with inversion of the anomeric configuration.<br />
<br />
== Catalytic Residues ==<br />
The [[catalytic nucleophile]] in GH29 was first identified in the ''Sulfolobus solfataricus'' α-L-fucosidase, Ssα-fuc, as Asp242 in the sequence VYF<u>'''D'''</u>WWI via chemical rescue of an inactive mutant with sodium azide <cite>6</cite>. Concomitantly the [[catalytic nucleophile]] of ''Thermotoga maritima'' α-L-fucosidase, Tmα-fuc, was confirmed to be Asp224 in the sequence LWN<u>'''D'''</u>MGW through trapping of the 2-deoxy-2-fluorofucosyl-enzyme [[intermediate]] and subsequent peptide mapping via LC-MS/MS technologies, as well as by chemical rescue of an inactive mutant <cite>7</cite>. The trapping of the 2-deoxy-2-fluorofucosyl-enzyme intermediate in Tmα-fuc was corroborated by crystallographic studies <cite>8</cite>. The [[catalytic nucleophile]] of the human enzyme FucA1 has recently been identified as being Asp225 <cite>9</cite>.<br />
<br />
Whereas the [[catalytic nucleophile]] in GH29 has been shown to be a conserved aspartate residue, the identity of the [[general acid/base]] is still controversial. Structural and mutagenesis studies of Tmα-fuc provided strong evidence for the variant Glu266 being the [[general acid/base]] <cite>8</cite>. In the crystal structure the carboxyl function of this residue is 5.5 Å away from that of the [[catalytic nucleophile]] Asp224, a distance commonly observed in retaining glycosidases proceeding ''via'' a [[classical Koshland double-displacement mechanism]]. Although multiple sequence alignments show that Glu266 is not conserved within GH29, the residue is structurally conserved in two 3-D structures of α-L-fucosidases from ''Bacteroides thetaiotaomicron VPI-5482'', recently deposited in the [http://www.pdb.org/ Protein Data Bank] (PDB accession numbers [{{PDBlink}}3eyp 3eyp] and [{{PDBlink}}3gza 3gza]). Studies of Ssα-fuc demonstrated that mutation of the Glu residue corresponding in sequence to Tmα-fuc Glu266 barely impaired the catalytic activity of the enzyme, whereas a Glu58Gly mutant had a 4000 fold lower ''k<sub>cat</sub>/K<sub>M</sub>'' and could be chemically rescued <cite>10</cite>. In the crystal structure of Tmα-fuc in complex with fucose <cite>8</cite>, the residue corresponding to Ssα-fuc Glu58, Glu66, is found 7.5 Å away from the [[catalytic nucleophile]] Asp224 and hydrogen bonded to the C-3 hydroxyl group of fucose, which altogether makes this residue an unlikely candidate for the function of the [[general acid/base]]. A recent study of the human α-L-fucosidase FucA1, carefully done combining bioinformatics, structural modelling, mutagenesis, chemical rescue with azide and <sup>1</sup>H NMR spectral analysis, identified Glu289 as the [[general acid/base]] <cite>9</cite>. The equivalent residue in Tmα-fuc, Glu281, as inferred from sequence alignment of FucA1 and Tmα-fuc, points to the interior of the (β/α)<sub>8</sub> barrel and lies about 15 Å apart form the catalytic centre.<br />
<br />
Altogether it appears that family GH29 is a quite exceptional CAZy family in that multiple sequence alignments do not allow an unambiguous assignment of the [[general acid/base]].<br />
<br />
== Three-dimensional structures ==<br />
Very few structures are available for GH29 enzymes. The first crystal structure to be solved is that of the α-L-fucosidase from ''T. maritima'', Tmα-fuc ([http://www.rcsb.org/pdb/explore/explore.do?structure 1HL8 PDB 1hl8]). The simultaneous solution of the structures of an enzyme-product complex ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1ODU PDB 1odu]) and of a glycosyl-enzyme intermediate ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1HL9 PDB 1hl9]) allowed the unambiguous identification of the [[general acid/base]] <cite>8</cite>, as described above. Tmα-fuc assembles as a hexamer and displays a two-domain fold, composed of a catalytic (β/α)<sub>8</sub>-like domain and a C-terminal β-sandwich domain. The two key active site residues are located at the C-terminal ends of strands β-strands 4 (nucleophile) and 6 (acid/base).<br />
Crystallization experiments for the ''S. solfataricus'' α-L-fucosidase, Ssα-fuc, were not very fruitful, but a small angle scattering study has been reported <cite>11</cite>, which suggests a nonameric assembly of the enzyme in solution. Two crystal structures, arising from Structural Genomics initiatives, have been deposited in the [http://www.pdb.org/ Protein Data Bank] for α-L-fucosidases from ''Bacteroides thetaiotaomicron VPI-5482'', with accession numbers [{{PDBlink}}3eyp 3eyp] and [{{PDBlink}}3gza 3gza]. <br />
The catalytic domain of Tmα-fuc does not adopt the canonical TIM-barrel (β/α)<sub>8</sub> fold, as it lacks helices α5 and α6. Helix α5 is also missing in the structure of one of the ''B. thetaiotaomicron VPI-5482'' α-L-fucosidases, BT3798 ([{{PDBlink}}3gza 3gza]), whereas α-L-fucosidase BT2192 ([{{PDBlink}}3eyp 3eyp]) from the same organism adopts the canonical TIM-barrel fold. The three structures differ furthermore by the insertion/deletion of a considerable number of additional α-helices, 3<sub>10</sub> helices, and extended surface loop regions. The closest structural homologues of GH29 enzymes within the CAZy classification can be found in families [[GH13]] and [[GH27]].<br />
<br />
== Fucosynthases ==<br />
Transglycosylation activity had been observed in 1987 for human liver α-fucosidase <cite>2</cite>. The first successful transformation of an α-fucosidase into an α-transfucosidase by directed evolution has been reported for ''Thermotoga maritima'' α-fucosidase <cite>12</cite>. α-fucosidases mutated in the [[catalytic nucleophile]] from both ''Sulfolobus solfataricus'' and ''Thermotoga maritima'' were successfully transformed into fucosynthases by the use of β-L-fucopyranosyl azide as donor substrate <cite>13</cite><br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: Retention of anomeric stereochemistry suggested for human liver α-fucosidase by the formation of methyl-α-L-fucoside using methanol as an alternative glycone acceptor <cite>2</cite>. Later confirmed by <sup>1</sup>H NMR for α-L-fucosidase from ''Thermus sp.'' <cite>3</cite>.<br />
; First [[catalytic nucleophile]] identification : ''Sulfolobus solfataricus'' α-L-fucosidase by azide rescue of an inactivated mutant <cite>6</cite> and confirmed shortly thereafter by labeling of the nucleophile and peptide mapping <cite>7</cite>.<br />
; First [[general acid/base]] residue identification : ''Thermotoga maritima'' α-fucosidase by X-ray structural analysis and mutagenesis <cite>8</cite>.<br />
; First 3-D structure : ''Thermotoga maritima'' α-fucosidase, free enzyme ([{{PDBlink}}1hl8 PDB 1hl8]), product complex ([{{PDBlink}}1odu PDB 1odu]) and glycosyl-enzyme intermediate ([{{PDBlink}}1hl9 PDB 1hl9]) <cite>8</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=2894306<br />
#2 pmid=3828350<br />
#3 pmid=12441672<br />
#4 pmid=12042250<br />
#5 pmid=12569098<br />
#6 pmid=12911294<br />
#7 pmid=12975375<br />
#8 pmid=14715651<br />
#9 pmid=19072333<br />
#10 pmid=15835922<br />
#11 pmid=15207718 <br />
#12 pmid=17240986 <br />
#13 Cobucci-Ponzano B, Conte F, Bedini E, Corsaro MM, Parrilli M, Sulzenbacher G, Lipski A, Dal Piaz F, Lepore L, Rossi M, and Moracci, M. ''β-glycosyl azides as substrates for α-glycosynthases: preparation of efficient α-L-fucosynthases''. Chem Biol 2009 Oct; 16(10) 1097-108. [http://linkinghub.elsevier.com/retrieve/pii/S1074552109003238 DOI: 10.1016/j.chembiol.2009.09.013] <br />
<br />
</biblio> <br />
<br />
[[Category:Glycoside Hydrolase Families|GH029]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_29&diff=3498Glycoside Hydrolase Family 292010-01-15T22:14:52Z<p>Steve Withers: </p>
<hr />
<div><!-- CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Gerlind Sulzenbacher^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH 29'''<br />
|-<br />
|'''Clan''' <br />
|none<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH29.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The [[glycoside hydrolases]] of this family are exo-acting α-fucosidases from archaeal, bacterial and eukaryotic origin. No other activities have been observed for GH29 family members. So far the only other CAZY family containing α-fucosidases is family [[GH95]]. The human enzyme FucA1 is of medical interest because its deficiency leads to fucosidosis, an autosomal recessive lysosomal storage disease <cite>1</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
GH29 α-fucosidases are [[retaining]] enzymes following a [[classical Koshland double-displacement mechanism]], as first proposed in 1987 for human liver α-fucosidase ''via'' burst kinetics experiments and using methanol as an alternative glycone acceptor to produce methyl-α-L-fucoside <cite>2</cite>. This has been further confirmed by <sup>1</sup>H NMR monitoring of the reaction catalyzed by a α-L-fucosidase from ''Thermus sp.'' <cite>3</cite>, and a α-L-fucosidase from the marine mollusc ''Pecten maximus'' <cite>4</cite>, as well as by COSY and <sup>1</sup>H-<sup>13</sup>C NMR spectroscopy analysis of the interglycosidic linkage of disaccharides formed by the transglycosylation action of ''Sulfolobus solfataricus'' α-L-fucosidase, Ssα-fuc <cite>5</cite>. [[GH95]] α-fucosidases, in contrast, operate with inversion of the anomeric configuration.<br />
<br />
== Catalytic Residues ==<br />
The [[catalytic nucleophile]] in GH29 was first identified in the ''Sulfolobus solfataricus'' α-L-fucosidase, Ssα-fuc, as Asp242 in the sequence VYF<u>'''D'''</u>WWI via chemical rescue of an inactive mutant with sodium azide <cite>6</cite>. Concomitantly the [[catalytic nucleophile]] of ''Thermotoga maritima'' α-L-fucosidase, Tmα-fuc, was confirmed to be Asp224 in the sequence LWN<u>'''D'''</u>MGW through trapping of the 2-deoxy-2-fluorofucosyl-enzyme [[intermediate]] and subsequent peptide mapping via LC-MS/MS technologies, as well as by chemical rescue of an inactive mutant <cite>7</cite>. The trapping of the 2-deoxy-2-fluorofucosyl-enzyme intermediate in Tmα-fuc was corroborated by crystallographic studies <cite>8</cite>. The [[catalytic nucleophile]] of the human enzyme FucA1 has recently been identified as being Asp225 <cite>9</cite>.<br />
<br />
Whereas the [[catalytic nucleophile]] in GH29 has been shown to be a conserved aspartate residue, the identity of the [[general acid/base]] is still controversial. Structural and mutagenesis studies of Tmα-fuc provided strong evidence for the variant Glu266 being the [[general acid/base]] <cite>8</cite>. In the crystal structure the carboxyl function of this residue is 5.5 Å apart from that of the [[catalytic nucleophile]] Asp224, a distance commonly observed in retaining glycosidases proceeding ''via'' a [[classical Koshland double-displacement mechanism]]. Although multiple sequence alignments show that Glu266 is not conserved within GH29, the residue is structurally conserved in two 3-D structures of α-L-fucosidases from ''Bacteroides thetaiotaomicron VPI-5482'', recently deposited in the [http://www.pdb.org/ Protein Data Bank] (PDB accession numbers [{{PDBlink}}3eyp 3eyp] and [{{PDBlink}}3gza 3gza]). Studies of Ssα-fuc demonstrated that mutation of the Glu residue corresponding in sequence to Tmα-fuc Glu266 scarcely impaired the catalytic activity of the enzyme, whereas a Glu58Gly mutant yielded a 4000-fold reduction of ''k<sub>cat</sub>/K<sub>M</sub>'' and could be chemically rescued <cite>10</cite>. In the crystal structure of Tmα-fuc in complex with fucose <cite>8</cite>, the residue corresponding to Ssα-fuc Glu58, Glu66, is found 7.5 Å distant form the [[catalytic nucleophile]] Asp224 and hydrogen bond to the C-3 hydroxyl group of fucose, which altogether makes this residue an unlikely candidate for the function of the [[general acid/base]]. A recent study of the human α-L-fucosidase FucA1, carefully done combining bioinformatics, structural modelling, mutagenesis, chemical rescue with azide and <sup>1</sup>H NMR spectral analysis, identified Glu289 as the [[general acid/base]] <cite>9</cite>. The equivalent residue in Tmα-fuc, Glu281, as inferred from sequence alignment of FucA1 and Tmα-fuc, points to the interior of the (β/α)<sub>8</sub> barrel and lies about 15 Å apart form the catalytic centre.<br />
