https://www.cazypedia.org/api.php?action=feedcontributions&feedformat=atom&user=Kyle+RobinsonCAZypedia - User contributions [en-ca]2026-05-04T19:59:06ZUser contributionsMediaWiki 1.35.10https://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15496Glycoside Hydrolase Family 332020-06-23T17:48:45Z<p>Kyle Robinson: </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 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: Glu230, Tyr342, Asp59. 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 C1 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15495Glycoside Hydrolase Family 332020-06-23T17:47:55Z<p>Kyle Robinson: </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 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: Glu230, Tyr342, Asp59. 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 C1 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15494Glycoside Hydrolase Family 332020-06-23T17:46:02Z<p>Kyle Robinson: </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 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15493Glycoside Hydrolase Family 342020-06-23T17:44:34Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNAc, NeuNA) are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them in an exo- or endo- acting fashion are found in families [[GH33]], GH34, and [[GH83]] or [[GH53]] respectively.<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-fluorosialosyl fluorides <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 intermediate, 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 Tyr406 by use of 3-fluorosialosyl 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 Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 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 report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be directly involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9 of Neu5Ac. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant neuraminidase to bind tightly to red blood cells by kinetic and structural analysis of enzyme 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 the role of acid/base catalyst for Asp151 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 />
== Three-dimensional structures ==<br />
These viral neuraminidases are members of Clan GH-E, along with families [[GH33]], [[GH83]] and [[GH93]]. 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 />
== Family Firsts ==<br />
;First stereochemistry determination: Determined for influenza neuraminidase 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15492Glycoside Hydrolase Family 342020-06-23T17:44:11Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNAc, NeuNA) are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them in an exo- or endo- acting fashion are found in families [[GH33]], GH34, and [[GH83]] or [[GH53]] respectively.<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-fluorosialosyl fluorides <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 intermediate, 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 Tyr406 by use of 3-fluorosialosyl 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 Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 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 report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be directly involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9 of Neu5Ac. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant neuraminidase to bind tightly to red blood cells by kinetic and structural analysis of enzyme 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 the role of acid/base catalyst for Asp151 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 viral neuraminidases are members of Clan GH-E, along with families [[GH33]], [[GH83]] and [[GH93]]. 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 />
== Family Firsts ==<br />
;First stereochemistry determination: Determined for influenza neuraminidase 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15491Glycoside Hydrolase Family 342020-06-23T17:38:30Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNAc, NeuNA) are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them in an exo- or endo- acting fashion are found in families [[GH33]], GH34, and [[GH83]] or [[GH53]] respectively.<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-fluorosialosyl fluorides <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 intermediate, 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 Tyr406 by use of 3-fluorosialosyl 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 Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 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 report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be directly involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9 of Neu5Ac. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant neuraminidase to bind tightly to red blood cells by kinetic and structural analysis of enzyme 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 the role of acid/base catalyst for Asp151 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 viral neuraminidases are members of Clan GH-E, along with families [[GH33]], [[GH83]] and [[GH93]]. 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 neuraminidase 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15490Glycoside Hydrolase Family 342020-06-23T17:35:06Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNAc, NeuNA) are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them in an exo- or endo- acting fashion are found in families [[GH33]], GH34, and [[GH83]] or [[GH53]] respectively.<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-fluorosialosyl fluorides <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 intermediate, 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 Tyr406 by use of 3-fluorosialosyl 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 Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 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 report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant neuraminidase to bind tightly to red blood cells by kinetic and structural analysis of enzyme 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 the role of acid/base catalyst for Asp151 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 viral neuraminidases are members of Clan GH-E, along with families [[GH33]], [[GH83]] and [[GH93]]. 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 neuraminidase 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15489Glycoside Hydrolase Family 342020-06-23T17:28:34Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNAc, NeuNA) are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them in an exo- or endo- acting fashion are found in families [[GH33]], GH34, and [[GH83]] or [[GH53]] respectively.<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-fluorosialosyl fluorides <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 intermediate, 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 Tyr406 by use of 3-fluorosialosyl 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 Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 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 report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging <cite> GhateAir1998 </cite>. More recently Zhu and Wilson investigated why mutations to Asp151 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 Asp151 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 viral neuraminidases are members of Clan GH-E, along with families [[GH33]], [[GH83]] and [[GH93]]. 