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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 ?-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-? 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-?-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 ?-azide product was formed in the presence of sodium azide in both mutants. The Asp-His dyad is conserved within ?-N-acetylglucosaminidases but otherwise absent in ?-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 ?-N-acetylglucosaminidases, which are involved in bacterial cell wall turnover, spore germination, and induction of ?-lactamase.

SUBMITTER: Litzinger S

PROVIDER: S-EPMC2975192 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Structural and kinetic analysis of Bacillus subtilis N-acetylglucosaminidase reveals a unique Asp-His dyad mechanism.

Litzinger Silke S Fischer Stefanie S Polzer Patrick P Diederichs Kay K Welte Wolfram W Mayer Christoph C

The Journal of biological chemistry 20100907 46

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) fo ...[more]

PMID: 20826810

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Project description:The side chains of histidine and aspartate residues form a hydrogen bond in the active sites of many enzymes. In serine proteases, the His...Asp hydrogen bond of the catalytic triad is known to contribute greatly to catalysis, perhaps via the formation of a low-barrier hydrogen bond. In bovine pancreatic ribonuclease A (RNase A), the His...Asp dyad is composed of His119 and Asp121. Previously, site-directed mutagenesis was used to show that His119 has a fundamental role, to act as an acid during catalysis of RNA cleavage [Thompson, J. E., and Raines, R. T. (1994) J. Am. Chem. Soc. 116, 5467-5468]. Here, Asp121 was replaced with an asparagine or alanine residue. The crystalline structures of the two variants were determined by X-ray diffraction analysis to a resolution of 1.6 A with an R-factor of 0.18. Replacing Asp121 with an asparagine or alanine residue does not perturb the overall conformation of the enzyme. In the structure of D121N RNase A, Ndelta rather than Odelta of Asn121 faces His119. This alignment in the crystalline state is unlikely to exist in solution because catalysis by the D121N variant is not compromised severely. The steady-state kinetic parameters for catalysis by the wild-type and variant enzymes were determined for the cleavage of uridylyl(3'-->5')adenosine and poly(cytidylic acid), and for the hydrolysis of uridine 2',3'-cyclic phosphate. Replacing Asp121 decreases the values of kcat/Km and kcat for cleavage by 10-fold (D121N) and 10(2)-fold (D121A). Replacing Asp121 also decreases the values of kcat/Km and kcat for hydrolysis by 10(0. 5)-fold (D121N) and 10-fold (D121A) but has no other effect on the pH-rate profiles for hydrolysis. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. Apparently, the major role of Asp121 is to orient the proper tautomer of His119 for catalysis. Thus, the mere presence of a His...Asp dyad in an enzymic active site is not a mandate for its being crucial in effecting catalysis.

Project description:Various microbial biomasses have been employed as biosorbents. Bacterial biomass has added advantages because of easy in production at a low cost. The study investigated the biosorption of iron from aqueous solutions by Bacillus subtilis. An optimum biosorption capacity of 7.25 mg of the metal per gram of the biosorbent was obtained by the Inductive Coupled Plasma Optical Emission Spectroscopy (ICP-OES) under the experimental conditions of initial metal concentration of 100 mg/l, pH 4.5, and biomass dose of 1 g/l at 30°C for 24 hrs. The data showed the best fit with the Freundlich isotherm model while following pseudo-first-order kinetics. Scanning Electron Microscope (SEM) and Energy Dispersive X-ray (EDX) analysis confirmed iron biosorption as precipitates on the bacterial surface, and as a peak in the EDX spectrum. The functional hydroxyl, carboxyl, and amino groups that are involved in biosorption were revealed by the Fourier Transform Infrared spectroscopy (FTIR). The amorphous nature of the biosorbent for biosorption was indicated by the X-ray Diffraction (XRD) analysis. The biomass of B. subtilis exhibited a point zero charge (pHpzc) at 2.0.Various microbial biomasses have been employed as biosorbents. Bacterial biomass has added advantages because of easy in production at a low cost. The study investigated the biosorption of iron from aqueous solutions by Bacillus subtilis. An optimum biosorption capacity of 7.25 mg of the metal per gram of the biosorbent was obtained by the Inductive Coupled Plasma Optical Emission Spectroscopy (ICP-OES) under the experimental conditions of initial metal concentration of 100 mg/l, pH 4.5, and biomass dose of 1 g/l at 30°C for 24 hrs. The data showed the best fit with the Freundlich isotherm model while following pseudo-first-order kinetics. Scanning Electron Microscope (SEM) and Energy Dispersive X-ray (EDX) analysis confirmed iron biosorption as precipitates on the bacterial surface, and as a peak in the EDX spectrum. The functional hydroxyl, carboxyl, and amino groups that are involved in biosorption were revealed by the Fourier Transform Infrared spectroscopy (FTIR). The amorphous nature of the biosorbent for biosorption was indicated by the X-ray Diffraction (XRD) analysis. The biomass of B. subtilis exhibited a point zero charge (pHpzc) at 2.0.

Dataset Information

Structural and kinetic analysis of Bacillus subtilis N-acetylglucosaminidase reveals a unique Asp-His dyad mechanism.

Publications

Structural and kinetic analysis of Bacillus subtilis N-acetylglucosaminidase reveals a unique Asp-His dyad mechanism.

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