CAZypedia - User contributions [en-ca]

User:Junho Lee

2016-04-29T20:12:59Z

Junho Lee:

Junho Lee is currently a pharmacy undergraduate student at the University of British Columbia. He has also studied Biochemesitry and Music performance with specialization in French Horn at the University of British Columbia. He was given the opportunity to contribute to [[GH33]].

[[Category:Contributors|Lee, Junho]]

User:Junho Lee

2013-07-17T07:46:57Z

Junho Lee:

Junho Lee is currently a biochemistry undergraduate student at the University of British Columbia. He has also studied Music performance with specialization in French Horn at the University of British Columbia. Recently, he was given the opportunity to contribute to [[GH33]].

[[Category:Contributors|Lee, Junho]]

User:Junho Lee

2013-07-15T23:23:44Z

Junho Lee:

'''Hi''' this is ''Junho''.

[[Category:Contributors|Lee, Junho]]

Glycoside Hydrolase Family 33

2013-07-15T23:17:56Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-E
|-
|'''Mechanism'''
|Retaining
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
===Nucleophile===
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>.

===Acid Base Catalyst===
An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.

From <cite>Amaya2004</cite>:
<blockquote>
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."
</blockquote>

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Moustafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T23:12:51Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-E
|-
|'''Mechanism'''
|Retaining
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.

From <cite>Amaya2004</cite>:
<blockquote>
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."
</blockquote>

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Moustafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T23:12:20Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-E
|-
|'''Mechanism'''
|Retaining
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. The mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.

From <cite>Amaya2004</cite>:
<blockquote>
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."
</blockquote>

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Moustafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T23:06:21Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-E
|-
|'''Mechanism'''
|Retaining
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose.

From <cite>Amaya2004</cite>:
<blockquote>
"It has been argued previously that Asp59 might not function as acid catalyst since its relatively high solvent exposure would suggest it may have a pKa that is too low to allow it to function in such a role according to the known pH dependence <cite>Chong1992 Burmeister1993</cite>. However, this study, which is the first showing a Michaelis complex for a sialidase, reveals that the position of the aromatic side chain of Tyr119 and the binding of the aglycone (lactose or methylumbelliferyl) of the substrate significantly decrease this solvent exposure. Further, the binding of the anionic substrate itself would be expected to substantially raise the pKa of this residue as a consequence of the electrostatic interactions between the two carboxyl groups."
</blockquote>

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Moustafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:54:13Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-E
|-
|'''Mechanism'''
|Retaining
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Moustafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:50:21Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-E
|-
|'''Mechanism'''
|Retaining
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watts2003</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Moustafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:45:56Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-E
|-
|'''Mechanism'''
|Retaining
|-
|'''Active site residues'''
|Known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watson2003</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Moustafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:45:12Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-E
|-
|'''Mechanism'''
|Retaining
|-
|'''Active site residues'''
|Tyr and Glu
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watson2003</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Moustafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:42:14Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-E
|-
|'''Mechanism'''
|Retaining
|-
|'''Active site residues'''
|Tyr and Glu
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watson2003</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Mosutafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:40:51Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-E
|-
|'''Mechanism'''
|Retaining
|-
|'''Active site residues'''
|Tyr and Glu
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watson2003</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Comfort2007</cite>.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Sinnott1990</cite>.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>He1999</cite>.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>StickWilliams</cite>.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Mosutafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:39:30Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-E
|-
|'''Mechanism'''
|Retaining
|-
|'''Active site residues'''
|Tyr and Glu
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2008</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watson2003</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Comfort2007</cite>.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Sinnott1990</cite>.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>He1999</cite>.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>StickWilliams</cite>.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Mosutafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:38:10Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Junho Lee^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|retaining/inverting
|-
|'''Active site residues'''
|known/not known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2008</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watson2003</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Comfort2007</cite>.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Sinnott1990</cite>.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>He1999</cite>.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>StickWilliams</cite>.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Mosutafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:37:53Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Tom Wennekes^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|retaining/inverting
|-
|'''Active site residues'''
|known/not known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2008</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watson2003</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Comfort2007</cite>.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Sinnott1990</cite>.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>He1999</cite>.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>StickWilliams</cite>.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Mosutafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:37:16Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Tom Wennekes^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|retaining/inverting
|-
|'''Active site residues'''
|known/not known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2008</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watson2003</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Comfort2007</cite>.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Sinnott1990</cite>.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>He1999</cite>.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>StickWilliams</cite>.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Mosutafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:35:10Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Tom Wennekes^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|retaining/inverting
|-
|'''Active site residues'''
|known/not known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi''trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2008</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watson2003</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3-OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.

== Three-dimensional structures ==

All members of the sialidase superfamily, including the members of GH34 and GH83, display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold. All bacterial sialidases generally have four to five aspartate boxes (Asp-box, Ser/Thr-x-Asp-x-Gly-x-Thr-Trp/Phe; where x represents any amino acid) within the catalytic domain, and these repeated sequences are found in identical positions in the beta sheet fold, far from the active site.