<br />
Altogether it appears that family GH29 is a quite exceptional CAZy family in that multiple sequence alignments do not allow an unambiguous assignment of the [[general acid/base]].<br />
<br />
== Three-dimensional structures ==<br />
Very few structures are available for GH29 enzyme. The first crystal structure being solved is the one of the α-L-fucosidase from ''T. maritima'', Tmα-fuc ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1HL8 PDB 1hl8]). The simultaneous solution of the structures of an enzyme-product complex ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1ODU PDB 1odu]) and of a glycosyl-enzyme intermediate ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1HL9 PDB 1hl9]) allowed the unambiguous identification of the [[general acid/base]] <cite>8</cite>, as described above. Tmα-fuc assembles as a hexamer and displays a two-domain fold, composed of a catalytic (β/α)<sub>8</sub>-like domain and a C-terminal β-sandwich domain. The two key active site residues are located at the C-terminal ends of strands β-strands 4 (nucleophile) and 6 (acid/base).<br />
Crystallization experiments for the ''S. solfataricus'' α-L-fucosidase, Ssα-fuc, were not very fruitful, but a small angle scattering study has been reported <cite>11</cite>, which suggests a nonameric assembly of the enzyme in solution. Two crystal structures, arising from Structural Genomics initiatives, have been deposited in the [http://www.pdb.org/ Protein Data Bank] for α-L-fucosidases from ''Bacteroides thetaiotaomicron VPI-5482'', with accession numbers [{{PDBlink}}3eyp 3eyp] and [{{PDBlink}}3gza 3gza]. <br />
The catalytic domain of Tmα-fuc does not adopt the canonical TIM-barrel (β/α)<sub>8</sub> fold, as it lacks helices α5 and α6. Helix α5 is missing as well in the structure of one of the ''B. thetaiotaomicron VPI-5482'' α-L-fucosidases, BT3798 ([{{PDBlink}}3gza 3gza]), whereas α-L-fucosidase BT2192 ([{{PDBlink}}3eyp 3eyp]) from the same organism adopts the canonical TIM-barrel fold. The three structures differ furthermore by the insertion/deletion of a considerable number of additional α-helices, 3<sub>10</sub> helices, and extended surface loop regions. The closest structural homologues of GH29 enzymes within the CAZy classification can be found in families [[GH13]] and [[GH27]].<br />
<br />
== Fucosynthases ==<br />
Transglycosylation activity had been observed in 1987 for human liver α-fucosidase <cite>2</cite>. The first successful transformation of an α-fucosidase into an α-transfucosidase by directed evolution has been reported for ''Thermotoga maritima'' α-fucosidase <cite>12</cite>. α-fucosidases mutated in the [[catalytic nucleophile]] from both ''Sulfolobus solfataricus'' and ''Thermotoga maritima'' could be successfully transformed into fucosynthases by the use of β-L-fucopyranosyl azide as donor substrate <cite>13</cite><br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: Retention of anomeric stereochemistry suggested for human liver α-fucosidase by the formation of methyl-α-L-fucoside using methanol as an alternative glycone acceptor <cite>2</cite>. Later confirmed by <sup>1</sup>H NMR for α-L-fucosidase from ''Thermus sp.'' <cite>3</cite>.<br />
; First [[catalytic nucleophile]] identification : ''Sulfolobus solfataricus'' α-L-fucosidase by azide rescue of an inactivated mutant <cite>6</cite> and confirmed shortly thereafter by labeling of the nucleophile and peptide mapping <cite>7</cite>.<br />
; First [[general acid/base]] residue identification : ''Thermotoga maritima'' α-fucosidase by X-ray structural analysis and mutagenesis <cite>8</cite>.<br />
; First 3-D structure : ''Thermotoga maritima'' α-fucosidase, free enzyme ([{{PDBlink}}1hl8 PDB 1hl8]), product complex ([{{PDBlink}}1odu PDB 1odu]) and glycosyl-enzyme intermediate ([{{PDBlink}}1hl9 PDB 1hl9]) <cite>8</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=2894306<br />
#2 pmid=3828350<br />
#3 pmid=12441672<br />
#4 pmid=12042250<br />
#5 pmid=12569098<br />
#6 pmid=12911294<br />
#7 pmid=12975375<br />
#8 pmid=14715651<br />
#9 pmid=19072333<br />
#10 pmid=15835922<br />
#11 pmid=15207718 <br />
#12 pmid=17240986 <br />
#13 Cobucci-Ponzano B, Conte F, Bedini E, Corsaro MM, Parrilli M, Sulzenbacher G, Lipski A, Dal Piaz F, Lepore L, Rossi M, and Moracci, M. ''β-glycosyl azides as substrates for α-glycosynthases: preparation of efficient α-L-fucosynthases''. Chem Biol 2009 Oct; 16(10) 1097-108. [http://linkinghub.elsevier.com/retrieve/pii/S1074552109003238 DOI: 10.1016/j.chembiol.2009.09.013] <br />
<br />
</biblio> <br />
<br />
[[Category:Glycoside Hydrolase Families|GH029]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_29&diff=3497Glycoside Hydrolase Family 292010-01-15T22:10:02Z<p>Steve Withers: </p>
<hr />
<div><!-- CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{UnderConstruction}}<br />
* [[Author]]: ^^^Gerlind Sulzenbacher^^^<br />
* [[Responsible Curator]]: ^^^Steve Withers^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH 29'''<br />
|-<br />
|'''Clan''' <br />
|none<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH29.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
<br />
== Substrate specificities ==<br />
The [[glycoside hydrolases]] of this family are exo-acting α-fucosidases from archaeal, bacterial and eukaryotic origin. No other activities have been observed for GH29 family members. So far the only other CAZY family containing α-fucosidases is family [[GH95]]. The human enzyme FucA1 is of medical interest because its deficiency leads to fucosidosis, an autosomal recessive lysosomal storage disease <cite>1</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
GH29 α-fucosidases are [[retaining]] enzymes following a [[classical Koshland double-displacement mechanism]], as first proposed in 1987 for human liver α-fucosidase ''via'' burst kinetics experiments and using methanol as an alternative glycone acceptor to produce methyl-α-L-fucoside <cite>2</cite>. This has been further confirmed by <sup>1</sup>H NMR monitoring of the reaction catalyzed by a α-L-fucosidase from ''Thermus sp.'' <cite>3</cite>, and a α-L-fucosidase from the marine mollusc ''Pecten maximus'' <cite>4</cite>, as well as by COSY and <sup>1</sup>H-<sup>13</sup>C NMR spectroscopy analysis of the interglycosidic linkage of disaccharides formed by the transglycosylation action of ''Sulfolobus solfataricus'' α-L-fucosidase, Ssα-fuc <cite>5</cite>. [[GH95]] α-fucosidases, in contrast, operate with inversion of the anomeric configuration.<br />
<br />
== Catalytic Residues ==<br />
The [[catalytic nucleophile]] in GH29 was first identified in the ''Sulfolobus solfataricus'' α-L-fucosidase, Ssα-fuc, as Asp242 in the sequence VYF<u>'''D'''</u>WWI via chemical rescue of an inactive mutant with sodium azide <cite>6</cite>. Concomitantly the [[catalytic nucleophile]] of ''Thermotoga maritima'' α-L-fucosidase, Tmα-fuc, was confirmed to be Asp224 in the sequence LWN<u>'''D'''</u>MGW through trapping of the 2-deoxy-2-fluorofucosyl-enzyme [[intermediate]] and subsequent peptide mapping via LC-MS/MS technologies, as well as by chemical rescue of an inactive mutant <cite>7</cite>. The trapping of the 2-deoxy-2-fluorofucosyl-enzyme intermediate in Tmα-fuc was corroborated by crystallographic studies <cite>8</cite>. The [[catalytic nucleophile]] of the human enzyme FucA1 has recently been identified as being Asp225 <cite>9</cite>.<br />
<br />
Whereas the [[catalytic nucleophile]] in GH29 has been shown to be a conserved aspartate residue, the identity of the [[general acid/base]] is still controversial. Structural and mutagenesis studies of Tmα-fuc provided strong evidence for the variant Glu266 being the [[general acid/base]] <cite>8</cite>. In the crystal structure the carboxyl function of this residue is 5.5 Å apart from that of the [[catalytic nucleophile]] Asp224, a distance commonly observed in retaining glycosidases proceeding ''via'' a [[classical Koshland double-displacement mechanism]]. Although multiple sequence alignments show that Glu266 is not conserved within GH29, the residue is structurally conserved in two 3-D structures of α-L-fucosidases from ''Bacteroides thetaiotaomicron VPI-5482'', recently deposited in the [http://www.pdb.org/ Protein Data Bank] (PDB accession numbers [{{PDBlink}}3eyp 3eyp] and [{{PDBlink}}3gza 3gza]). Studies of Ssα-fuc demonstrated that mutation of the Glu residue corresponding in sequence to Tmα-fuc Glu266 scarcely impaired the catalytic activity of the enzyme, whereas a Glu58Gly mutant yielded a 4000-fold reduction of ''k<sub>cat</sub>/K<sub>M</sub>'' and could be chemically rescued <cite>10</cite>. In the crystal structure of Tmα-fuc in complex with fucose <cite>8</cite>, the residue corresponding to Ssα-fuc Glu58, Glu66, is found 7.5 Å distant form the [[catalytic nucleophile]] Asp224 and hydrogen bond to the C-3 hydroxyl group of fucose, which altogether makes this residue an unlikely candidate for the function of the [[general acid/base]]. A recent study of the human α-L-fucosidase FucA1, carefully done combining bioinformatics, structural modelling, mutagenesis, chemical rescue with azide and <sup>1</sup>H NMR spectral analysis, identified Glu289 as the [[general acid/base]] <cite>9</cite>. The equivalent residue in Tmα-fuc, Glu281, as inferred from sequence alignment of FucA1 and Tmα-fuc, points to the interior of the (β/α)<sub>8</sub> barrel and lies about 15 Å apart form the catalytic centre.<br />
<br />
Altogether it appears that family GH29 is a quite exceptional CAZy family in that multiple sequence alignments do not allow an unambiguous assignment of the [[general acid/base]].<br />
<br />
== Three-dimensional structures ==<br />
Very few structures are available for GH29 enzyme. The first crystal structure being solved is the one of the α-L-fucosidase from ''T. maritima'', Tmα-fuc ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1HL8 PDB 1hl8]). The simultaneous solution of the structures of an enzyme-product complex ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1ODU PDB 1odu]) and of a glycosyl-enzyme intermediate ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1HL9 PDB 1hl9]) allowed the unambiguous identification of the [[general acid/base]] <cite>8</cite>, as described above. Tmα-fuc assembles as a hexamer and displays a two-domain fold, composed of a catalytic (β/α)<sub>8</sub>-like domain and a C-terminal β-sandwich domain. The two key active site residues are located at the C-terminal ends of strands β-strands 4 (nucleophile) and 6 (acid/base).<br />
Crystallization experiments for the ''S. solfataricus'' α-L-fucosidase, Ssα-fuc, were not very fruitful, but a small angle scattering study has been reported <cite>11</cite>, which suggests a nonameric assembly of the enzyme in solution. Two crystal structures, arising from Structural Genomics initiatives, have been deposited in the [http://www.pdb.org/ Protein Data Bank] for α-L-fucosidases from ''Bacteroides thetaiotaomicron VPI-5482'', with accession numbers [{{PDBlink}}3eyp 3eyp] and [{{PDBlink}}3gza 3gza]. <br />
The catalytic domain of Tmα-fuc does not adopt the canonical TIM-barrel (β/α)<sub>8</sub> fold, as it lacks helices α5 and α6. Helix α5 is missing as well in the structure of one of the ''B. thetaiotaomicron VPI-5482'' α-L-fucosidases, BT3798 ([{{PDBlink}}3gza 3gza]), whereas α-L-fucosidase BT2192 ([{{PDBlink}}3eyp 3eyp]) from the same organism adopts the canonical TIM-barrel fold. The three structures differ furthermore by the insertion/deletion of a considerable number of additional α-helices, 3<sub>10</sub> helices, and extended surface loop regions. The closest structural homologues of GH29 enzymes within the CAZy classification can be found in families [[GH13]] and [[GH27]].<br />
<br />
== Fucosynthases ==<br />
