Glycoside Hydrolase Family 97

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• Author: ^^^Tracey Gloster^^^
• Responsible Curator: ^^^Gideon Davies^^^

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Glycoside Hydrolase Family GH97
Clan	Not assigned
Mechanism	Retaining and Inverting
Active site residues	Inferred
CAZy DB link
http://www.cazy.org/GH97.html

Substrate specificities

Family 97 glycoside hydrolases hydrolyse α-linked D-glycosides; the two enzymes from this family that have been characterised to date have α-glucosidase (EC 3.2.1.20) and α-galactosidase (EC 3.2.1.22) activity [1]. The α-glucosidase from Bacteroides thetaiotaomicron has been characterised in the most detail, and has been demonstrated to hydrolyse substrates ranging from maltose to maltoheptaose in length, and those containing α-1,6-, α-1,3- and α-1,2-, as well as α-1,4-linkages [2, 3].

Kinetics and Mechanism

Family GH97 is unusual as it contains both retaining and inverting enzymes, as shown unequivocally by NMR [1] and HPLC [3] analyses, by characterization of two enzymes from Bacteroides thetaiotaomicron. Catalysis is dependent on the presence of calcium, which coordinates the C2-OH group of the substrate in the -1 subsite, as well as four glutamate residues in the active site [1]. One of the glutamate residues coordinated by the calcium ion is predicted to be the general acid/base residue, which may receive acid assistance from the calcium during hydrolysis.

Catalytic Residues

The constellation of glutamate residues (five in total, four of which coordinate the calcium ion) in the active site of the only (inverting) GH97 enzyme whose structure has been solved to date, has made the assignment of catalytic residues difficult. Analysis of sequence alignments, structural alignments with family GH27, and a mutant tested with substrates with different leaving group abilities, has provided a likely candidate for the general acid residue in this inverting enzyme or as the general acid/base in the retaining members of the family [1]. It is possible that the calcium ion provides acid assistance to the acid/base during hydrolysis. The general base for the inverting enzyme has also been predicted on the basis of its position in the active site; in addition it coordinates a water molecule that is situated in a position primed for nucleophilic attack, and mutation causes the enzyme to be virtually inactive. Although a structure has not been solved for a retaining enzyme, the catalytic nucleophile for the retaining enzymes has been predicted based on sequence alignments, and similarity to GH27 enzymes, to be an aspartate residue [1].

Three-dimensional structures

There has been one structure solved, using X-ray crystallography, for a member of family GH97 (which inverts stereochemistry). This is an enzyme from Bacteroides thetaiotaomicron, SusB, which is involved in the degradation of starch in the human gut. The tertiary structure of the GH97 enzyme revealed three domains; an N-terminal β-super-sandwich domain, followed by a canonical (β/α)8 barrel (which houses the catalytic domain) and a C-terminal β-sheet domain [1, 3]. There have also been complexes solved with the inhibitors acarbose [3], deoxynojirimycin and castanospermine [1]. Structural alignments show similarity to families GH27 and GH36 (as predicted previously by a bioinformatics study [4]).

Family Firsts

First sterochemistry determination

Two GH97 members from Bacteroides thetaiotaomicron were shown to differ in stereochemical outcome, by NMR [1] and HPLC [3] analyses, demonstrating that the family contains enzymes that hydrolyse with both retention and inversion of anomeric stereochemistry.

First catalytic nucleophile identification

Not proven unequivocally (although has been predicted using structure and sequence alignments, see [1]).

First general acid/base residue identification

Not proven unequivocally (although has been predicted using structure and sequence alignments, see [1]).

First 3-D structure

A GH97 member from Bacteroides thetaiotaomicron, SusB, was solved using X-ray crystallography by two groups [1, 3].

References

1. Gloster TM, Turkenburg JP, Potts JR, Henrissat B, and Davies GJ. (2008). Divergence of catalytic mechanism within a glycosidase family provides insight into evolution of carbohydrate metabolism by human gut flora. Chem Biol. 2008;15(10):1058-67. DOI:10.1016/j.chembiol.2008.09.005 |
2. Smith KA and Salyers AA. (1991). Characterization of a neopullulanase and an alpha-glucosidase from Bacteroides thetaiotaomicron 95-1. J Bacteriol. 1991;173(9):2962-8. DOI:10.1128/jb.173.9.2962-2968.1991 |
3. Kitamura M, Okuyama M, Tanzawa F, Mori H, Kitago Y, Watanabe N, Kimura A, Tanaka I, and Yao M. (2008). Structural and functional analysis of a glycoside hydrolase family 97 enzyme from Bacteroides thetaiotaomicron. J Biol Chem. 2008;283(52):36328-37. DOI:10.1074/jbc.M806115200 |
4. Naumoff DG (2005). GH97 is a new family of glycoside hydrolases, which is related to the alpha-galactosidase superfamily. BMC Genomics. 2005;6:112. DOI:10.1186/1471-2164-6-112 |