Difference between revisions of "Glycoside Hydrolase Family 33"

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Author: Kyle Robinson and Junho Lee
Responsible Curator: Steve Withers

Glycoside Hydrolase Family GH33
Clan	GH-E
Mechanism	Retaining
Active site residues	Known
CAZy DB link
http://www.cazy.org/GH33.html

Substrate specificities

Sialic acids, often known as N-acetylneuraminic acids (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment [1]. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an α(2,3) or α(2,6) linkage to galactose, N-acetylgalactosamine, and N-acetylglucosamine or an α(2,8) linkage to another sialic acid [2, 3, 4]. These α(2,8) linked sialic acids can occur in polymeric form as polysialic acids, in the brain, as well as in capsular polysaccharides of some bacteria. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, GH34, and GH83 families are exosialidases while GH53 is an endosialidase [5].

The GH33 family includes most bacterial and simple eukaryotic sialidases, trans-sialidases [6] and intramolecular trans-sialidases [7, 8]. 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 [2]. 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 [9]. 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 [2].

Kinetics and Mechanism

GH33 sialidases and trans-sialidases hydrolyse, or transfer sialic acids to acceptor sugars, with retention of anomeric configuration [10, 11]. The general mechanism is depicted here. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a covalent glycosyl-enzyme intermediate was observed on T. cruzi trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue and the labelled amino acid residue identified as Tyr342 by peptide mapping. Subsequent crystal structures provided further insight into the mechanism of the protozoan sialidase [6, 12]. Kinetic analysis of TcTS confirmed the expected ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry [13]. Subsequent structural studies of two strictly hydrolytic sialidases from T. rangelli [14] and C. perfringens [15] also characterised their covalent intermediates.

Catalytic Residues

Nucleophile

The catalytic machinery is conserved throughout this family and, as shown by studies on TcTS, three key residues are involved: Glu230, Tyr342, Asp59. Since Watts first trapped the covalent 3F-sialyl enzyme intermediate on Tyr342 in 2003, many confirmatory structures have been solved. It is likely that tyrosine has evolved as the catalytic nucleophile, rather than the carboxylate moiety found in most retaining glycosidases, in order to minimize repulsive charge-charge interactions with the C1 carboxylate of the sialic acids. The neutral tyrosine can more readily approach the anomeric centre, and is rendered more nucleophilic by an invariant glutamate, Glu230 for TcTS, that serves as a base catalyst for deprotonation/reprotonation of the tyrosine hydroxyl. See Chan et al [16] for a detailed analysis of the role of this residue in the GH33 sialidase from M. viridifaciens.

Acid Base Catalyst

The third mechanistically relevant conserved residue in the active site is Asp59, which serves as the acid/base catalyst, protonating the glycosidic oxygen as bond cleavage occurs. Following formation of the intermediate, Asp59 then serves as a base catalyst, activating the incoming water molecule (for simple hydrolytic pathways) or glycosyl acceptor (in the case of trans-sialidases) towards trans-glycosylation of the glycosyl-enzyme intermediate. This second attack leads to a double inversion at the anomeric center and a net retention of stereochemistry observed in the released product, Neu5Ac [6, 13, 15].

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 [6], which is accepted as the canonical neuraminidase fold. The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst [5].

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 [17]. 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 [18, 19].

Family Firsts

First stereochemistry determination: First determined by proton NMR by Friebolin et al [10, 11].
First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. [12].
First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al [20].
First 3-D structure: First determined for the Salmonella typhimurium enzyme by Crennell and Taylor [20].

