Glycoside Hydrolase Family 33

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Author: ^^^Junho Lee^^^
Responsible Curator: ^^^Steve Withers^^^

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

Nucleophile

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: First determined by proton NMR by Friebolin et al Biochem. Int. (1981) 3, 321-326.
First catalytic nucleophile identification: NMR structures of Crennell and others were strongly suggestive, but mechanism was not clear. First definitively shown for the T. cruzi trans-sialidase by Watts et al through paptide mapping after labelling with 2,3-difluorosialic acid. Watts et al J. Am. Chem. Soc. (2003) 125, 7532-7533.
First general acid/base residue identification: Identified by X-ray crystallography by Crennell et al Proc. Natl. Acad. Sci. USA (1993) 90, 9852.
First 3-D structure: First determined for the Salmonella typhimurium enzyme by Crennell and Taylor Proc. Natl. Acad. Sci. USA (1993) 90, 9852.

References

All Medline abstracts: PubMed

Friebolin 1981 Friebolin, H. et al Biochem. Int. (1981) 3, 321-326.