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Structural and biochemical characterization of glycoside hydrolase family 79 ?-glucuronidase from Acidobacterium capsulatum.

ABSTRACT: We present the first structure of a glycoside hydrolase family 79 ?-glucuronidase from Acidobacterium capsulatum, both as a product complex with ?-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (?/?)(8)-barrel domain and a ?-domain with irregular Greek key motifs that is of unknown function. The enzyme showed ?-glucuronidase activity and trace levels of ?-glucosidase and ?-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of ?-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the ?-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the ?-glucosidase activity is actually 3 times higher and the ?-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-?-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.

SUBMITTER: Michikawa M

PROVIDER: S-EPMC3340155 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Structural and biochemical characterization of glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum.

Michikawa Mari M Ichinose Hitomi H Momma Mitsuru M Biely Peter P Jongkees Seino S Yoshida Makoto M Kotake Toshihisa T Tsumuraya Yoichi Y Withers Stephen G SG Fujimoto Zui Z Kaneko Satoshi S

The Journal of biological chemistry 20120224 17

We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutage ...[more]

PMID: 22367201

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Project description:BackgroundEnzymatic degradation of chitin has attracted substantial attention because chitin is an abundant renewable natural resource, second only to lignocellulose, and because of the promising applications of N-acetylglucosamine in the bioethanol, food and pharmaceutical industries. However, the low activity and poor tolerance to salts and N-acetylglucosamine of most reported β-N-acetylglucosaminidases limit their applications. Mining for novel enzymes from new microorganisms is one way to address this problem.ResultsA glycoside hydrolase family 20 (GH 20) β-N-acetylglucosaminidase (GlcNAcase) was identified from Microbacterium sp. HJ5 harboured in the saline soil of an abandoned salt mine and was expressed in Escherichia coli. The purified recombinant enzyme showed specific activities of 1773.1 ± 1.1 and 481.4 ± 2.3 μmol min-1 mg-1 towards p-nitrophenyl β-N-acetylglucosaminide and N,N'-diacetyl chitobiose, respectively, a V max of 3097 ± 124 μmol min-1 mg-1 towards p-nitrophenyl β-N-acetylglucosaminide and a K i of 14.59 mM for N-acetylglucosamine inhibition. Most metal ions and chemical reagents at final concentrations of 1.0 and 10.0 mM or 0.5 and 1.0% (v/v) had little or no effect (retaining 84.5 - 131.5% activity) on the enzyme activity. The enzyme can retain more than 53.6% activity and good stability in 3.0-20.0% (w/v) NaCl. Compared with most GlcNAcases, the activity of the enzyme is considerably higher and the tolerance to salts and N-acetylglucosamine is much better. Furthermore, the enzyme had higher proportions of aspartic acid, glutamic acid, alanine, glycine, random coils and negatively charged surfaces but lower proportions of cysteine, lysine, α-helices and positively charged surfaces than its homologs. These molecular characteristics were hypothesised as potential factors in the adaptation for salt tolerance and high activity of the GH 20 GlcNAcase.ConclusionsBiochemical characterization revealed that the GlcNAcase had novel salt-GlcNAc tolerance and high activity. These characteristics suggest that the enzyme has versatile potential in biotechnological applications, such as bioconversion of chitin waste and the processing of marine materials and saline foods. Molecular characterization provided an understanding of the molecular-function relationships for the salt tolerance and high activity of the GH 20 GlcNAcase.

Dataset Information

Structural and biochemical characterization of glycoside hydrolase family 79 ?-glucuronidase from Acidobacterium capsulatum.

Publications

Structural and biochemical characterization of glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum.

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