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Glycoside Hydrolase Family 34
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|Glycoside Hydrolase Family GH34|
|Active site residues||Tyr/Glu and Asp|
|CAZy DB link|
All enzymes in GH34 are Neuraminidases (also known as sialidases; EC. 22.214.171.124) 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 (Neu5Ac, NANA, NeuNAc, NeuNA) are nine-carbon monosaccharides that bear a carboxylate residue at the C-1 position. Enzymes that cleave them in an exo- or endo- acting fashion are found in families GH33, GH34, and GH83 or GH53 respectively.
Kinetics and Mechanism
Sialidases of the GH34 family have been shown to cleave sialic acid residues with retention of anomeric stereochemistry . 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-fluorosialosyl fluorides  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 intermediate, releasing Neu5Ac with net retention of anomeric stereochemistry.
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 Tyr406 by use of 3-fluorosialosyl fluorides to trap the covalent intermediate, followed by peptide mapping . An X-ray crystal structure confirmed the identity and covalency and shows carboxylate residue Glu277 to be appropriately positioned to act as the general acid/base pair for activation of Tyr406 during glycosylation and deglycosylation of the covalent enzyme intermediate complex [2, 3]. This role had been discussed previously  though not favoured. In the same report Asp151 was presented as a candidate for the acid/base catalyst based upon its interaction with OH2 in the complex with sialic acid. However, enthusiasm was tempered by the previous kinetic analysis of a series of enzyme mutants by Lentz et al , on which basis they suggested Glu276 to be directly involved in catalysis - though subsequent structures instead showed that the glutamate residue in fact interacts with OH8 and OH9 of Neu5Ac. Further mutant analysis failed to identify a more suitable candidate, but their use of an acetate buffer (hence enabling possible rescue) rendered interpretation challenging . More recently Zhu and Wilson investigated why mutations to Asp151 allowed the mutant neuraminidase to bind tightly to red blood cells by kinetic and structural analysis of enzyme mutants . Their demonstration that both kcat and Km are reduced substantially for cleavage of aryl sialoside substrates by these mutants strongly supports the role of acid/base catalyst for Asp151 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.
These viral neuraminidases are members of Clan GH-E, along with families GH33, GH83 and GH93. 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 . 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 . 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].
- First stereochemistry determination
- Determined for influenza neuraminidase by NMR by the von Itzstein group .
- 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  and by Vavricka .
- First general acid/base residue identification
- Inferred from X-ray structure below .
- First 3-D structure
- Influenza neuraminidase determined by Colman group .
- 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. 1992;207(1):335-43. DOI:10.1111/j.1432-1033.1992.tb17055.x |
- Kim JH, Resende R, Wennekes T, Chen HM, Bance N, Buchini S, Watts AG, Pilling P, Streltsov VA, Petric M, Liggins R, Barrett S, McKimm-Breschkin JL, Niikura M, and Withers SG. (2013). Mechanism-based covalent neuraminidase inhibitors with broad-spectrum influenza antiviral activity. Science. 2013;340(6128):71-5. DOI:10.1126/science.1232552 |
- Vavricka CJ, Liu Y, Kiyota H, Sriwilaijaroen N, Qi J, Tanaka K, Wu Y, Li Q, Li Y, Yan J, Suzuki Y, and Gao GF. (2013). Influenza neuraminidase operates via a nucleophilic mechanism and can be targeted by covalent inhibitors. Nat Commun. 2013;4:1491. DOI:10.1038/ncomms2487 |
- Varghese JN, McKimm-Breschkin JL, Caldwell JB, Kortt AA, and Colman PM. (1992). The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor. Proteins. 1992;14(3):327-32. DOI:10.1002/prot.340140302 |
- Lentz MR, Webster RG, and Air GM. (1987). Site-directed mutation of the active site of influenza neuraminidase and implications for the catalytic mechanism. Biochemistry. 1987;26(17):5351-8. DOI:10.1021/bi00391a020 |
- Ghate AA and Air GM. (1998). Site-directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design. Eur J Biochem. 1998;258(2):320-31. DOI:10.1046/j.1432-1327.1998.2580320.x |
- Zhu X, McBride R, Nycholat CM, Yu W, Paulson JC, and Wilson IA. (2012). Influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic Acid receptors. J Virol. 2012;86(24):13371-83. DOI:10.1128/JVI.01426-12 |
- Shtyrya YA, Mochalova LV, and Bovin NV. (2009). Influenza virus neuraminidase: structure and function. Acta Naturae. 2009;1(2):26-32. | Google Books | Open Library
- von Itzstein M (2007). The war against influenza: discovery and development of sialidase inhibitors. Nat Rev Drug Discov. 2007;6(12):967-74. DOI:10.1038/nrd2400 |
- Varghese JN, Laver WG, and Colman PM. (1983). Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 A resolution. Nature. 1983;303(5912):35-40. DOI:10.1038/303035a0 |