title: Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.
abstract: Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan.|Laboratory of Supramolecular Crystallography, Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nakamichi
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan.
authors.forename: Sayoko
authors.initials: S
authors.lastname: Oiki
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan.
authors.forename: Bunzo
authors.initials: B
authors.lastname: Mikami
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan.|Department of Life Science, Faculty of Science and Engineering, Setsunan University, Neyagawa, Osaka, 572-8508, Japan.
authors.forename: Kousaku
authors.initials: K
authors.lastname: Murata
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan. whasimot@kais.kyoto-u.ac.jp.
authors.forename: Wataru
authors.initials: W
authors.lastname: Hashimoto
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013292:Streptococcus agalactiae / Q000737:chemistry* / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D006025:Glycosaminoglycans; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine; D006026:Glycoside Hydrolases; C120983:unsaturated glucuronyl hydrolase
keywords: Glycosaminoglycan; Streptococcus; Unsaturated glucuronyl hydrolase; X-ray crystallography