Transglycosylation activity had been observed in 1987 for human liver α-fucosidase <cite>2</cite>. The first successful transformation of an α-fucosidase into an α-transfucosidase by directed evolution has been reported for ''Thermotoga maritima'' α-fucosidase <cite>12</cite>. α-fucosidases mutated in the [[catalytic nucleophile]] from both ''Sulfolobus solfataricus'' and ''Thermotoga maritima'' could be successfully transformed into fucosynthases by the use of β-L-fucopyranosyl azide as donor substrate <cite>13</cite><br />
<br />
== Family Firsts ==<br />
;First stereochemistry determination: Retention of anomeric stereochemistry suggested for human liver α-fucosidase by the formation of methyl-α-L-fucoside using methanol as an alternative glycone acceptor <cite>2</cite>. Later confirmed by <sup>1</sup>H NMR for α-L-fucosidase from ''Thermus sp.'' <cite>3</cite>.<br />
; First [[catalytic nucleophile]] identification : ''Sulfolobus solfataricus'' α-L-fucosidase by azide rescue of an inactivated mutant <cite>6</cite>.<br />
; First [[general acid/base]] residue identification : ''Thermotoga maritima'' α-fucosidase by X-ray structural analysis and mutagenesis <cite>8</cite>.<br />
; First 3-D structure : ''Thermotoga maritima'' α-fucosidase, free enzyme ([{{PDBlink}}1hl8 PDB 1hl8]), product complex ([{{PDBlink}}1odu PDB 1odu]) and glycosyl-enzyme intermediate ([{{PDBlink}}1hl9 PDB 1hl9]) <cite>8</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=2894306<br />
#2 pmid=3828350<br />
#3 pmid=12441672<br />
#4 pmid=12042250<br />
#5 pmid=12569098<br />
#6 pmid=12911294<br />
#7 pmid=12975375<br />
#8 pmid=14715651<br />
#9 pmid=19072333<br />
#10 pmid=15835922<br />
#11 pmid=15207718 <br />
#12 pmid=17240986 <br />
#13 Cobucci-Ponzano B, Conte F, Bedini E, Corsaro MM, Parrilli M, Sulzenbacher G, Lipski A, Dal Piaz F, Lepore L, Rossi M, and Moracci, M. ''β-glycosyl azides as substrates for α-glycosynthases: preparation of efficient α-L-fucosynthases''. Chem Biol 2009 Oct; 16(10) 1097-108. [http://linkinghub.elsevier.com/retrieve/pii/S1074552109003238 DOI: 10.1016/j.chembiol.2009.09.013] <br />
<br />
</biblio> <br />
<br />
[[Category:Glycoside Hydrolase Families|GH029]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=User:Brian_Rempel&diff=3328User:Brian Rempel2010-01-12T19:28:07Z<p>Steve Withers: </p>
<hr />
<div>Brian Rempel received his undergraduate degree from the University of Alberta (B.Sc. Hons. 2003) and went on to do his graduate training in the lab of Dr. Stephen G. Withers at the University of British Columbia (Ph.D., 2009). His graduate work focused on the synthesis and enzymatic evaluation of activated fluorosugars as covalent inactivators of glycosidases. His work was funded by both NSERC and the Michael Smith Foundation for Health Research during this time. He is currently teaching organic chemistry at the University of Alberta, Augustana campus in Camrose, Alberta.<br />
<br />
<br />
''Representative publications:''<br />
<biblio><br />
#Rempel2008 pmid=18499865<br />
#Rempel2005 pmid=15862278<br />
</biblio><br />
<br />
[[Category:Contributors|Rempel, Brian]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Catalytic_acid/base&diff=1821Catalytic acid/base2009-08-31T14:31:54Z<p>Steve Withers: /* Overview */</p>
<hr />
<div>* Author: [[User:SpencerWilliams|Spencer Williams]]<br />
* Responsible Curator: [[User:SpencerWilliams|Spencer Williams]]<br />
----<br />
== Overview ==<br />
<br />
The term '''catalytic acid/base''' refers to an amino acid residue in a [[glycoside hydrolase]] or a related enzyme that participates in the mechanism of hydrolysis by removing or adding a proton. The mechanism may be a [[retaining]] or [[inverting]] mechanism.<br />
<br />
[[Image:Retaining_glycosidase_mechanism_1.png|centre]]<br />
<br />
==Methods for indentifying the catalytic acid/base==<br />
<br />
== References ==<br />
<biblio><br />
<br />
</biblio><br />
[[Category:Definitions and explanations]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=CAZypedia:Community_Portal&diff=1820CAZypedia:Community Portal2009-08-31T14:28:12Z<p>Steve Withers: /* Nomenclature of sugar ring faces */</p>
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<div>'''''Please write your ideas/suggestions/etc. for the improvement of CAZypedia on this page. Community participation is essential for the continued development of this resource!'''''<br />
<br />
'''PLEASE NOTE: If you leave a comment here, please sign it by adding four tildes (<nowiki>~~~~</nowiki>) after your post. Thanks!''' [[User:Harry Brumer|Harry Brumer]] 05:21, 29 July 2009 (CEST)<br />
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<br />
<hr><br />
== Nomenclature of sugar ring faces ==<br />
* Confusion about sugar ring faces; GH people seem to prefer calling the faces based on the sides relative to the anomeric carbon (C1), but lectin (or sugar-binding protein) people usually call them according to a definition of faces of ring compounds (PMID: 16592816). They are unfortunately "upside down" (D. Ross). [[User:Shinya Fushinobu|ShinyaFushinobu]] 07:06, 31 August 2009 (UTC)<br />
** Thanks for the comment. I've passed this on to [[User:Steve Withers]] and [[User:Spencer Williams]], who are maintaining the lexicon for their comments. [[User:Harry Brumer|Harry Brumer]] 07:45, 31 August 2009 (UTC)<br />
** Dear Shinya, the nomenclature of Wimmer et al. works well for many compound classes, but has limitations with sugars. In particular, there is potential conflict with the use of the terms alpha and beta, which for carbohydrates is used for the anomeric configuration. When using the Wimmer nomenclature, for D-sugars the alpha face is the same as that which the alpha anomeric substituent is oriented. However, for L-sugars, the nomenclature for faces and the anomeric orientations are opposed. I appreciate that different communities have different 'lexicons'; in the case of GHs teh anomeric configuration is frequently discussed in close concert with the face of the molecule and so the use of the anomeric configuration to define faces would appear more convenient. As a point of interest - do you happen to know whether IUPAC or IUBMB has adopted a formal definition of the face of a molecule? The closest I have seen for sugars is the top and bottom faces defined in the "CONFORMATIONAL NOMENCLATURE FOR FIVE- AND SIX-MEMBERED RING FORMS OF MONOSACCHARIDES AND THEIR DERIVATIVES (Pure & Appi. Chem., 1981, Vol.53, pp.1901—1905).[[User:Spencer Williams|Spencer Williams]] 12:07, 31 August 2009 (UTC)<br />
I must confess I was not aware of this other nomenclature.. Personally I do not see that we can use other than the IUPAC carbohydrate nomenclature or all hell would break loose. However, perhaps when the CBM section is included we should make a note there about the lectin nomenclature (or even now in the Lexicon).[[User:Steve Withers|Steve Withers]] 14:28, 31 August 2009 (UTC)<br />
<br />
== GH category page, order of families ==<br />
* It would be nice if the glycoside hydrolase pages under the category link were ordered numerically; perhaps a tabulated series of links could be created in numercial order? Spencer Williams<br />
** This is an issue with the way the Category tags are listed in alphabetical order. One work-around could be to modify the tag in each page with a sort key. See http://meta.wikimedia.org/wiki/Help:Category#Sort_key [[User:Harry Brumer|Harry Brumer]] 05:21, 29 July 2009 (CEST)<br />
** '''Fixed:''' added sort keys in the format "GH001" to all existing pages during the first week of August 2009 (at Cellulase GRC). [[User:Harry Brumer|Harry Brumer]] 20:42, 6 August 2009 (UTC)</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_2&diff=1609Glycoside Hydrolase Family 22009-08-13T23:44:55Z<p>Steve Withers: </p>
<hr />
<div>* [[Author]]: [[User:Withers|Stephen Withers]]<br />
* [[Responsible Curator]]: [[User:Withers|Stephen Withers]]<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH2'''<br />
|-<br />
|'''Clan''' <br />
|GH-A<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH2.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
This family contains beta-galactosidases, beta-glucuronidases, beta-mannosidases, exo-beta-glucosaminidases and, in plants, a mannosylglycoprotein endo-beta-mannosidase. The enzymes are found across a broad spectrum of life forms, but are concentrated in bacteria. The most famous enzyme in this family is the ''E. coli'' (lacZ) beta-galactosidase of lac operon fame. Not only did this enzyme play a key role in developing the understanding of operon structure and control of gene expression, but also it continues to play a key role as a cell biological probe. Another matter of note is that this remains the largest protein monomer to be sequenced entirely at the peptide level <cite>1</cite>. ''E. coli'' also contains a second, vestigial beta-galactosidase (ebg) whose activity has been shown to evolve in lacZ<sup>-</sup> strains of ''E. coli'' grown under selective pressure with lactose as sole carbon source <cite>2</cite>. Another reasonably well-studied GH2 enzyme is the ''E. coli'' beta-glucuronidase, whose activity is used to detect the presence of ''E. coli'' (http://www.cfsan.fda.gov/~ebam/bam-4.html), though interestingly not the nasty 0157 strain. The principal enzyme of medical interest in GH2 is the lysosomal beta-glucuronidase whose deficiency leads to Sly syndrome <cite>3</cite>. The only other human GH2 enzyme is the lysosomal beta-mannosidase.<br />
<br />
== Kinetics and Mechanism ==<br />
Family 2 beta-glycosidases are retaining enzymes, as first shown by XXXXX <cite>4</cite> and follow a classical Koshland double-displacement mechanism. The best studied enzyme kinetically must be the ''E. coli'' (lacZ) beta-galactosidase, for which a key set of studies defining the two- step mechanism and elucidating rate-limiting steps was published by the groups of Yon and Sinnott in the early 1970’s <cite>5</cite>, <cite>6</cite>, <cite>7</cite>. Indeed the approaches developed on that system laid the foundations for many subsequent studies on other glycosidases. An analysis of the roles of each substrate hydroxyl in catalysis, based upon kinetic studies with modified sugars has also been published <cite>8</cite>. Some GH2 glycosidases require Mg2+ for activity and in ''E. coli'' beta-galactosidase this Mg2+ requirement is associated with the binding of the cation in the active site such that it places the acid/base catalyst appropriately. Others, such as the human beta-glucuronidase, have no such metal ion requirement.<br />
<br />
== Catalytic Residues ==<br />
The catalytic nucleophile in GH2 was first correctly identified in the ''E. coli'' (lacZ) beta-galactosidase as Glu537 in the sequence ILC'''<u>E</u>'''YAH through trapping of the 2-deoxy-2-fluorogalactosyl-enzyme intermediate and subsequent peptide mapping via HPLC techniques using radiolabeled tracers <cite>9</cite>. Earlier studies, carefully done using conduritol C cis-epoxide as affinity label, had identified Glu461 as the labeled residue, <cite>10</cite> on which basis a series of beautifully executed kinetic studies on mutants modified at this position that appeared initially to support this conclusion were performed <cite>11</cite>. However doubts were when similar kinetic analysis of nucleophile mutants of the GH1 Agrobacterium sp. b-glucosidase yielded quite different results, leading to the above labeling study <cite>9</cite>. The acid/base catalyst was then identified as Glu461 by re-interpretation <cite>9</cite> of the published kinetic results on mutants at that position <cite>11</cite>, which had included azide rescue experiments. These conclusions were fully supported by subsequent 3-dimensional structural analyses (below).<br />
<br />
== Three-dimensional structures ==<br />