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 neuraminidase 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15488Glycoside Hydrolase Family 342020-06-23T17:27:01Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNAc, NeuNA) are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them in an exo- or endo- acting fashion are found in families [[GH33]], GH34, and [[GH83]] or [[GH53]] respectively.<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-fluorosialosyl fluorides <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 intermediate, 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 Tyr406 by use of 3-fluorosialosyl 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 Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 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 report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures instead showed that the glutamate 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 Asp151 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 Asp151 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 viral neuraminidases are members of Clan GH-E, along with families [[GH33]], [[GH83]] and [[GH93]]. 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 neuraminidase 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15487Glycoside Hydrolase Family 342020-06-23T17:25:37Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNAc, NeuNA) are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them in an exo- or endo- acting fashion are found in families [[GH33]], GH34, and [[GH83]] or [[GH53]] respectively.<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-fluorosialosyl fluorides <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 intermediate, 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 Tyr406 by use of 3-fluorosialosyl 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 Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 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 report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al <cite> Lentz1987 </cite>, on which basis they suggested Glu276 to be involved in catalysis - though subsequent structures showed that this glutamate 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 Asp151 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 Asp151 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 viral neuraminidases are members of Clan GH-E, along with families [[GH33]], [[GH83]] and [[GH93]]. 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 neuraminidase 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15486Glycoside Hydrolase Family 342020-06-23T17:18:20Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNAc, NeuNA) are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them in an exo- or endo- acting fashion are found in families [[GH33]], GH34, and [[GH83]] or [[GH53]] respectively.<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-fluorosialosyl fluorides <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 intermediate, 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 Tyr406 by use of 3-fluorosialosyl 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 Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 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 report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 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 Asp151 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 Asp151 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 viral neuraminidases are members of Clan GH-E, along with families [[GH33]], [[GH83]] and [[GH93]]. 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 neuraminidase 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15484Glycoside Hydrolase Family 342020-06-23T17:13:48Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNAc, NeuNA) are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them in an exo- or endo- acting fashion are found in families [[GH33]], GH34, and [[GH83]] or [[GH53]] respectively.<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-fluorosialosyl fluorides <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 intermediate, 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 Tyr406 by use of 3-fluorosialosyl 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 Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 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 OH2 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 Asp151 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 Asp151 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 viral neuraminidases are members of Clan GH-E, along with families [[GH33]], [[GH83]] and [[GH93]]. 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 neuraminidase 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15483Glycoside Hydrolase Family 342020-06-23T17:05:54Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNAc, NeuNA) 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 />
== 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-fluorosialosyl fluorides <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 intermediate, 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-fluorosialosyl 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15482Glycoside Hydrolase Family 342020-06-23T17:02:20Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNAc, NeuNA) 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 />
== 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 intermediate, 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15481Glycoside Hydrolase Family 342020-06-23T17:01:34Z<p>Kyle Robinson: </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 (Neu5Ac, NANA, NeuNA) 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 />
== 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 intermediate, 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15480Glycoside Hydrolase Family 342020-06-23T16:57:07Z<p>Kyle Robinson: </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 />
== 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15479Glycoside Hydrolase Family 342020-06-23T16:54:33Z<p>Kyle Robinson: </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 />
== 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_34&diff=15478Glycoside Hydrolase Family 342020-06-23T16:50:50Z<p>Kyle Robinson: </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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15477Glycoside Hydrolase Family 332020-06-23T16:48:43Z<p>Kyle Robinson: </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 pmid15527797<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15476Glycoside Hydrolase Family 332020-06-23T16:44:24Z<p>Kyle Robinson: </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 pmid15527797<br />
<br />
</biblio><br />
<br />