References

All Medline abstracts: PubMed

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+* [[Author]]: [[User:Kyle Robinson|Kyle Robinson]] and [[User:Junho Lee|Junho Lee]]
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+* [[Responsible Curator]]:  [[User:Steve Withers|Steve Withers]]
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 |-
 |'''Clan'''
-|GH-x
+|GH-E
 |-
 |'''Mechanism'''
-|retaining/inverting
+|Retaining
 |-
 |'''Active site residues'''
-|known/not known
+|Known
 |-
 |{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
@@ Line 29: / Line 29: @@
 == 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>.
+Sialic acids, often known as ''N''-acetylneuraminic acids (Neu5Ac, NANA, NeuNAc, NeuNA), are a family of nine carbon monosaccharides with a carboxylate group in the carbon 1 position that occupy the terminal position of the glycans, glycoproteins, glycolipids, and polysaccharides in cells and play important roles in interactions of the cell with its environment <cite>Varki1997</cite>. More than 50 sialic acid derivatives have been detected in eukaryotic and prokaryotic species; the most frequently detected sialic acids have an  α(2,3) or α(2,6) linkage to galactose, ''N''-acetylgalactosamine, and ''N''-acetylglucosamine or an α(2,8) linkage to another sialic acid <cite>Kim2011 Varki2007 Vimir2004</cite>. These α(2,8) linked sialic acids can occur in polymeric form as polysialic acids, in the brain, as well as in capsular polysaccharides of some bacteria. Sialic acids are hydrolyzed by sialidases (E.C. 3.2.1.18), and these enzymes are categorized into four different glycoside hydrolase(GH) families: GH33, [[GH34]], and [[GH83]] families are exosialidases while [[GH53]] is an endosialidase <cite>Buschiazzo2008</cite>.
-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>.
+The GH33 family includes most bacterial and simple eukaryotic sialidases, trans-sialidases <cite> Amaya2004</cite> and intramolecular trans-sialidases <cite> Li1990 Tailford2015 </cite>. Members of GH33 exhibit different preferences for the three most common sialic acid linkage types listed above, despite similar protein structure. For example, sialidases from ''Salmonella typhimurium'' LT2, ''Vibrio Cholerae'', and ''Clostridium septicum'', ''Clostridium sordellii'', ''Clostridium chauvoei'', ''Clostridium tertium'' demonstrate a higher hydrolysis activity towards α(2,3) linked substrates than α(2,6) linked substrates, while sialidases from ''Corynebacteriumm diphtheria'' and ''Micromonospora viridifaciens'' prefer to hydrolyze substrates with α(2,6) linkages <cite> Kim2011</cite>. One organism may produce sialidase isoenzymes with different substrate preferences. ''Pasteurella multocida'' produces two sialidases with different substrate preferences: NanH, an extracellular enzyme favouring α(2,3)-linked sialyllactose over α(2,6)-linked sialyllactose and NanB, a membrane bound enzyme that prefers α(2,6)-linked substrates over α(2,3)-linked substrates  <cite> Mizan2000</cite>. Similarly, membrane-bound NanA of ''Salmonella pneumoniae'' displays similar hydrolysis rates for sialyllactoses with α(2,3)-, α(2,6)- and α(2,8)-linkages whereas  extracellular NanB from the same organism prefers α(2,3) linkage over substrates with the other two linkage types <cite> Kim2011</cite>.
 == 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.
+GH33 sialidases and trans-sialidases hydrolyse, or transfer sialic acids to acceptor sugars, with retention of anomeric configuration <cite> Friebolin1980 Friebolin1981</cite>. The general mechanism is depicted [[Glycoside_hydrolases#Alternative_nucleophiles|here]]. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a covalent glycosyl-enzyme intermediate was observed on ''T. cruzi'' trans-sialidase(TcTS) by mass spectrometry using a fluorinated sialic acid analogue and the labelled amino acid residue identified as Tyr342 by peptide mapping. Subsequent crystal structures provided further insight into the mechanism of the protozoan sialidase <cite> Amaya2004 Watts2003</cite>. Kinetic analysis of TcTS confirmed the expected ping-pong double-displacement mechanism, and a covalent intermediate was demonstrated, without use of a fluorinated derivative, by use of mass spectrometry <cite> Damager2008</cite>. Subsequent structural studies of two strictly hydrolytic sialidases from ''T. rangelli'' <cite> Watts2006</cite> and ''C. perfringens'' <cite> Newstead2008</cite> also characterised their covalent intermediates.
