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Mechanistic studies of family 4 glycosidases GlvA and BGlu Li, Yunsong


The glycosidases are a group of enzymes that can hydrolyze the O-glycosidic bond between two or more carbohydrates or between a carbohydrate moiety and a noncarbohydrate moiety. Based on their substrate specificities, this group of enzymes was classified into the enzyme classification scheme as EC 3.2.1.-. In an alternative enzyme scheme based on sequence similarity, the glycosidases have been assigned to 87 families at the most recent count. The family 4 glycoside hydrolases is a unique family, not only because the enzymes in this family all require NAD and divalent metal ion as cofactors, but also because this family contains a wide range of substrate specificities. As a consequence the catalytic mechanisms and different substrate specificities are of considerable interest. Two enzymes in this family, GlvA from Bacillus subtilis 168 and Bglu from Bacillus halodurans are the focus of this study. The substrates of GlvA are 6-phospho-α-glucosides and the substrates of Bglu are 6-phospho-β-glucosides. NMR analysis of the cleavage of 4'-nitrophenyl (β-D-glucopyranoside-6-phosphate by Bglu in the presence of 3 M methanol revealed that the product formed is the methyl-β-glucoside; indicating that Bglu is a retaining glycosidase. This is the first determination of stereochemical outcome for an enzyme in family 4. Hydrolysis of 4'-nitrophenyl β-D-glucopyranoside-6-phosphate by Bglu in deuterium oxide exclusively yields [2-²H]-glucose-6-phosphate, thus the hydrogen at the 2 position is substituted by deuterium during cleavage. Further, reaction of Bglu with 2- deoxy-2-fluoro-6-phospho-β-D-glucosyl fluoride did not result in trapping of a glycosylenzyme intermediate. Based on these results, a novel mechanism was proposed. In order to further study the proposed mechanism, some isotopically labeled substrates for GlvA were synthesized for kinetic isotope effect (KIE) measurements, along with samples of the natural substrate, maltose-6'-phosphate.

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