Three-dimensional structures are available for five Family GH2 enzymes currently, the first solved being that of the ''E. coli'' (lacZ) beta-galactosidase in a tour de force of X-ray crystallography at that time, given its huge size (4 x 125,000 Da)<cite>12</cite>. The enzyme is multidomain, but as members of Clan GHA the catalytic domains a classical (a/b)8 TIM barrel fold with the two key active site glutamic acids being approximately 200 residues apart in sequence and located at the C-terminal ends of b-strands 4 (acid/base) and 7 (nucleophile).<br />
<br />
== Family Firsts ==<br />
;First sterochemistry determination: (pmid=TBA)<br />
;First catalytic nucleophile identification: ''E. coli'' (lacZ) beta-galactosidase by 2-fluorogalactose labeling <cite>9</cite><br />
;First general acid/base residue identification: ''E. coli'' (lacZ) beta-galactosidase by re-interpretation of kinetic studies with mutants <cite>9 11</cite><br />
;First 3-D structure: ''E. coli'' (lacZ) beta-galactosidase <cite>12</cite><br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=97298<br />
#2 pmid=10234816<br />
#3 pmid=4265197<br />
#4 pmid=TBA<br />
#5 pmid=4578762<br />
#6 pmid=4721624<br />
#7 pmid=4691347<br />
#8 pmid=1417731<br />
#9 pmid=1350782<br />
#10 pmid=6420154<br />
#11 pmid=1969405<br />
#12 pmid=8008071<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH002]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1608Glycoside Hydrolase Family 312009-08-13T23:07:13Z<p>Steve Withers: /* Kinetics and Mechanism */</p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
CAZy Family GH31 is one of the two major families, along with GH13, that contain &alpha;-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal &alpha;-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to &alpha;-glucosidases, GH31 also contains &alpha;-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic [[Alpha-glucan lyases|&alpha;-glucan lyases]]. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.<br />
<br />
== Kinetics and Mechanism ==<br />
Family GH 31 enzymes are retaining &alpha;-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement. <cite>#1</cite> GH31 enzymes (except for the &alpha;-glucan lyases) are believed to follow the classical double displacement mechanism. <cite>#2</cite> This has been strongly supported by labelinging of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide <cite>#3</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>#4</cite> and human lysosomal &alpha;-glucosidase <cite>#5</cite>. Later studies on an &alpha;-glucosidase from ''Aspergillus niger'' <cite>#6</cite> and an &alpha;-xylosidase from ''Escherichia coli'' <cite>#7</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates.<br />
<br />
The &alpha;-glucan lyases from GH31 cleave &alpha;-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose (see [[Alpha-glucan lyases|LEXICON]]). Detailed mechanistic studies have been carried out on ''Gracilariopsis'' &alpha;-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>#8 #9</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme intermediate by 5-fluoro-&beta;-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two &alpha;-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-&alpha;-D-glucopyranosyl fluoride.<br />
<br />
== Catalytic Residues ==<br />
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>#2 #7 #9</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the catalytic nucleophile, corresponding to Asp224 of ''Aspergillus niger'' &alpha;-glucosidase within the sequence IDM <cite>#3 #5</cite>. This was confirmed by using the more reliable 5-fluoro-&alpha;-D-glucopyransyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>#6</cite>. The general acid/base residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' &alpha;-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>#10</cite>. This was subsequently confirmed by the crystallographic studies on &alpha;-xylosidase (YicI) from ''Escherichia coli'' <cite>#7</cite> and successfully engineering YicI into the first &alpha;-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>#11</cite>. The catalytic nucleophile in ''Gracilariopsis'' &alpha;-1,4-glucan lyase has been identified as Asp553 in the sequence QDM, through the use of 5-fluoro-&beta;-L-idopyranosyl fluoride <cite>#8</cite>. This corresponds precisely to the position of the catalytic nucleophile in the GH31 glycosidases. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the catalytic nucleophile as it departs <cite>#9</cite>.<br />
<br />
== Three-dimensional structures ==<br />
The first crystal structure of a GH31 enzyme was that of the &alpha;-xylosidase YicI from ''Escherichia coli'' , published in 2005 <cite>#7</cite>. To date two other crystal structures of GH31 enzymes have been published, one being the ''Sulfolobus solfataricus'' &alpha;-glucosidase (MalA) <cite>#12</cite> and the other being the N-terminal domain of human intestinal maltase-glucoamylase <cite>#13</cite>. All of these structures feature a (-&beta;/&alpha;)<sub>8</sub> barrel in the catalytic domain.<br />
<br />
== Family Firsts ==<br />
<br />
;'''First stereochemical outcome'''<br />
<br />
:Determined for several &alpha;-glucosidases by a combination of polarimetric and reducing end measurements <cite>#1</cite><br />
<br />
;'''First catalytic nucleophile identification'''<br />
<br />
:Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling <cite>#4</cite><br />
<br />
;'''First general acid/base residue identification'''<br />
<br />
:''Schizosaccharomyces pombe'' &alpha;-glucosidase by sequence comparison and kinetic studies of the mutants <cite>#10</cite><br />
<br />
;'''First three-dimensional structure of GH31 enzymes'''<br />
<br />
:''Escherichia coli'' &alpha;-xylosidase (YicI) <cite>#7</cite><br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=376499<br />
<br />
#2 pmid=9620260<br />
<br />
#3 pmid=7766184<br />
<br />
#4 pmid=776963<br />
<br />
#5 pmid=1856189<br />
<br />
#6 pmid=11583585<br />
<br />
#7 pmid=15501829<br />
<br />
#8 pmid=11982345<br />
<br />
#9 pmid=14596624<br />
<br />
#10 pmid=11298744<br />
<br />
#11 pmid=16478160<br />
<br />
#12 pmid=16580018<br />
<br />
#13 pmid=18036614<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1607Glycoside Hydrolase Family 312009-08-13T23:06:01Z<p>Steve Withers: /* Catalytic Residues */</p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
CAZy Family GH31 is one of the two major families, along with GH13, that contain &alpha;-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal &alpha;-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to &alpha;-glucosidases, GH31 also contains &alpha;-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic [[Alpha-glucan lyases|&alpha;-glucan lyases]]. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.<br />
<br />
== Kinetics and Mechanism ==<br />
Family GH 31 enzymes are retaining &alpha;-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement. <cite>#1</cite> GH31 enzymes (except for the &alpha;-glucan lyases) are believed to follow the classical double displacement mechanism. <cite>#2</cite> This has been strongly supported by labelinging of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide <cite>#3</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>#4</cite> and human lysosomal &alpha;-glucosidase <cite>#5</cite>. Later studies on an &alpha;-glucosidase from ''Aspergillus niger'' <cite>#6</cite> and an &alpha;-xylosidase from ''Escherichia coli'' <cite>#7</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates.<br />
<br />
The &alpha;-glucan lyases from GH31 cleave &alpha;-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose (see [[Alpha-glucan lyases|LEXICON]] for more detail). Detailed mechanistic studies have been carried out on ''Gracilariopsis'' &alpha;-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>#8 #9</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme intermediate by 5-fluoro-&beta;-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two &alpha;-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-&alpha;-D-glucopyranosyl fluoride.<br />
<br />
== Catalytic Residues ==<br />
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>#2 #7 #9</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the catalytic nucleophile, corresponding to Asp224 of ''Aspergillus niger'' &alpha;-glucosidase within the sequence IDM <cite>#3 #5</cite>. This was confirmed by using the more reliable 5-fluoro-&alpha;-D-glucopyransyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>#6</cite>. The general acid/base residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' &alpha;-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>#10</cite>. This was subsequently confirmed by the crystallographic studies on &alpha;-xylosidase (YicI) from ''Escherichia coli'' <cite>#7</cite> and successfully engineering YicI into the first &alpha;-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>#11</cite>. The catalytic nucleophile in ''Gracilariopsis'' &alpha;-1,4-glucan lyase has been identified as Asp553 in the sequence QDM, through the use of 5-fluoro-&beta;-L-idopyranosyl fluoride <cite>#8</cite>. This corresponds precisely to the position of the catalytic nucleophile in the GH31 glycosidases. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the catalytic nucleophile as it departs <cite>#9</cite>.<br />
<br />
== Three-dimensional structures ==<br />
The first crystal structure of a GH31 enzyme was that of the &alpha;-xylosidase YicI from ''Escherichia coli'' , published in 2005 <cite>#7</cite>. To date two other crystal structures of GH31 enzymes have been published, one being the ''Sulfolobus solfataricus'' &alpha;-glucosidase (MalA) <cite>#12</cite> and the other being the N-terminal domain of human intestinal maltase-glucoamylase <cite>#13</cite>. All of these structures feature a (-&beta;/&alpha;)<sub>8</sub> barrel in the catalytic domain.<br />
<br />
== Family Firsts ==<br />
<br />
;'''First stereochemical outcome'''<br />
<br />
:Determined for several &alpha;-glucosidases by a combination of polarimetric and reducing end measurements <cite>#1</cite><br />
<br />
;'''First catalytic nucleophile identification'''<br />
<br />
:Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling <cite>#4</cite><br />
<br />
;'''First general acid/base residue identification'''<br />
<br />
:''Schizosaccharomyces pombe'' &alpha;-glucosidase by sequence comparison and kinetic studies of the mutants <cite>#10</cite><br />
<br />
;'''First three-dimensional structure of GH31 enzymes'''<br />
<br />
:''Escherichia coli'' &alpha;-xylosidase (YicI) <cite>#7</cite><br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=376499<br />
<br />
#2 pmid=9620260<br />
<br />
#3 pmid=7766184<br />
<br />
#4 pmid=776963<br />
<br />
#5 pmid=1856189<br />
<br />
#6 pmid=11583585<br />
<br />
#7 pmid=15501829<br />
<br />
#8 pmid=11982345<br />
<br />
#9 pmid=14596624<br />
<br />
#10 pmid=11298744<br />
<br />
#11 pmid=16478160<br />
<br />
#12 pmid=16580018<br />
<br />
#13 pmid=18036614<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1606Glycoside Hydrolase Family 312009-08-13T23:05:34Z<p>Steve Withers: /* Catalytic Residues */</p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
CAZy Family GH31 is one of the two major families, along with GH13, that contain &alpha;-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal &alpha;-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to &alpha;-glucosidases, GH31 also contains &alpha;-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic [[Alpha-glucan lyases|&alpha;-glucan lyases]]. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.<br />
<br />
== Kinetics and Mechanism ==<br />
Family GH 31 enzymes are retaining &alpha;-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement. <cite>#1</cite> GH31 enzymes (except for the &alpha;-glucan lyases) are believed to follow the classical double displacement mechanism. <cite>#2</cite> This has been strongly supported by labelinging of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide <cite>#3</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>#4</cite> and human lysosomal &alpha;-glucosidase <cite>#5</cite>. Later studies on an &alpha;-glucosidase from ''Aspergillus niger'' <cite>#6</cite> and an &alpha;-xylosidase from ''Escherichia coli'' <cite>#7</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates.<br />