+== Catalytic Residues ==
+===Nucleophile===
+The catalytic machinery is conserved throughout this family and, as shown by studies on TcTS, three key residues are involved: Glu230, Tyr342, Asp59. Since Watts first trapped the covalent 3F-sialyl enzyme intermediate on Tyr342 in 2003, many confirmatory structures have been solved. It is likely that tyrosine has evolved as the catalytic nucleophile, rather than the carboxylate moiety found in most retaining glycosidases, in order to minimize repulsive charge-charge interactions with the C1 carboxylate of the sialic acids. The neutral tyrosine can more readily approach the anomeric centre, and is rendered more nucleophilic by an invariant glutamate, Glu230 for TcTS, that serves as a base catalyst for deprotonation/reprotonation of the tyrosine hydroxyl. See Chan et al <cite> ChanBennet2012 </cite> for a detailed analysis of the role of this residue in the GH33 sialidase from ''M. viridifaciens''.
-== Catalytic Residues ==
+===Acid Base Catalyst===
-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>.
+The third mechanistically relevant conserved residue in the active site is Asp59, which serves as the acid/base catalyst, protonating the glycosidic oxygen as bond cleavage occurs. Following formation of the intermediate, Asp59 then serves as a base catalyst, activating the incoming water molecule (for simple hydrolytic pathways) or glycosyl acceptor (in the case of trans-sialidases) towards trans-glycosylation of the glycosyl-enzyme intermediate. This second attack leads to a double inversion at the anomeric center and a net retention of stereochemistry observed in the released product, Neu5Ac <cite> Amaya2004 Newstead2008 Damager2008 </cite>.
-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.
+All members of the sialidase superfamily, including the members of [[GH34]] and [[GH83]], display a 6 bladed beta-propeller sheet catalytic domain <cite> Amaya2004</cite>, which is accepted as the canonical neuraminidase fold.  The catalytic site structure is strictly conserved in all three families and contains an arginine triad which binds to the carboxylate in the C1 position of the sialic acid, a Tyr/Glu nucleophilic pair, and an aspartic acid that acts as the acid/base catalyst <cite> Buschiazzo2008</cite>.
+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 stereochemistry determination: First determined by proton NMR by Friebolin et al <cite>Friebolin1980 Friebolin1981</cite>.
-;First catalytic nucleophile identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>Sinnott1990</cite>.
+;First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the ''T. cruzi'' trans-sialidase by Watts et al through peptide mapping after labelling with 2,3-difluorosialic acid. <cite> Watts2003 </cite>.
-;First general acid/base residue identification: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>He1999</cite>.
+;First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al <cite> Crennell1993 </cite>.
-;First 3-D structure: Cite some reference here, with a ''short'' (1-2 sentence) explanation <cite>StickWilliams</cite>.
+;First 3-D structure: First determined for the ''Salmonella typhimurium'' enzyme by Crennell and Taylor <cite> Crennell1993 </cite>.
 == References ==
 <biblio>
@@ Line 66: / Line 68: @@
 #Chong1992 pmid=1628657
 #Burmeister1993 pmid=8069621
+#Moustafa2004 pmid=15226294
+#Watson2005 pmid=16206228
+#Gaskell1995 pmid=8591030
+#Friebolin1981 Friebolin, H. et al (1981) 1H-NMR Spectroscopic evidence for the release of N-acetyl-alpha-D-neuraminic acid as the first product of sialidase action. Biochem. Int. 3, 321-326.
+#Friebolin1980 pmid=6253376
+#Crennell1993 pmid=8234325
+#Tailford2015 pmid=26154892
+#Li1990 pmid=2254319
+#ChanBennet2012 pmid=22133027
+#WatsonBennet2004 pmid=15527797
 </biblio>
 [[Category:Glycoside Hydrolase Families|GH033]]