Glycoside Hydrolase Family 33

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• Author: ^^^Tom Wennekes^^^
• Responsible Curator: ^^^Steve Withers^^^

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Glycoside Hydrolase Family GH33
Clan	GH-x
Mechanism	retaining/inverting
Active site residues	known/not known
CAZy DB link
https://www.cazy.org/GH33.html

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. 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].

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 cite> Mizan2000. 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. Considerable debate had occurred over whether an ionic or covalent intermediate was formed. However, a glycosyl-enzyme intermediate was observed on T. cruzitrans-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, 7]. 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 [8]. Subsequent structural studies of two strictly hydrolytic sialidases from T. rangelli [9] and C. perfringens [10] 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 [7]. 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, 10]. 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 [11].

Three-dimensional structures

Content is to be added here.

Family Firsts

First stereochemistry determination

Cite some reference here, with a short (1-2 sentence) explanation [12].

First catalytic nucleophile identification

Cite some reference here, with a short (1-2 sentence) explanation [13].

First general acid/base residue identification

Cite some reference here, with a short (1-2 sentence) explanation [14].

First 3-D structure

Cite some reference here, with a short (1-2 sentence) explanation [15].

References

1. Varki A (1997). Sialic acids as ligands in recognition phenomena. FASEB J. 1997;11(4):248-55. DOI:10.1096/fasebj.11.4.9068613 |
2. Kim S, Oh DB, Kang HA, and Kwon O. (2011). Features and applications of bacterial sialidases. Appl Microbiol Biotechnol. 2011;91(1):1-15. DOI:10.1007/s00253-011-3307-2 |
3. Varki A (2007). Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature. 2007;446(7139):1023-9. DOI:10.1038/nature05816 |
4. Vimr ER, Kalivoda KA, Deszo EL, and Steenbergen SM. (2004). Diversity of microbial sialic acid metabolism. Microbiol Mol Biol Rev. 2004;68(1):132-53. DOI:10.1128/MMBR.68.1.132-153.2004 |
5. Buschiazzo A and Alzari PM. (2008). Structural insights into sialic acid enzymology. Curr Opin Chem Biol. 2008;12(5):565-72. DOI:10.1016/j.cbpa.2008.06.017 |
6. Amaya MF, Watts AG, Damager I, Wehenkel A, Nguyen T, Buschiazzo A, Paris G, Frasch AC, Withers SG, and Alzari PM. (2004). Structural insights into the catalytic mechanism of Trypanosoma cruzi trans-sialidase. Structure. 2004;12(5):775-84. DOI:10.1016/j.str.2004.02.036 |
7. 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. 2003;125(25):7532-3. DOI:10.1021/ja0344967 |
8. 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. 2008;47(11):3507-12. DOI:10.1021/bi7024832 |
10. 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. 2008;283(14):9080-8. DOI:10.1074/jbc.M710247200 |
16. 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. 2000;182(24):6874-83. DOI:10.1128/JB.182.24.6874-6883.2000 |
17. 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. 2006;281(7):4149-55. DOI:10.1074/jbc.M510677200 |