Difference between revisions of "Glycoside Hydrolase Family 34"

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Author: ^^^Kyle Robinson^^^
Responsible Curator: ^^^Steve Withers^^^

Glycoside Hydrolase Family GH34
Clan	GH-E
Mechanism	retaining
Active site residues	Tyr/Glu and Asp
CAZy DB link
https://www.cazy.org/GH34.html

Substrate specificities

All enzymes in GH34 are Neuraminidases (also known as sialidases; EC. 3.2.1.18) that are found, along with the sialic acid-binding protein Hemagglutinin, on the surface of influenza viruses that are pathogenic to mammalian or avian species. These are the H and N of H1N1. Viruses bind to the cell surface and enter via interaction of the hemagglutinin with cell surface sialic acids. The function of the neuraminidase is to cleave sialic acid from the cell surface after budding of progeny virus, to assist viral spread to other cells. GH34 neuraminidases have therefore been major drug design targets, and are very effectively inhibited by the drugs Tamiflu (Oseltamivir) and Relenza (Zanamivir).

The cell surface sialic acids they cleave are linked α(2,3) or α(2,6) to galactose or N-acetyl galactosamine residues that terminate glycolipid or glycoprotein structures. Sialic acids are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them are found in families GH33, GH34, GH83 and, along with GH93 arabinanases constitute Clan GH-E.

Kinetics and Mechanism

Sialidases of the GH34 family have been shown to cleave sialic acid residues with retention of anomeric stereochemistry [1]. The mechanism of this hydrolysis has been extensively studied, and for years was thought to occur through a sialosyl cation intermediate. However, trapping of the covalent enzyme intermediate of the viral neuraminidase using 3-fluoro-glycosylfluorides [2] confirmed a covalent structure. Therefore, the mechanism of these neuraminidases is a classic Koshland type double-displacement involving a covalent sialyl-enzyme intermediate on a Tyrosine residue. This is followed by base-catalysed attack of water on the sialyl enzyme, releasing Neu5Ac with net retention of anomeric stereochemistry.

Catalytic Residues

The catalytic machinery of this family of viral sialidases includes two key residues: an acid/base glutamate residue and a catalytic tyrosine nucleophile. The catalytic nucleophile of the Influenza A neuraminidase was identified as Y406 by use of 3-fluoro-sialosyl fluorides to trap the covalent intermediate, followed by peptide mapping [2]. An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue E277 to be appropriately positioned to act as the general acid/base pair for activation of Y406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex [2, 3]. This role had been discussed previously [4] though not favoured. In the same paper Asp151 was considered as a candidate for the acid/base catalyst based upon its interaction with C2OH in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of mutants by Lentz et al [5], on which basis they suggested Glu276, though subsequent structures showed that this residue in fact interacts with OH8 and OH9. Further mutant analysis failed to identify a candidate, but their use of an acetate buffer (hence possible rescue) render interpretation challenging [6]. More recently Zhu and Wilson investigated why mutations to D151 allowed the mutant NA to bind tightly to Red Blood Cells by kinetic and structural analysis of mutants [7]. Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports a role as acid/base catalyst for D151 since this is the classical kinetic signature of acid/base mutants. The lowered Km arises from selective slowing of the second step as explained in the CAZy Lexicon under General Acid/Base.

Three-dimensional structures

These are members of Clan GH-E, along with GH33, 83 and 93. Three dimensional crystallographic structures have been described for neuraminidases of all subtypes of Influenza A (N1-N10) and B (see Structures section of CAZy for PDBs). These structures consist of cytoplasmic, transmembrane, ‘head’ and ‘stem’ domains. Homotetramers of these enzymes form mushroom-like structures on the surface of the virus [8]. Each of these enzymes displays the sialidase 6-blade beta-propeller fold in the catalytic ‘head’ domain, as well as a calcium-binding domain common to this class of glycoside hydrolase. The viral neuraminidases have eight highly conserved residues in the active site that form key stabilizing interactions (hydrogen bonding, hydrophobic interactions, charge-charge interactions) with the bound substrates, and an additional ten conserved residues that are thought to be key structural factors for these enzymes [9]. An arginine triad interacts with the carboxylate residue of active site-bound sialic acid. Nearby is the nucleophilic tyrosine residue Tyr406 and its partner glutamate Glu277, which serves as an acid/base for deprotonation/reprotonation of Tyr406 during turnover. Also nearby is the probable acid/base Asp151 for protonation of the glycosidic oxygen and deprotonation of incoming water. Structures of trapped 3-fluorosialosyl enzyme intermediates are available ([2, 3].

Family Firsts

First stereochemistry determination: Determined for influenza NA by NMR by the von Itzstein group [1].
First catalytic nucleophile identification: Implied by X-ray structures, although intermediate was thought to be a carbocation. Determined definitively using a 2,3-difluorosialic acid in the Withers group [2] and by Vavricka [3].
First general acid/base residue identification: Inferred from X-ray structure below [10].
First 3-D structure: Influenza neuraminidase determined by Colman group [10].

References

All Medline abstracts: PubMed

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	The cell surface sialic acids they cleave are linked α(2,3) or α(2,6) to galactose or N-acetyl galactosamine residues that terminate glycolipid or glycoprotein structures. Sialic acids are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them are found in families GH33, GH34, GH83 and, along with GH93 arabinanases constitute Clan GH-E.		The cell surface sialic acids they cleave are linked α(2,3) or α(2,6) to galactose or N-acetyl galactosamine residues that terminate glycolipid or glycoprotein structures. Sialic acids are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them are found in families GH33, GH34, GH83 and, along with GH93 arabinanases constitute Clan GH-E.
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	== Kinetics and Mechanism ==		== Kinetics and Mechanism ==