title: Structure-based conversion of the coenzyme requirement of a short-chain dehydrogenase/reductase involved in bacterial alginate metabolism.
abstract: The alginate-assimilating bacterium, Sphingomonas sp. strain A1, degrades the polysaccharides to monosaccharides through four alginate lyase reactions. The resultant monosaccharide, which is nonenzymatically converted to 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), is further metabolized to 2-keto-3-deoxy-D-gluconate by NADPH-dependent reductase A1-R in the short-chain dehydrogenase/reductase (SDR) family. A1-R-deficient cells produced another DEH reductase, designated A1-R', with a preference for NADH. Here, we show the identification of a novel NADH-dependent DEH reductase A1-R' in strain A1, structural determination of A1-R' by x-ray crystallography, and structure-based conversion of a coenzyme requirement in SDR enzymes, A1-R and A1-R'. A1-R' was purified from strain A1 cells and enzymatically characterized. Except for the coenzyme requirement, there was no significant difference in enzyme characteristics between A1-R and A1-R'. Crystal structures of A1-R' and A1-R'·NAD(+) complex were determined at 1.8 and 2.7 Å resolutions, respectively. Because of a 64% sequence identity, overall structures of A1-R' and A1-R were similar, although a difference in the coenzyme-binding site (particularly the nucleoside ribose 2' region) was observed. Distinct from A1-R, A1-R' included a negatively charged, shallower binding site. These differences were caused by amino acid residues on the two loops around the site. The A1-R' mutant with the two A1-R-typed loops maintained potent enzyme activity with specificity for NADPH rather than NADH, demonstrating that the two loops determine the coenzyme requirement, and loop exchange is a promising method for conversion of coenzyme requirement in the SDR family.
authors.affiliation: From the Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, and.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Takase
authors.affiliation: the Laboratory of Applied Structural Biology, 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: From the Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, and.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Kawai
authors.affiliation: From the Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, and.
authors.forename: Kousaku
authors.initials: K
authors.lastname: Murata
authors.affiliation: From the Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, and whasimot@kais.kyoto-u.ac.jp.
authors.forename: Wataru
authors.initials: W
authors.lastname: Hashimoto
mesh_terms: D000464:Alginates / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D020723:Glucuronic Acid / Q000737:chemistry / Q000378:metabolism; D006603:Hexuronic Acids / Q000737:chemistry / Q000378:metabolism; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020578:Sphingomonas / Q000201:enzymology*
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D006603:Hexuronic Acids; D009249:NADP; D020723:Glucuronic Acid; D010088:Oxidoreductases
keywords: Alginate Lyase; Bacterial Metabolism; Enzyme Kinetics; Nicotinamide Adenine Dinucleotide (NADH); Reductase; Site-directed Mutagenesis; X-ray Crystallography