title: Molecular identification of unsaturated uronate reductase prerequisite for alginate metabolism in Sphingomonas sp. A1.
abstract: In Sphingomonas sp. A1, alginate is degraded by alginate lyases to its constituent monosaccharides, which are nonenzymatically converted to an alpha-keto acid, namely, 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH). The properties of the DEH-metabolizing enzyme and its gene in strain A1 were characterized. In the presence of alginate, strain A1 cells inducibly produced an NADPH-dependent DEH reductase (A1-R) in their cytoplasm. Molecular cloning of the enzyme gene indicated that A1-R belonged to the short-chain dehydrogenase/reductase superfamily and catalyzed the conversion of DEH to 2-keto-3-deoxy-d-gluconic acid most efficiently at around pH 7.0 and 50 degrees C. Crystal structures of A1-R and its complex with NADP were determined at around 1.6A resolution by X-ray crystallography. The enzyme consists of three layers (alpha/beta/alpha), with a coenzyme-binding Rossmann fold. NADP is surrounded by positively charged residues, and Gly-38 and Arg-39 are crucial for NADP binding. Site-directed mutagenesis studies suggest that Ser-150, Tyr-164, and Lys-168 located around the Rossmann fold constitute the catalytic triad. To our knowledge, this is the first report on molecular cloning and structure determination of a bacterial DEH reductase responsible for alginate metabolism.
authors.affiliation: Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Takase
authors.forename: Akihito
authors.initials: A
authors.lastname: Ochiai
authors.forename: Bunzo
authors.initials: B
authors.lastname: Mikami
authors.forename: Wataru
authors.initials: W
authors.lastname: Hashimoto
authors.forename: Kousaku
authors.initials: K
authors.lastname: Murata
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000464:Alginates / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D005942:Gluconates / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D000464:Alginates; D005942:Gluconates; D011994:Recombinant Proteins; D009249:NADP; D010088:Oxidoreductases; C023280:uronate dehydrogenase; D000445:Aldehyde Oxidoreductases; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase; C030691:gluconic acid