<br />
The &alpha;-glucan lyases from GH31 cleave &alpha;-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose (see [[Alpha-glucan lyases|LEXICON]] for more detail). Detailed mechanistic studies have been carried out on ''Gracilariopsis'' &alpha;-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>#8 #9</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme intermediate by 5-fluoro-&beta;-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two &alpha;-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-&alpha;-D-glucopyranosyl fluoride.<br />
<br />
== Catalytic Residues ==<br />
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>#2 #7 #9</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the catalytic nucleophile, corresponding to Asp224 of ''Aspergillus niger'' &alpha;-glucosidase within the sequence IDM <cite>#3 #5</cite>. This was confirmed by using the more reliable 5-fluoro-&alpha;-D-glucopyransyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>#6</cite>. The general acid/base residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' &alpha;-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>#10</cite>. This was subsequently confirmed by the crystallographic studies on &alpha;-xylosidase (YicI) from ''Escherichia coli'' <cite>#7</cite> and successfully engineering YicI into the first &alpha;-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>#11</cite>. Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 The catalytic nucleophile in ''Gracilariopsis'' &alpha;-1,4-glucan lyase has been identified as Asp553 in the sequence QDM, through the use of 5-fluoro-&beta;-L-idopyranosyl fluoride <cite>#8</cite>. Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 This corresponds precisely to the position of the catalytic nucleophile in the GH31 glycosidases. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the catalytic nucleophile as it departs <cite>#9</cite>.<br />
<br />
== Three-dimensional structures ==<br />
The first crystal structure of a GH31 enzyme was that of the &alpha;-xylosidase YicI from ''Escherichia coli'' , published in 2005 <cite>#7</cite>. To date two other crystal structures of GH31 enzymes have been published, one being the ''Sulfolobus solfataricus'' &alpha;-glucosidase (MalA) <cite>#12</cite> and the other being the N-terminal domain of human intestinal maltase-glucoamylase <cite>#13</cite>. All of these structures feature a (-&beta;/&alpha;)<sub>8</sub> barrel in the catalytic domain.<br />
<br />
== Family Firsts ==<br />
<br />
;'''First stereochemical outcome'''<br />
<br />
:Determined for several &alpha;-glucosidases by a combination of polarimetric and reducing end measurements <cite>#1</cite><br />
<br />
;'''First catalytic nucleophile identification'''<br />
<br />
:Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling <cite>#4</cite><br />
<br />
;'''First general acid/base residue identification'''<br />
<br />
:''Schizosaccharomyces pombe'' &alpha;-glucosidase by sequence comparison and kinetic studies of the mutants <cite>#10</cite><br />
<br />
;'''First three-dimensional structure of GH31 enzymes'''<br />
<br />
:''Escherichia coli'' &alpha;-xylosidase (YicI) <cite>#7</cite><br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=376499<br />
<br />
#2 pmid=9620260<br />
<br />
#3 pmid=7766184<br />
<br />
#4 pmid=776963<br />
<br />
#5 pmid=1856189<br />
<br />
#6 pmid=11583585<br />
<br />
#7 pmid=15501829<br />
<br />
#8 pmid=11982345<br />
<br />
#9 pmid=14596624<br />
<br />
#10 pmid=11298744<br />
<br />
#11 pmid=16478160<br />
<br />
#12 pmid=16580018<br />
<br />
#13 pmid=18036614<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1605Glycoside Hydrolase Family 312009-08-13T23:00:01Z<p>Steve Withers: /* Kinetics and Mechanism */</p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
CAZy Family GH31 is one of the two major families, along with GH13, that contain &alpha;-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal &alpha;-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to &alpha;-glucosidases, GH31 also contains &alpha;-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic [[Alpha-glucan lyases|&alpha;-glucan lyases]]. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.<br />
<br />
== Kinetics and Mechanism ==<br />
Family GH 31 enzymes are retaining &alpha;-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement. <cite>#1</cite> GH31 enzymes (except for the &alpha;-glucan lyases) are believed to follow the classical double displacement mechanism. <cite>#2</cite> This has been strongly supported by labelinging of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide <cite>#3</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>#4</cite> and human lysosomal &alpha;-glucosidase <cite>#5</cite>. Later studies on an &alpha;-glucosidase from ''Aspergillus niger'' <cite>#6</cite> and an &alpha;-xylosidase from ''Escherichia coli'' <cite>#7</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates.<br />
<br />
The &alpha;-glucan lyases from GH31 cleave &alpha;-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose (see [[Alpha-glucan lyases|LEXICON]] for more detail). Detailed mechanistic studies have been carried out on ''Gracilariopsis'' &alpha;-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>#8 #9</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme intermediate by 5-fluoro-&beta;-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two &alpha;-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-&alpha;-D-glucopyranosyl fluoride.<br />
<br />
== Catalytic Residues ==<br />
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>#2 #7 #9</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the catalytic nucleophile, corresponding to Asp224 of ''Aspergillus niger'' &alpha;-glucosidase within the sequence IDM <cite>#3 #5</cite>. This was confirmed by using the more reliable 5-fluoro-&alpha;-D-glucopyransyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>#6</cite>. The general acid/base residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' &alpha;-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>#10</cite>. This was subsequently confirmed by the crystallographic studies on &alpha;-xylosidase (YicI) from ''Escherichia coli'' <cite>#7</cite> and successfully engineering YicI into the first &alpha;-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>#11</cite>. The equivalent residue, Asp553, in ''Gracilariopsis'' &alpha;-1,4-glucan lyase has also been identified as the catalytic nucleophile through the use of 5-fluoro-&beta;-L-idopyranosyl fluoride <cite>#8</cite>. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the catalytic nucleophile as it departs <cite>#9</cite>.<br />
<br />
== Three-dimensional structures ==<br />
The first crystal structure of a GH31 enzyme was that of the &alpha;-xylosidase YicI from ''Escherichia coli'' , published in 2005 <cite>#7</cite>. To date two other crystal structures of GH31 enzymes have been published, one being the ''Sulfolobus solfataricus'' &alpha;-glucosidase (MalA) <cite>#12</cite> and the other being the N-terminal domain of human intestinal maltase-glucoamylase <cite>#13</cite>. All of these structures feature a (-&beta;/&alpha;)<sub>8</sub> barrel in the catalytic domain.<br />
<br />
== Family Firsts ==<br />
<br />
;'''First stereochemical outcome'''<br />
<br />
:Determined for several &alpha;-glucosidases by a combination of polarimetric and reducing end measurements <cite>#1</cite><br />
<br />
;'''First catalytic nucleophile identification'''<br />
<br />
:Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling <cite>#4</cite><br />
<br />
;'''First general acid/base residue identification'''<br />
<br />
:''Schizosaccharomyces pombe'' &alpha;-glucosidase by sequence comparison and kinetic studies of the mutants <cite>#10</cite><br />
<br />
;'''First three-dimensional structure of GH31 enzymes'''<br />
<br />
:''Escherichia coli'' &alpha;-xylosidase (YicI) <cite>#7</cite><br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=376499<br />
<br />
#2 pmid=9620260<br />
<br />
#3 pmid=7766184<br />
<br />
#4 pmid=776963<br />
<br />
#5 pmid=1856189<br />
<br />
#6 pmid=11583585<br />
<br />
#7 pmid=15501829<br />
<br />
#8 pmid=11982345<br />
<br />
#9 pmid=14596624<br />
<br />
#10 pmid=11298744<br />
<br />
#11 pmid=16478160<br />
<br />
#12 pmid=16580018<br />
<br />
#13 pmid=18036614<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1604Glycoside Hydrolase Family 312009-08-13T22:16:11Z<p>Steve Withers: /* Family Firsts */</p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
CAZy Family GH31 is one of the two major families, along with GH13, that contain &alpha;-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal &alpha;-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to &alpha;-glucosidases, GH31 also contains &alpha;-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic [[Alpha-glucan lyases|&alpha;-glucan lyases]]. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.<br />
<br />
== Kinetics and Mechanism ==<br />
Family GH 31 enzymes are retaining &alpha;-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement. <cite>#1</cite> GH31 enzymes (except for the &alpha;-glucan lyases) are believed to follow the classical double displacement mechanism. <cite>#2</cite> This has been strongly supported by labelinging of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide <cite>#3</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>#4</cite> and human lysosomal &alpha;-glucosidase <cite>#5</cite>. Later studies on an &alpha;-glucosidase from ''Aspergillus niger'' <cite>#6</cite> and an &alpha;-xylosidase from ''Escherichia coli'' <cite>#7</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates.<br />
<br />
The &alpha;-glucan lyases from GH31 cleave &alpha;-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose. Detailed mechanistic studies have been carried out on ''Gracilariopsis'' &alpha;-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>#8 #9</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme intermediate by 5-fluoro-&beta;-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two &alpha;-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-&alpha;-D-glucopyranosyl fluoride.<br />
<br />
== Catalytic Residues ==<br />
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>#2 #7 #9</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the catalytic nucleophile, corresponding to Asp224 of ''Aspergillus niger'' &alpha;-glucosidase within the sequence IDM <cite>#3 #5</cite>. This was confirmed by using the more reliable 5-fluoro-&alpha;-D-glucopyransyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>#6</cite>. The general acid/base residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' &alpha;-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>#10</cite>. This was subsequently confirmed by the crystallographic studies on &alpha;-xylosidase (YicI) from ''Escherichia coli'' <cite>#7</cite> and successfully engineering YicI into the first &alpha;-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>#11</cite>. The equivalent residue, Asp553, in ''Gracilariopsis'' &alpha;-1,4-glucan lyase has also been identified as the catalytic nucleophile through the use of 5-fluoro-&beta;-L-idopyranosyl fluoride <cite>#8</cite>. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the catalytic nucleophile as it departs <cite>#9</cite>.<br />
<br />
== Three-dimensional structures ==<br />
The first crystal structure of a GH31 enzyme was that of the &alpha;-xylosidase YicI from ''Escherichia coli'' , published in 2005 <cite>#7</cite>. To date two other crystal structures of GH31 enzymes have been published, one being the ''Sulfolobus solfataricus'' &alpha;-glucosidase (MalA) <cite>#12</cite> and the other being the N-terminal domain of human intestinal maltase-glucoamylase <cite>#13</cite>. All of these structures feature a (-&beta;/&alpha;)<sub>8</sub> barrel in the catalytic domain.<br />
