title: Structural and kinetic analysis of Bacillus subtilis N-acetylglucosaminidase reveals a unique Asp-His dyad mechanism.
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.
authors.affiliation: Department of Molecular Microbiology, University of Konstanz, 78457 Konstanz, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Litzinger
authors.forename: Stefanie
authors.initials: S
authors.lastname: Fischer
authors.forename: Patrick
authors.initials: P
authors.lastname: Polzer
authors.forename: Kay
authors.initials: K
authors.lastname: Diederichs
authors.forename: Wolfram
authors.initials: W
authors.lastname: Welte
authors.forename: Christoph
authors.initials: C
authors.lastname: Mayer
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D019810:Sodium Azide / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D001224:Aspartic Acid; D006639:Histidine; D019810:Sodium Azide; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine
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