[[Category:Glycoside Hydrolase Families|GH033]]</div>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15475Glycoside Hydrolase Family 332020-06-23T16:39:35Z<p>Kyle Robinson: </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''. 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15474Glycoside Hydrolase Family 332020-06-23T16:36:30Z<p>Kyle Robinson: </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. 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15473Glycoside Hydrolase Family 332020-06-23T16:21:06Z<p>Kyle Robinson: </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: 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=Glycoside_Hydrolase_Family_33&diff=15472Glycoside Hydrolase Family 332020-06-23T16:18:58Z<p>Kyle Robinson: </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 />
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<!-- 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 />
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<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 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>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=User:Kyle_Robinson&diff=10948User:Kyle Robinson2015-08-13T05:15:46Z<p>Kyle Robinson: </p>
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Kyle Robinson began his studies at [http://www.mcgill.ca McGill University], where he completed his B.Sc. while studying under Prof. Paul Wiseman. He has since been pursuing his PhD at the [http://www.ubc.ca University of British Columbia] under the supervision of [[User:Steve_Withers|Prof. Stephen Withers]]. His research focuses on the design of selective mechanism-based inhibitors for glycosyl hydrolases, specifically, sialidases and trans-sialidases from [[Glycoside Hydrolase Family 33]].<br />
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[[Category:Contributors|Robinson,Kyle]]</div>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=User:Kyle_Robinson&diff=10941User:Kyle Robinson2015-08-11T03:31:33Z<p>Kyle Robinson: </p>
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Kyle Robinson began his studies at [https://en.wikipedia.org/wiki/McGill_University McGill University], where he completed his B.Sc. while studying under Prof. Paul Wiseman. He has since been pursuing his PhD at the University of British Columbia under the supervision of Prof. [[User:Steve_Withers|Stephen Withers]]. His research focuses on the design of selective mechanism-based inhibitors for glycosyl hydrolases, specifically, sialidases and trans-sialidases from [[Glycoside Hydrolase Family 33]].<br />
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[[Category:Contributors|Robinson,Kyle]]</div>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=File:Profile.jpg&diff=10940File:Profile.jpg2015-08-11T03:29:31Z<p>Kyle Robinson: </p>
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<div></div>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=User:Kyle_Robinson&diff=10938User:Kyle Robinson2015-08-10T21:12:28Z<p>Kyle Robinson: </p>
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<div><br />
Kyle Robinson began his studies at [https://en.wikipedia.org/wiki/McGill_University McGill University], where he completed his B.Sc. while studying under Prof. Paul Wiseman. He has since been pursuing his PhD at the University of British Columbia under the supervision of Prof. [https://www.cazypedia.org/index.php/User:Steve_Withers Stephen Withers]. His research focuses on the design of selective mechanism-based inhibitors for glycosyl hydrolases, specifically, sialidases and trans-sialidases from [https://en.wikipedia.org/wiki/Glycoside_hydrolase_family_33 Family 33].<br />
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[[Category:Contributors|Robinson,Kyle]</div>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=User:Kyle_Robinson&diff=10937User:Kyle Robinson2015-08-10T21:06:15Z<p>Kyle Robinson: </p>
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<div>[[Image:profile.jpg|200px|right]]<br />
Kyle Robinson began his studies at [https://en.wikipedia.org/wiki/McGill_University McGill University], where he completed his B.Sc. while studying under Prof. Paul Wiseman. He has since been pursuing his PhD at the University of British Columbia under the supervision of Prof. [https://www.cazypedia.org/index.php/User:Steve_Withers Stephen Withers]. His research focuses on the design of selective mechanism-based inhibitors for glycosyl hydrolases, specifically, sialidases and trans-sialidases from [https://en.wikipedia.org/wiki/Glycoside_hydrolase_family_33 Family 33].<br />
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[[Category:Contributors|Robinson,Kyle]</div>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=User:Kyle_Robinson&diff=10936User:Kyle Robinson2015-08-10T20:38:30Z<p>Kyle Robinson: </p>
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Kyle Robinson began his studies at [https://en.wikipedia.org/wiki/McGill_University McGill University], where he completed his B.Sc. while studying under Prof. Paul Wiseman. He has since been pursuing his PhD at the University of British Columbia under the supervision of Prof. [https://www.cazypedia.org/index.php/User:Steve_Withers Stephen Withers]. His research focuses on the design of selective mechanism-based inhibitors for glycosyl hydrolases, specifically, sialidases and trans-sialidases from [https://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_33 Family 33].<br />
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[[Category:Contributors|Robinson,Kyle]]</div>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=User:Kyle_Robinson&diff=10935User:Kyle Robinson2015-08-10T20:14:49Z<p>Kyle Robinson: </p>
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Kyle Robinson began his studies at [https://en.wikipedia.org/wiki/McGill_University McGill University], where he completed his B.Sc. while studying under Prof. Paul Wiseman. He has since been pursuing his PhD at the University of British Columbia under the supervision of Prof. [https://www.cazypedia.org/index.php/User:Steve_Withers Stephen Withers]. His research focuses on the design of selective mechanism-based inhibitors for glycosyl hydrolases, specifically, sialidases and trans-sialidases from [https://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_33 Family 33].<br />
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[[Category:Contributors|Robinson,Kyle]]</div>Kyle Robinsonhttps://www.cazypedia.org/index.php?title=User:Kyle_Robinson&diff=10934User:Kyle Robinson2015-08-10T20:13:33Z<p>Kyle Robinson: </p>
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Kyle Robinson began his studies at [https://en.wikipedia.org/wiki/McGill_University, McGill University], where he completed his B.Sc. while studying under Prof. Paul Wiseman. He has since been pursuing his PhD at the University of British Columbia under the supervision of Prof. [https://www.cazypedia.org/index.php/User:Steve_Withers, Stephen Withers]. His research focuses on the design of selective mechanism-based inhibitors for glycosyl hydrolases, specifically, sialidases and trans-sialidases from [https://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_33, Family 33].<br />
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