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>.

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>.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Comfort2007</cite>.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Sinnott1990</cite>.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>He1999</cite>.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>StickWilliams</cite>.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
#Mosutafa2004 pmid=15226294
#Watson2005 pmid=16206228

#Gaskell1995 pmid=8591030
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:28:56Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Tom Wennekes^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|retaining/inverting
|-
|'''Active site residues'''
|known/not known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi''trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2008</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watson2003</cite>.

An Asp in the active site within hydrogen bonding distance of the glycosidic oxygen acts as the acid catalyst for the initial glycosyl-enzyme formation. After glycosyl-enzyme formation, the same Asp acts as the general base catalyst for nucleophilic attack by the 3 OH of lactose. The role of Asp as the acid catalyst has been contested in the early 90s due to the low pKa values arising from the exposure to the solvent <cite> Amaya2004 Chong1992 Burmeister1993</cite>. However, Michaelis complex of ''T. cruzi'' demonstrated that the pKa value of Asp can be raised significantly to perform the role as an acid catalyst because of the position of the Tyr119 aromatic ring and the binding of the aglycone of the substrate that decrease the solvent exposure of said Asp residue <cite> Amaya2004</cite>.
== Three-dimensional structures ==
Content is to be added here.

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Comfort2007</cite>.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Sinnott1990</cite>.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>He1999</cite>.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>StickWilliams</cite>.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
#Chong1992 pmid=1628657
#Burmeister1993 pmid=8069621
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:24:30Z

Junho Lee:

{{UnderConstruction}}
* [[Author]]: ^^^Tom Wennekes^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH33'''
|-
|'''Clan'''
|GH-x
|-
|'''Mechanism'''
|retaining/inverting
|-
|'''Active site residues'''
|known/not known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH33.html
|}
</div>


== Substrate specificities ==
Sialic acids, often known as ''N''-acetylneuraminic acid (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acids <cite>Kim2011 Varki2007 Vimir2004</cite>. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases <cite> Amaya2004</cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.

== Kinetics and Mechanism ==
Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on ''T. cruzi''trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue, and the subsequent crystal structures helped to determine the mechanism of the bacterial sialidases <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS revealed a ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2008</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.

== Catalytic Residues ==
The strictly conserved tyrosine in the active site acts as the nucleophile attacking the anomeric centre of the sialic acid <cite> Amaya2004</cite>. Peptide mapping of ''T. cruzi'' with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 <cite> Watts2003</cite>. The nucleophilic character of Tyr342 is enhanced by interaction with a nearby invariant glutamate, which acts as a base catalyst. Tyrosine has likely evolved as the catalytic nucleophile rather than the carboxylate group normally found in glycosidases in order to minimize charge repulsion with the carboxylate at C1 of the sialic acid <cite> Amaya2004 Newstead2008</cite>. The 20 fold increase of Km when the Tyr is mutated to Asp also supports the need to minimize the Coulombic repulsion between the enzyme and the substrate <cite> Watson2003</cite>.

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

== Family Firsts ==
;First stereochemistry determination: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Comfort2007</cite>.
;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Sinnott1990</cite>.
;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>He1999</cite>.
;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>StickWilliams</cite>.

== References ==
<biblio>
#Varki1997 pmid=9068613
#Kim2011 pmid=21544654
#Varki2007 pmid=17460663
#Vimir2004 pmid=15007099
#Buschiazzo2008 pmid=18625334
#Amaya2004 pmid=15130470
#Mizan2000 pmid=11092845
#Watts2003 pmid=12812490
#Damager2008 pmid=18284211
#Watts2006 pmid=16298994
#Newstead2008 pmid=18218621
</biblio>

[[Category:Glycoside Hydrolase Families|GH033]]

Glycoside Hydrolase Family 33

2013-07-15T22:16:05Z

Junho Lee:

Glycoside Hydrolase Family 33

2013-07-15T22:02:46Z

Junho Lee:

Glycoside Hydrolase Family 33

2013-07-15T21:57:09Z

Junho Lee:

Glycoside Hydrolase Family 33

2013-07-15T21:56:12Z

Junho Lee:

Glycoside Hydrolase Family 33

2013-07-15T21:55:42Z

Junho Lee:

Glycoside Hydrolase Family 33

2013-07-15T21:55:09Z

Junho Lee:

Glycoside Hydrolase Family 33

2013-07-15T21:49:56Z

Junho Lee:

Glycoside Hydrolase Family 33

2013-07-15T21:38:57Z

Junho Lee:

Glycoside Hydrolase Family 33

2013-07-15T21:34:43Z

Junho Lee:

User:Junho Lee

2013-07-15T21:26:54Z

Junho Lee:

'''Hi''' this is ''Junho''.

[[Category:Contributors|Lee, Junho]]

User:Junho Lee

2013-07-15T21:21:28Z

Junho Lee: Created page with "Hi this is Junho."

Hi this is Junho.