<br />
== Family Firsts ==<br />
<br />
;'''First stereochemical outcome'''<br />
<br />
:Determined for several &alpha;-glucosidases by a combination of polarimetric and reducing end measurements <cite>#1</cite><br />
<br />
;'''First catalytic nucleophile identification'''<br />
<br />
:Rabbit intestinal sucrase/isomaltase via conduritol B epoxide labeling <cite>#4</cite><br />
<br />
;'''First general acid/base residue identification'''<br />
<br />
:''Schizosaccharomyces pombe'' &alpha;-glucosidase by sequence comparison and kinetic studies of the mutants <cite>#10</cite><br />
<br />
;'''First three-dimensional structure of GH31 enzymes'''<br />
<br />
:''Escherichia coli'' &alpha;-xylosidase (YicI) <cite>#7</cite><br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=376499<br />
<br />
#2 pmid=9620260<br />
<br />
#3 pmid=7766184<br />
<br />
#4 pmid=776963<br />
<br />
#5 pmid=1856189<br />
<br />
#6 pmid=11583585<br />
<br />
#7 pmid=15501829<br />
<br />
#8 pmid=11982345<br />
<br />
#9 pmid=14596624<br />
<br />
#10 pmid=11298744<br />
<br />
#11 pmid=16478160<br />
<br />
#12 pmid=16580018<br />
<br />
#13 pmid=18036614<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1603Glycoside Hydrolase Family 312009-08-13T22:13:46Z<p>Steve Withers: /* References */</p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
CAZy Family GH31 is one of the two major families, along with GH13, that contain &alpha;-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal &alpha;-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to &alpha;-glucosidases, GH31 also contains &alpha;-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic [[Alpha-glucan lyases|&alpha;-glucan lyases]]. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.<br />
<br />
== Kinetics and Mechanism ==<br />
Family GH 31 enzymes are retaining &alpha;-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement. <cite>#1</cite> GH31 enzymes (except for the &alpha;-glucan lyases) are believed to follow the classical double displacement mechanism. <cite>#2</cite> This has been strongly supported by labelinging of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide <cite>#3</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>#4</cite> and human lysosomal &alpha;-glucosidase <cite>#5</cite>. Later studies on an &alpha;-glucosidase from ''Aspergillus niger'' <cite>#6</cite> and an &alpha;-xylosidase from ''Escherichia coli'' <cite>#7</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates.<br />
<br />
The &alpha;-glucan lyases from GH31 cleave &alpha;-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose. Detailed mechanistic studies have been carried out on ''Gracilariopsis'' &alpha;-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>#8 #9</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme intermediate by 5-fluoro-&beta;-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two &alpha;-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-&alpha;-D-glucopyranosyl fluoride.<br />
<br />
== Catalytic Residues ==<br />
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>#2 #7 #9</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the catalytic nucleophile, corresponding to Asp224 of ''Aspergillus niger'' &alpha;-glucosidase within the sequence IDM <cite>#3 #5</cite>. This was confirmed by using the more reliable 5-fluoro-&alpha;-D-glucopyransyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>#6</cite>. The general acid/base residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' &alpha;-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>#10</cite>. This was subsequently confirmed by the crystallographic studies on &alpha;-xylosidase (YicI) from ''Escherichia coli'' <cite>#7</cite> and successfully engineering YicI into the first &alpha;-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>#11</cite>. The equivalent residue, Asp553, in ''Gracilariopsis'' &alpha;-1,4-glucan lyase has also been identified as the catalytic nucleophile through the use of 5-fluoro-&beta;-L-idopyranosyl fluoride <cite>#8</cite>. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the catalytic nucleophile as it departs <cite>#9</cite>.<br />
<br />
== Three-dimensional structures ==<br />
The first crystal structure of a GH31 enzyme was that of the &alpha;-xylosidase YicI from ''Escherichia coli'' , published in 2005 <cite>#7</cite>. To date two other crystal structures of GH31 enzymes have been published, one being the ''Sulfolobus solfataricus'' &alpha;-glucosidase (MalA) <cite>#12</cite> and the other being the N-terminal domain of human intestinal maltase-glucoamylase <cite>#13</cite>. All of these structures feature a (-&beta;/&alpha;)<sub>8</sub> barrel in the catalytic domain.<br />
<br />
== Family Firsts ==<br />
;First sterochemistry determination: Cite some reference here, with a ''short'' explanation <cite>1</cite>.<br />
;First catalytic nucleophile identification: <br />
;First general acid/base residue identification: <br />
;First 3-D structure: <br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=376499<br />
<br />
#2 pmid=9620260<br />
<br />
#3 pmid=7766184<br />
<br />
#4 pmid=776963<br />
<br />
#5 pmid=1856189<br />
<br />
#6 pmid=11583585<br />
<br />
#7 pmid=15501829<br />
<br />
#8 pmid=11982345<br />
<br />
#9 pmid=14596624<br />
<br />
#10 pmid=11298744<br />
<br />
#11 pmid=16478160<br />
<br />
#12 pmid=16580018<br />
<br />
#13 pmid=18036614<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1602Glycoside Hydrolase Family 312009-08-13T22:13:19Z<p>Steve Withers: /* References */</p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
CAZy Family GH31 is one of the two major families, along with GH13, that contain &alpha;-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal &alpha;-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to &alpha;-glucosidases, GH31 also contains &alpha;-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic [[Alpha-glucan lyases|&alpha;-glucan lyases]]. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.<br />
<br />
== Kinetics and Mechanism ==<br />
Family GH 31 enzymes are retaining &alpha;-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement. <cite>#1</cite> GH31 enzymes (except for the &alpha;-glucan lyases) are believed to follow the classical double displacement mechanism. <cite>#2</cite> This has been strongly supported by labelinging of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide <cite>#3</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>#4</cite> and human lysosomal &alpha;-glucosidase <cite>#5</cite>. Later studies on an &alpha;-glucosidase from ''Aspergillus niger'' <cite>#6</cite> and an &alpha;-xylosidase from ''Escherichia coli'' <cite>#7</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates.<br />
<br />
The &alpha;-glucan lyases from GH31 cleave &alpha;-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose. Detailed mechanistic studies have been carried out on ''Gracilariopsis'' &alpha;-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>#8 #9</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme intermediate by 5-fluoro-&beta;-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two &alpha;-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-&alpha;-D-glucopyranosyl fluoride.<br />
<br />
== Catalytic Residues ==<br />
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>#2 #7 #9</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the catalytic nucleophile, corresponding to Asp224 of ''Aspergillus niger'' &alpha;-glucosidase within the sequence IDM <cite>#3 #5</cite>. This was confirmed by using the more reliable 5-fluoro-&alpha;-D-glucopyransyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>#6</cite>. The general acid/base residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' &alpha;-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>#10</cite>. This was subsequently confirmed by the crystallographic studies on &alpha;-xylosidase (YicI) from ''Escherichia coli'' <cite>#7</cite> and successfully engineering YicI into the first &alpha;-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>#11</cite>. The equivalent residue, Asp553, in ''Gracilariopsis'' &alpha;-1,4-glucan lyase has also been identified as the catalytic nucleophile through the use of 5-fluoro-&beta;-L-idopyranosyl fluoride <cite>#8</cite>. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the catalytic nucleophile as it departs <cite>#9</cite>.<br />
<br />
== Three-dimensional structures ==<br />
The first crystal structure of a GH31 enzyme was that of the &alpha;-xylosidase YicI from ''Escherichia coli'' , published in 2005 <cite>#7</cite>. To date two other crystal structures of GH31 enzymes have been published, one being the ''Sulfolobus solfataricus'' &alpha;-glucosidase (MalA) <cite>#12</cite> and the other being the N-terminal domain of human intestinal maltase-glucoamylase <cite>#13</cite>. All of these structures feature a (-&beta;/&alpha;)<sub>8</sub> barrel in the catalytic domain.<br />
<br />
== Family Firsts ==<br />
;First sterochemistry determination: Cite some reference here, with a ''short'' explanation <cite>1</cite>.<br />
;First catalytic nucleophile identification: <br />
;First general acid/base residue identification: <br />
;First 3-D structure: <br />
<br />
== References ==<br />
<biblio><br />
Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4<br />
<br />
#1 pmid=376499<br />
<br />
#2 pmid=9620260<br />
<br />
#3 pmid=7766184<br />
<br />
#4 pmid=776963<br />
<br />
#5 pmid=1856189<br />
<br />
#6 pmid=11583585<br />
<br />
#7 pmid=15501829<br />
<br />
#8 pmid=11982345<br />
<br />
#9 pmid=14596624<br />
<br />
#10 pmid=11298744<br />
<br />
#11 pmid=16478160<br />
<br />
#12 pmid=16580018<br />
<br />
#13 pmid=18036614<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1601Glycoside Hydrolase Family 312009-08-13T22:12:21Z<p>Steve Withers: /* Three-dimensional structures */</p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
CAZy Family GH31 is one of the two major families, along with GH13, that contain &alpha;-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal &alpha;-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to &alpha;-glucosidases, GH31 also contains &alpha;-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic [[Alpha-glucan lyases|&alpha;-glucan lyases]]. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.<br />
<br />
== Kinetics and Mechanism ==<br />
Family GH 31 enzymes are retaining &alpha;-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement. <cite>#1</cite> GH31 enzymes (except for the &alpha;-glucan lyases) are believed to follow the classical double displacement mechanism. <cite>#2</cite> This has been strongly supported by labelinging of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide <cite>#3</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>#4</cite> and human lysosomal &alpha;-glucosidase <cite>#5</cite>. Later studies on an &alpha;-glucosidase from ''Aspergillus niger'' <cite>#6</cite> and an &alpha;-xylosidase from ''Escherichia coli'' <cite>#7</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates.<br />
<br />
The &alpha;-glucan lyases from GH31 cleave &alpha;-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose. Detailed mechanistic studies have been carried out on ''Gracilariopsis'' &alpha;-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>#8 #9</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme intermediate by 5-fluoro-&beta;-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two &alpha;-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-&alpha;-D-glucopyranosyl fluoride.<br />
<br />
== Catalytic Residues ==<br />
