Glycoside Hydrolase Family 34

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

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Glycoside Hydrolase Family GH34
Clan	GH-E
Mechanism	retaining
Active site residues	Tyr/Glu
CAZy DB link
http://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 a-2,3 or a-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.

This is an example of how to make references to a journal article [1]. (See the References section below). Multiple references can go in the same place like this [1, 2]. You can even cite books using just the ISBN [3]. References that are not in PubMed can be typed in by hand [4].

Kinetics and Mechanism

Sialidases of the GH34 family have been shown to cleave sialic acid residues from host polysaccharides with retention of anomeric stereochemistry (REF). The mechanism of this hydrolysis has been extensively studied, and for years was thought to occur through a sialosyl cation intermediate (von Itzstein). However, trapping of the covalent enzyme intermediate of the viral neuraminidase using 3-fluoro-glycosylfluorides (Kim 2013) 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 (Withers 2013, REF). 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. This role had been discussed previously (Varghese Proteins 1992) though not favoured. In the same paper Asp151 is 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 1987 Biochemistry, on which basis they suggested Glu276, though subsequent structures showed this residue interacting 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 Ghate and Air (Eur J Biochem 1998). 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. 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. 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 (Bovin ActaNature 2009). 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 (von Itzstein 2007). 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 (Kim et al; Vavricka et al Gao 2013).

Family Firsts

First stereochemistry determination

Determined for influenza NA by NMR by the von Itzstein group Eur. J. Biochem. (1992) 207, 335-343. [1].

First catalytic nucleophile identification

Implied by X-ray structures, though intermediate thought to be a carbocation. Determined definitively using a 2,3-difluorosialic acid in the Withers group Kim et al Science (2013) 340, 71-75. [4].

First general acid/base residue identification

Inferred from X-ray structure below [2].

First 3-D structure

Influenza neuraminidase determined by Colman group: Varghese et al (1983) Nature 303, 35-40. [3].

References

1. Comfort DA, Bobrov KS, Ivanen DR, Shabalin KA, Harris JM, Kulminskaya AA, Brumer H, and Kelly RM. (2007). Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases. Biochemistry. 2007;46(11):3319-30. DOI:10.1021/bi061521n |
2. He S and Withers SG. (1997). Assignment of sweet almond beta-glucosidase as a family 1 glycosidase and identification of its active site nucleophile. J Biol Chem. 1997;272(40):24864-7. DOI:10.1074/jbc.272.40.24864 |
3. Robert V. Stick and Spencer J. Williams. (2009) Carbohydrates. Elsevier Science.

4. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006