Glycoside Hydrolase Family 98

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• Author: ^^^Fathima Shaikh^^^
• Responsible Curator: ^^^Al Boraston^^^

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Glycoside Hydrolase Family GH98
Clan	Not assigned
Mechanism	Inverting
Active site residues	Known
CAZy DB link
http://www.cazy.org/fam/GH98.html

Substrate specificities

The glycoside hydrolases of this family are endo-β-galactosidases. No other activities have been reported. Family 98 glycoside hydrolases are unique in their specificity towards cleavage of ABO blood group A and B trisaccharides from glycoconjugates [1, 2, 3]. The presence of an L-fucose residue on the substrate is essential for activity of some members of this family (EABase, Sp4GH98) [1, 2, 3]. The crystal structure of Sp3GH98 suggests that the L-fucose residue is not required as a specificity determinant in this enzyme [2].

Kinetics and Mechanism

Family GH98 galactosidases are inverting enzymes, as first shown by NMR monitoring of the Sp4GH98-catalyzed hydrolysis of the Lewis^Y tetrasaccharide [2]. EABase was also shown to act through an inverting enzyme by NMR monitoring of the EABase catalysed hydrolysis of an artificial substrate, DNP-A-trisaccharide [3]. These results are contrary to the initial predictions made by Rigden [4]. EABase follows normal Michaelis-Menten kinetics [3].

Catalytic Residues

The catalytic base, an aspartate and glutamate diad, and the catalytic acid, glutamate, were identified through the crystal structures of Sp3GH98 and Sp4GH98 in complex with the A trisaccharide and H disaccharide, respectively, and confirmed by site-directed mutagenesis [2]. Similar results were obtained through kinetic studies of the corresponding acid and base mutants of EABase with the artificial substrate DNP-A-trisaccharide. Further biochemical evidence for the catalytic acid residue was obtained by comparison between the activity of the acid mutant and the pK_a of the leaving group of the substrate [3].

Three-dimensional structures

The first crystal structures from family 98 were the Sp3GH98 and Sp4GH98 enzymes from S. pneumoniae in complex with the A trisaccharide and H disaccharide respectively [2]. Both structures feature a (α/β)₈ barrel in the catalytic domain [2]

Family Firsts

First sterochemistry determination

Streptococcus pneumoniae TIGR4 endo-beta-galactosidase Sp4GH98 by NMR [2].

First catalytic base identification

Streptococcus pneumoniae SP3-BS71 endo-beta-galactosidase Sp3GH98 [2].

Streptococcus pneumoniae TIGR4 endo-beta-galactosidase Sp4GH98 [2].

First catalytic acid identification

Streptococcus pneumoniae SP3-BS71 endo-beta-galactosidase Sp3GH98 [2].

Streptococcus pneumoniae TIGR4 endo-beta-galactosidase Sp4GH98 [2].

First 3-D structures

Streptococcus pneumoniae SP3-BS71 endo-beta-galactosidase Sp3GH98 [2].

Streptococcus pneumoniae TIGR4 endo-beta-galactosidase Sp4GH98 [2].

References

1. Anderson KM, Ashida H, Maskos K, Dell A, Li SC, and Li YT. (2005). A clostridial endo-beta-galactosidase that cleaves both blood group A and B glycotopes: the first member of a new glycoside hydrolase family, GH98. J Biol Chem. 2005;280(9):7720-8. DOI:10.1074/jbc.M414099200 |
2. Higgins MA, Whitworth GE, El Warry N, Randriantsoa M, Samain E, Burke RD, Vocadlo DJ, and Boraston AB. (2009). Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases. J Biol Chem. 2009;284(38):26161-73. DOI:10.1074/jbc.M109.024067 |
3. Shaikh FA, Randriantsoa M, and Withers SG. (2009). Mechanistic analysis of the blood group antigen-cleaving endo-beta-galactosidase from Clostridium perfringens. Biochemistry. 2009;48(35):8396-404. DOI:10.1021/bi900991h |
4. Rigden DJ (2005). Analysis of glycoside hydrolase family 98: catalytic machinery, mechanism and a novel putative carbohydrate binding module. FEBS Lett. 2005;579(25):5466-72. DOI:10.1016/j.febslet.2005.09.011 |