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.
Highlights
Double displacement mechanism [1, 2]
We are studying a group of bacterial -N-acetylglucosaminidases, which hydrolyze the other glycosidic linkage in peptidoglycan, between GlcNAc and MurNAc, and are involved in turnover and recycling of the bacterial cell wall [5,6,7,8,9,10]
We present the structure of NagZ of Bacillus subtilis (BsNagZ), the first structure of a two-domain -N-acetylglucosaminidase, along with kinetic analyses, which provide evidence nucleophile by trapping the glycosyl enzyme intermediate for participation of the side chains using slow substrates, proteolytic digestion, and subsequent of the conserved Asp and His residues during catalysis
Summary
Reside on the N-terminal domain of BsNagZ are directly involved in the mechanism of the -N-acetylglucosaminidases subfamily of family 3 glycosidases. We present the structure of NagZ of Bacillus subtilis (BsNagZ), the first structure of a two-domain -N-acetylglucosaminidase, along with kinetic analyses, which provide evidence nucleophile by trapping the glycosyl enzyme intermediate for participation of the side chains using slow substrates, proteolytic digestion, and subsequent of the conserved Asp and His residues during catalysis. Results indicate that the histidine, instead of a glutamate, acts as for some -glucosidases of family 3, good evidence is provided acid/base catalyst, which undergoes hydrogen bonding with the by structural or kinetic analyses for a glutamate acting as the aspartate residue, thereby forming a catalytic dyad that protoacid/base catalyst, e.g. exo--glucanase of Hordeum vulgare nates the glycosidic oxygen in the first (glycosylation) step and (14 –16), -glucosidase of Flavobacterium meningosepticum deprotonates and activates water for nucleophilic attack of the [17, 18], and the -glucosylceramidase of Paenibacillus sp. Residues Asp232 and His234 (shown in orange) are in H-bond distance to each other and His234, acid/base catalyst, as well as Asp318, the catalytic nucleophile (shown in orange), are H-bonding the PUGNAc inhibitor
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