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>#2 #7 #9</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the catalytic nucleophile, corresponding to Asp224 of ''Aspergillus niger'' &alpha;-glucosidase within the sequence IDM <cite>#3 #5</cite>. This was confirmed by using the more reliable 5-fluoro-&alpha;-D-glucopyransyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>#6</cite>. The general acid/base residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' &alpha;-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>#10</cite>. This was subsequently confirmed by the crystallographic studies on &alpha;-xylosidase (YicI) from ''Escherichia coli'' <cite>#7</cite> and successfully engineering YicI into the first &alpha;-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>#11</cite>. The equivalent residue, Asp553, in ''Gracilariopsis'' &alpha;-1,4-glucan lyase has also been identified as the catalytic nucleophile through the use of 5-fluoro-&beta;-L-idopyranosyl fluoride <cite>#8</cite>. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the catalytic nucleophile as it departs <cite>#9</cite>.<br />
<br />
== Three-dimensional structures ==<br />
The first crystal structure of a GH31 enzyme was that of the &alpha;-xylosidase YicI from ''Escherichia coli'' , published in 2005 <cite>#7</cite>. To date two other crystal structures of GH31 enzymes have been published, one being the ''Sulfolobus solfataricus'' &alpha;-glucosidase (MalA) <cite>#12</cite> and the other being the N-terminal domain of human intestinal maltase-glucoamylase <cite>#13</cite>. All of these structures feature a (-&beta;/&alpha;)<sub>8</sub> barrel in the catalytic domain.<br />
<br />
== Family Firsts ==<br />
;First sterochemistry determination: Cite some reference here, with a ''short'' explanation <cite>1</cite>.<br />
;First catalytic nucleophile identification: <br />
;First general acid/base residue identification: <br />
;First 3-D structure: <br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=16580018<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1600Glycoside Hydrolase Family 312009-08-13T22:08:48Z<p>Steve Withers: /* Catalytic Residues */</p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
CAZy Family GH31 is one of the two major families, along with GH13, that contain &alpha;-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal &alpha;-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to &alpha;-glucosidases, GH31 also contains &alpha;-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic [[Alpha-glucan lyases|&alpha;-glucan lyases]]. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.<br />
<br />
== Kinetics and Mechanism ==<br />
Family GH 31 enzymes are retaining &alpha;-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement. <cite>#1</cite> GH31 enzymes (except for the &alpha;-glucan lyases) are believed to follow the classical double displacement mechanism. <cite>#2</cite> This has been strongly supported by labelinging of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide <cite>#3</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>#4</cite> and human lysosomal &alpha;-glucosidase <cite>#5</cite>. Later studies on an &alpha;-glucosidase from ''Aspergillus niger'' <cite>#6</cite> and an &alpha;-xylosidase from ''Escherichia coli'' <cite>#7</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates.<br />
<br />
The &alpha;-glucan lyases from GH31 cleave &alpha;-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose. Detailed mechanistic studies have been carried out on ''Gracilariopsis'' &alpha;-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>#8 #9</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme intermediate by 5-fluoro-&beta;-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two &alpha;-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-&alpha;-D-glucopyranosyl fluoride.<br />
<br />
== Catalytic Residues ==<br />
Measurements of pH profiles suggested that two essential residues were involved in catalysis <cite>#2 #7 #9</cite>. Earlier active site-labeling studies employing conduritol B epoxide identified an invariant Asp residue as the catalytic nucleophile, corresponding to Asp224 of ''Aspergillus niger'' &alpha;-glucosidase within the sequence IDM <cite>#3 #5</cite>. This was confirmed by using the more reliable 5-fluoro-&alpha;-D-glucopyransyl fluoride reagent followed by subsequent peptide mapping by LC/MS-MS <cite>#6</cite>. The general acid/base residue was first tentatively assigned as Asp647 in the ''Schizosaccharomyces pombe'' &alpha;-glucosidase based on sequence comparison and kinetic analysis of the mutants <cite>#10</cite>. This was subsequently confirmed by the crystallographic studies on &alpha;-xylosidase (YicI) from ''Escherichia coli'' <cite>#7</cite> and successfully engineering YicI into the first &alpha;-thioglycoligase by mutating the corresponding general acid/base residue D482 <cite>#11</cite>. The equivalent residue, Asp553, in ''Gracilariopsis'' &alpha;-1,4-glucan lyase has also been identified as the catalytic nucleophile through the use of 5-fluoro-&beta;-L-idopyranosyl fluoride <cite>#8</cite>. However, the identity of the base which deprotonates H-2 in the second elimination step is not clear, though one of the most probable candidates was proposed to be the carboxyl group of the catalytic nucleophile as it departs <cite>#9</cite>.<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
<br />
== Family Firsts ==<br />
;First sterochemistry determination: Cite some reference here, with a ''short'' explanation <cite>1</cite>.<br />
;First catalytic nucleophile identification: <br />
;First general acid/base residue identification: <br />
;First 3-D structure: <br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=16580018<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1599Glycoside Hydrolase Family 312009-08-13T22:05:15Z<p>Steve Withers: /* Kinetics and Mechanism */</p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
CAZy Family GH31 is one of the two major families, along with GH13, that contain &alpha;-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal &alpha;-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to &alpha;-glucosidases, GH31 also contains &alpha;-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic [[Alpha-glucan lyases|&alpha;-glucan lyases]]. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.<br />
<br />
== Kinetics and Mechanism ==<br />
Family GH 31 enzymes are retaining &alpha;-glycosidases, as was first demonstrated by a combination of polarimetric and reducing sugar measurement. <cite>#1</cite> GH31 enzymes (except for the &alpha;-glucan lyases) are believed to follow the classical double displacement mechanism. <cite>#2</cite> This has been strongly supported by labelinging of the catalytic nucleophile of several GH31 enzymes using conduritol B epoxide <cite>#3</cite>, with early examples including rabbit intestinal sucrase/isomaltase <cite>#4</cite> and human lysosomal &alpha;-glucosidase <cite>#5</cite>. Later studies on an &alpha;-glucosidase from ''Aspergillus niger'' <cite>#6</cite> and an &alpha;-xylosidase from ''Escherichia coli'' <cite>#7</cite> used the more reliable 5-fluoroglycosyl fluoride trapping reagents, which form catalytically competent intermediates.<br />
<br />
The &alpha;-glucan lyases from GH31 cleave &alpha;-glucan polymers via an elimination mechanism to generate 1,5-anhydro-fructose. Detailed mechanistic studies have been carried out on ''Gracilariopsis'' &alpha;-1,4-glucan lyase revealing a mechanism that involves a nucleophilic displacement as the first step, followed by syn-elimination as the second step <cite>#8 #9</cite>. Evidence for the first step includes trapping of the catalytically competent glycosyl-enzyme intermediate by 5-fluoro-&beta;-L-idopyranosyl fluoride and the observation of secondary kinetic isotope effects (KIEs) on both H-1 and H-2 of two &alpha;-glucoside substrates. Direct proof of the second elimination step was provided by the observation of a small primary KIE on C-2 by using a substrate for which the elimination step is rate-limiting, 5-fluoro-&alpha;-D-glucopyranosyl fluoride.<br />
<br />
== Catalytic Residues ==<br />
<br />
<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
<br />
== Family Firsts ==<br />
;First sterochemistry determination: Cite some reference here, with a ''short'' explanation <cite>1</cite>.<br />
;First catalytic nucleophile identification: <br />
;First general acid/base residue identification: <br />
;First 3-D structure: <br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=16580018<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1598Glycoside Hydrolase Family 312009-08-13T22:03:00Z<p>Steve Withers: /* Substrate specificities */</p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
CAZy Family GH31 is one of the two major families, along with GH13, that contain &alpha;-glucosidases. These enzymes play important roles in primary metabolism (e.g. human sucrase/isomaltase, a target for diabetic drugs such as miglitol), in catabolism (e.g. human lysosomal &alpha;-glucosidase) and in glycoprotein processing (e.g. ER glucosidase II). In addition to &alpha;-glucosidases, GH31 also contains &alpha;-xylosidases, isomaltosyltransferases, maltase/glucoamylases and the mechanistically interesting, non-hydrolytic [[Alpha-glucan lyases|&alpha;-glucan lyases]]. These enzymes can be found in a wide range of organisms including archaea, bacteria, plants and animals. Interestingly the two mammalian digestive enzymes are both duplicated genes, each with dual specificities.<br />
<br />
== Kinetics and Mechanism == <br />
<br />
== Catalytic Residues ==<br />
<br />
<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
<br />
== Family Firsts ==<br />
;First sterochemistry determination: Cite some reference here, with a ''short'' explanation <cite>1</cite>.<br />
;First catalytic nucleophile identification: <br />
;First general acid/base residue identification: <br />
;First 3-D structure: <br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=16580018<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_31&diff=1597Glycoside Hydrolase Family 312009-08-13T20:59:47Z<p>Steve Withers: </p>
<hr />
<div>* [[Author]]: Ran Zhang<br />
* [[Responsible Curator]]: Steve Withers<br />
----<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH31'''<br />
|-<br />
|'''Clan''' <br />
|GH-D<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH31.html<br />
|}<br />
</div><br />
<br />
== Substrate specificities ==<br />
Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 <br />
<br />
== Kinetics and Mechanism == <br />
<br />
== Catalytic Residues ==<br />
<br />
<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
<br />
== Family Firsts ==<br />
;First sterochemistry determination: Cite some reference here, with a ''short'' explanation <cite>1</cite>.<br />
;First catalytic nucleophile identification: <br />
;First general acid/base residue identification: <br />
;First 3-D structure: <br />
<br />
== References ==<br />
<biblio><br />
#1 pmid=16580018<br />
<br />
</biblio><br />
<br />
<!-- DO NOT REMOVE THIS CATEGORY TAG! --><br />
[[Category:Glycoside Hydrolase Families|GH031]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_39&diff=1496Glycoside Hydrolase Family 392009-07-30T17:42:50Z<p>Steve Withers: /* Kinetics and Mechanism */</p>
<hr />
<div>* [[Author]]: [[User:Brian Rempel|Brian Rempel]]<br />
* [[Responsible Curator]]: [[User:Steve Withers|Stephen Withers]]<br />
----<br />
<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}}<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family 30'''<br />
|-<br />
|'''Clan''' <br />
|GH-A<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH39.html<br />
|}<br />
</div><br />
<br />
==Substrate Specificities==<br />
