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Glycoside Hydrolase Family 33

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Glycoside Hydrolase Family GH33
Clan GH-E
Mechanism Retaining
Active site residues Known
CAZy DB link

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 [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 acids [2, 3, 4]. Sialic acids are hydrolyzed by sialidases (E.C., 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].

GH33 includes most bacterial and simple eukaryotic sialidases and trans-sialidases [6]. 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 [7]. 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

Sialidases and trans-sialidases hydrolyse or transfer sialic acids with retention of the anomeric configuration. The general mechanism is depicted 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 [6, 8]. 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 [9]. Subsequent structural studies of two strictly hydrolytic sialidases from T. rangelli [10] and C. perfringens [11] 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 [6]. Peptide mapping of T. cruzi with the fluorinated sialic acid identified the catalytic nucleophile as Tyr342 [8]. 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 [6, 11].

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 [6]:

"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 [12, 13]. 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."

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. 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 [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 [14]. 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 [15, 16].

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.


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  2. Kim S, Oh DB, Kang HA, and Kwon O. (2011) Features and applications of bacterial sialidases. Appl Microbiol Biotechnol. 91, 1-15. DOI:10.1007/s00253-011-3307-2 | PubMed ID:21544654 | HubMed [Kim2011]
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  7. Mizan S, Henk A, Stallings A, Maier M, and Lee MD. (2000) Cloning and characterization of sialidases with 2-6' and 2-3' sialyl lactose specificity from Pasteurella multocida. J Bacteriol. 182, 6874-83. DOI:10.1128/jb.182.24.6874-6883.2000 | PubMed ID:11092845 | HubMed [Mizan2000]
  8. Watts AG, Damager I, Amaya ML, Buschiazzo A, Alzari P, Frasch AC, and Withers SG. (2003) Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: tyrosine is the catalytic nucleophile. J Am Chem Soc. 125, 7532-3. DOI:10.1021/ja0344967 | PubMed ID:12812490 | HubMed [Watts2003]
  9. Damager I, Buchini S, Amaya MF, Buschiazzo A, Alzari P, Frasch AC, Watts A, and Withers SG. (2008) Kinetic and mechanistic analysis of Trypanosoma cruzi trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis. Biochemistry. 47, 3507-12. DOI:10.1021/bi7024832 | PubMed ID:18284211 | HubMed [Damager2008]
  10. Watts AG, Oppezzo P, Withers SG, Alzari PM, and Buschiazzo A. (2006) Structural and kinetic analysis of two covalent sialosyl-enzyme intermediates on Trypanosoma rangeli sialidase. J Biol Chem. 281, 4149-55. DOI:10.1074/jbc.M510677200 | PubMed ID:16298994 | HubMed [Watts2006]
  11. Newstead SL, Potter JA, Wilson JC, Xu G, Chien CH, Watts AG, Withers SG, and Taylor GL. (2008) The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates. J Biol Chem. 283, 9080-8. DOI:10.1074/jbc.M710247200 | PubMed ID:18218621 | HubMed [Newstead2008]
  12. Chong AK, Pegg MS, Taylor NR, and von Itzstein M. (1992) Evidence for a sialosyl cation transition-state complex in the reaction of sialidase from influenza virus. Eur J Biochem. 207, 335-43. DOI:10.1111/j.1432-1033.1992.tb17055.x | PubMed ID:1628657 | HubMed [Chong1992]
  13. Burmeister WP, Henrissat B, Bosso C, Cusack S, and Ruigrok RW. (1993) Influenza B virus neuraminidase can synthesize its own inhibitor. Structure. 1, 19-26. DOI:10.1016/0969-2126(93)90005-2 | PubMed ID:8069621 | HubMed [Burmeister1993]
  14. Moustafa I, Connaris H, Taylor M, Zaitsev V, Wilson JC, Kiefel MJ, von Itzstein M, and Taylor G. (2004) Sialic acid recognition by Vibrio cholerae neuraminidase. J Biol Chem. 279, 40819-26. DOI:10.1074/jbc.M404965200 | PubMed ID:15226294 | HubMed [Moustafa2004]
  15. Gaskell A, Crennell S, and Taylor G. (1995) The three domains of a bacterial sialidase: a beta-propeller, an immunoglobulin module and a galactose-binding jelly-roll. Structure. 3, 1197-205. DOI:10.1016/s0969-2126(01)00255-6 | PubMed ID:8591030 | HubMed [Gaskell1995]
  16. Watson JN, Newstead S, Narine AA, Taylor G, and Bennet AJ. (2005) Two nucleophilic mutants of the Micromonospora viridifaciens sialidase operate with retention of configuration by two different mechanisms. Chembiochem. 6, 1999-2004. DOI:10.1002/cbic.200500114 | PubMed ID:16206228 | HubMed [Watson2005]
All Medline abstracts: PubMed | HubMed