Abstract
Three-dimensional structures of NagZ of Bacillus subtilis, the first structures of a two-domain β-N-acetylglucosaminidase of family 3 of glycosidases, were determined with and without the transition state mimicking inhibitor PUGNAc bound to the active site, at 1.84- and 1.40-Å resolution, respectively. The structures together with kinetic analyses of mutants revealed an Asp-His dyad involved in catalysis: His(234) of BsNagZ acts as general acid/base catalyst and is hydrogen bonded by Asp(232) for proper function. Replacement of both His(234) and Asp(232) with glycine reduced the rate of hydrolysis of the fluorogenic substrate 4'-methylumbelliferyl N-acetyl-β-D-glucosaminide 1900- and 4500-fold, respectively, and rendered activity pH-independent in the alkaline range consistent with a role of these residues in acid/base catalysis. N-Acetylglucosaminyl enzyme intermediate accumulated in the H234G mutant and β-azide product was formed in the presence of sodium azide in both mutants. The Asp-His dyad is conserved within β-N-acetylglucosaminidases but otherwise absent in β-glycosidases of family 3, which instead carry a "classical" glutamate acid/base catalyst. The acid/base glutamate of Hordeum vulgare exoglucanase (Exo1) superimposes with His(234) of the dyad of BsNagZ and, in contrast to the latter, protrudes from a second domain of the enzyme into the active site. This is the first report of an Asp-His catalytic dyad involved in hydrolysis of glycosides resembling in function the Asp-His-Ser triad of serine proteases. Our findings will facilitate the development of mechanism-based inhibitors that selectively target family 3 β-N-acetylglucosaminidases, which are involved in bacterial cell wall turnover, spore germination, and induction of β-lactamase.