This family contains two known enzyme activities: &beta;-xylosidase and &alpha;-iduronidase. Both enzyme activities cleave equatorial glycosidic bonds: the &alpha; designation of &alpha;-iduronidase is a consequence of the stereochemical designations used for carbohydrates in which the &alpha;/&beta; designation is related to the [[Absolute_configuration:_D/L_nomenclature|D/L designation]] defined by the stereochemistry at C5 in hexopyranoses <cite>#1</cite>. Enzyme from this family are currently found in bacteria and eukaryotes, although one gene sequence encoding a putative Family GH39 enzyme from archaea has been reported. The known &beta;-xylosidase enzymes for which an enzyme activity has been experimentally established all come from bacteria, while the &alpha;-iduronidase enzymes all come from eukaryotes. Additionally, while there is a reasonable degree of sequence similarity within the &beta;-xylosidases in GH39 and within the &alpha;-iduronidases in GH39, there is a much lower degree of homology between the &beta;-xylosidases and &alpha;-iduronidases <cite>#2</cite>. The best-studied enzymes are human &alpha;-iduronidase, whose deficiency causes Mucopolysaccharidosis I (also known as Hurler-Scheie syndrome), and the &beta;-xylosidase from ''Thermoanaerobacterium saccharolyticum''. <br />
<br />
==Kinetics and Mechanism==<br />
Family GH39 enzymes are retaining enzymes that follow the classic Koshland double-displacement mechanism. This has been demonstrated experimentally through NMR analysis of the first-formed sugar product produced by glycoside hydrolysis by the &beta;-xylosidase from ''Thermoanaerobacterium saccharolyticum'' <cite>#3</cite> and human &alpha;-iduronidase <cite>#4</cite>, and by covalent trapping of the enzymatic nucleophile (described below) for these two enzymes <cite>#2 #4</cite>. These enzymes do not appear to require any activator or cofactor for activity.<br />
<br />
==Catalytic Residues==<br />
The catalytic nucleophile was first identified in the &beta;-xylosidase from ''Thermoanaerobacterium saccharolyticum'' as Glu-277 in the sequence IILNSHFPNLPFHIT<u>'''E'''</u>Y by trapping of the 2-deoxy-2-fluoro-xylosyl-enzyme intermediate and subsequent peptide mapping by LC/MS-MS <cite>#2</cite>. A similar analysis performed on human &alpha;-iduronidase also successfully trapped the catalytic nucleophile and identified it as Glu-299 in the sequence IYND<u>'''E'''</u>AD <cite>#4</cite>, which confirmed previous theoretical predictions <cite>#5</cite>. The catalytic acid/base has been experimentally identified in the &beta;-xylosidase from ''Thermoanaerobacterium saccharolyticum'' as Glu-160 through trapping using the affinity label N-bromoacetyl-&beta;-D-xylopyranosylamine and analysis of variant proteins created by mutation of that site <cite>#6</cite>.<br />
<br />
==Three-dimensional structures==<br />
The three-dimensional structure of the &beta;-xylosidase from ''Thermoanaerobacterium saccharolyticum'' was first solved in 2004 <cite>#7</cite>. Since then, the three dimensional structure for another GH39 &beta;-xylosidase from ''Geobacillus stearothermophilus'' has also been solved <cite>#8 #9</cite>. No experimentally determined three dimensional structure exists for the &alpha;-iduronidase enzymes, although a computer-generated homology model has been reported <cite>#10</cite>. GH39 enzymes are members of the GHA clan fold, consistent with the classic (&alpha;/&beta;)<sub>8</sub> TIM barrel fold with the two key active site glutamic acids located at the C-terminal ends of &beta;-strands 4 (acid/base) and 7 (nucleophile). <br />
<br />
<br />
==Family Firsts==<br />
<br />
;'''First stereochemistry determination'''<br />
:''Thermoanaerobacterium saccharolyticum'' &beta;-xylosidase by NMR <cite>#3</cite><br />
<br />
;'''First catalytic nucleophile identification'''<br />
:''Thermoanaerobacterium saccharolyticum'' &beta;-xylosidase by 2-fluoroxylose labelling <cite>#4</cite><br />
<br />
;'''First general acid/base residue identification'''<br />
:''Thermoanaerobacterium saccharolyticum'' &beta;-xylosidase through labelling with N-bromoacetyl-&beta;-D-xylopyranosylamine and kinetic analysis of mutants generated at the identified position <cite>#5</cite><br />
<br />
;'''First 3-D structure of a GH39 enzyme'''<br />
:''Thermoanaerobacterium saccharolyticum'' &beta;-xylosidase <cite>#6</cite><br />
<br />
==References==<br />
<biblio><br />
#1 pmid=9042704<br />
#2 pmid=9761746<br />
#3 pmid=8612648<br />
#4 pmid=12834357<br />
$5 pmid=9134434<br />
#6 pmid=12146939<br />
#7 pmid=14659747<br />
#8 pmid=16212978<br />
#9 pmid=14993701<br />
#10 pmid=15862278<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_30&diff=1495Glycoside Hydrolase Family 302009-07-30T17:17:04Z<p>Steve Withers: /* References */</p>
<hr />
<div>* [[Author]]: [[User:Brian Rempel|Brian Rempel]]<br />
* [[Responsible Curator]]: [[User:Steve Withers|Stephen Withers]]<br />
----<br />
<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}}<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family 30'''<br />
|-<br />
|'''Clan''' <br />
|GH-A<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH30.html<br />
|}<br />
</div><br />
<br />
==Substrate specificities==<br />
This family contains three known enzyme activities: &beta;-glucosylceramidase, &beta;-1,6-glucanase, and &beta;-xylosidase. This family enzymes currently contains enzymes from only bacteria and eukaryotes. The best-studied enzyme is human &beta;-glucocerebrosidase whose deficiency causes Gauchers disease <cite>#1</cite>. This enzyme is responsible for hydrolyzing the &beta;-glucoside from the glycolipid glucosylceramide.<br />
<br />
==Kinetics and Mechanism==<br />
Family GH30 enzymes are retaining enzymes. Although this has never been formally demonstrated experimentally through NMR analysis of the first-formed sugar product, covalent trapping of the enzymatic nucleophile (described below) conclusively demonstrates that these enzymes follow the classic Koshland double-displacement mechanism. The &beta;-glucosylceramidases require an activator protein and negatively charged phospholipids for optimal activity, <cite>#2</cite> although the role of these activators is still not entirely clear. Neither the &beta;-1,6-glucanases <cite>#3</cite> nor the &beta;-xylosidases <cite>#4</cite> appear to require any activators.<br />
<br />
==Catalytic Residues==<br />
The catalytic nucleophile was first identified in human &beta;-glucocerebrosidase as Glu340 in the sequence FAS<u>'''E'''</u>A by trapping of the 2-deoxy-2-fluoro-glucosyl-enzyme intermediate and subsequent peptide mapping by LC/MS-MS <cite>#5</cite>. The catalytic nucleophile had been previously mistakenly identified as Asp443 using a tritiated bromoconduritol epoxide <cite>#6 #7</cite>, although subsequent kinetic analyses of site-directed mutants of Asp443 were not consistent with its role as the catalytic nucleophile <cite>#8</cite>. The catalytic acid/base of human &beta;-glucoerebrosidase has been predicted to be Glu-274 <cite>#9</cite>. While this identification has not been experimentally verified through analysis of variant proteins created by mutation of that site, it is consistent with structural studies (below).<br />
<br />
==Three-dimensional structures==<br />
The three-dimensional structure of human &beta;-glucocerebrosidase was first solved in 2003 <cite>#10</cite>, and since then a number of structures of this enzyme have been reported (reviewed in <cite>#11</cite>). GH30 enzymes are members of the GHA clan fold, consistent with the classic (&alpha;/&beta;)<sub>8</sub> TIM barrel fold with the two key active site glutamic acids located at the C-terminal ends of &beta;-strands 4 (acid/base) and 7 (nucleophile) <cite>#12</cite>.<br />
<br />
==Family Firsts==<br />
<br />
;'''First catalytic nucleophile identification'''<br />
:Human &beta;-glucocerebrosidase by 2-fluoroglucose labelling <cite>#1</cite><br />
;'''First 3-D structure of a GH30 enzyme'''<br />
:Human &beta;-glucocerebrosidase <cite>#2</cite><br />
<br />
==References==<br />
<biblio><br />
#1 pmid=19094956<br />
#2 pmid=2127241<br />
#3 pmid=12162562<br />
#4 pmid=11909624<br />
#5 pmid=7908905<br />
#6 pmid=3456607<br />
#7 pmid=2077872<br />
#8 pmid=8294487<br />
#9 pmid=9134434<br />
#10 pmid=12792654<br />
#11 pmid=18783340<br />
#12 pmid=7624375<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families]]</div>Steve Withershttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_30&diff=1494Glycoside Hydrolase Family 302009-07-30T17:13:09Z<p>Steve Withers: /* Three-dimensional structures */</p>
<hr />
<div>* [[Author]]: [[User:Brian Rempel|Brian Rempel]]<br />
* [[Responsible Curator]]: [[User:Steve Withers|Stephen Withers]]<br />
----<br />
<br />
<br />
<br />
<div style="float:right"><br />
{| {{Prettytable}}<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family 30'''<br />
|-<br />
|'''Clan''' <br />
|GH-A<br />
|-<br />
|'''Mechanism'''<br />
|retaining<br />
|-<br />
|'''Active site residues'''<br />
|known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |http://www.cazy.org/fam/GH30.html<br />
|}<br />
</div><br />
<br />
==Substrate specificities==<br />
This family contains three known enzyme activities: &beta;-glucosylceramidase, &beta;-1,6-glucanase, and &beta;-xylosidase. This family enzymes currently contains enzymes from only bacteria and eukaryotes. The best-studied enzyme is human &beta;-glucocerebrosidase whose deficiency causes Gauchers disease <cite>#1</cite>. This enzyme is responsible for hydrolyzing the &beta;-glucoside from the glycolipid glucosylceramide.<br />
<br />
==Kinetics and Mechanism==<br />
Family GH30 enzymes are retaining enzymes. Although this has never been formally demonstrated experimentally through NMR analysis of the first-formed sugar product, covalent trapping of the enzymatic nucleophile (described below) conclusively demonstrates that these enzymes follow the classic Koshland double-displacement mechanism. The &beta;-glucosylceramidases require an activator protein and negatively charged phospholipids for optimal activity, <cite>#2</cite> although the role of these activators is still not entirely clear. Neither the &beta;-1,6-glucanases <cite>#3</cite> nor the &beta;-xylosidases <cite>#4</cite> appear to require any activators.<br />
<br />
==Catalytic Residues==<br />
The catalytic nucleophile was first identified in human &beta;-glucocerebrosidase as Glu340 in the sequence FAS<u>'''E'''</u>A by trapping of the 2-deoxy-2-fluoro-glucosyl-enzyme intermediate and subsequent peptide mapping by LC/MS-MS <cite>#5</cite>. The catalytic nucleophile had been previously mistakenly identified as Asp443 using a tritiated bromoconduritol epoxide <cite>#6 #7</cite>, although subsequent kinetic analyses of site-directed mutants of Asp443 were not consistent with its role as the catalytic nucleophile <cite>#8</cite>. The catalytic acid/base of human &beta;-glucoerebrosidase has been predicted to be Glu-274 <cite>#9</cite>. While this identification has not been experimentally verified through analysis of variant proteins created by mutation of that site, it is consistent with structural studies (below).<br />
<br />
==Three-dimensional structures==<br />
The three-dimensional structure of human &beta;-glucocerebrosidase was first solved in 2003 <cite>#10</cite>, and since then a number of structures of this enzyme have been reported (reviewed in <cite>#11</cite>). GH30 enzymes are members of the GHA clan fold, consistent with the classic (&alpha;/&beta;)<sub>8</sub> TIM barrel fold with the two key active site glutamic acids located at the C-terminal ends of &beta;-strands 4 (acid/base) and 7 (nucleophile) <cite>#12</cite>.<br />
<br />
==Family Firsts==<br />
<br />
;'''First catalytic nucleophile identification'''<br />
:Human &beta;-glucocerebrosidase by 2-fluoroglucose labelling <cite>#1</cite><br />
;'''First 3-D structure of a GH30 enzyme'''<br />
:Human &beta;-glucocerebrosidase <cite>#2</cite><br />
<br />
==References==<br />
<biblio><br />
#1 pmid=19094956<br />
#2 pmid=2127241<br />
#3 pmid=12162562<br />
#4 pmid=11909624<br />
#5 pmid=7908905<br />
#6 pmid=3456607<br />
#7 pmid=2077872<br />
#8 pmid=8294487<br />
#9 pmid=12792654<br />
#10 pmid=18783340<br />
#11 pmid=7624375<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families]]</div>Steve Withers