Molecular identification of unsaturated uronate reductase prerequisite for alginate metabolism in Sphingomonas sp. A1 |
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Authors: | Ryuichi Takase Akihito Ochiai Bunzo Mikami Wataru Hashimoto Kousaku Murata |
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Institution: | 1. Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan;2. Laboratory of Applied Structural Biology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan |
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Abstract: | In Sphingomonas sp. A1, alginate is degraded by alginate lyases to its constituent monosaccharides, which are nonenzymatically converted to an α-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 °C. Crystal structures of A1-R and its complex with NADP were determined at around 1.6 Å resolution by X-ray crystallography. The enzyme consists of three layers (α/β/α), 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. |
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Keywords: | Strain A1 Sphingomonas sp strain A1 DEH 4-deoxy-l-erythro-5-hexoseulose uronic acid KDG 2-keto-3-deoxy-d-gluconic acid A1-R DEH-specific strain A1 reductase TLC thin-layer chromatography KPB potassium phosphate buffer LC liquid chromatography SDS&ndash PAGE sodium dodecyl sulfate&ndash polyacrylamide gel electrophoresis MS mass spectrometry ESI electrospray ionization NMR nuclear magnetic resonance A1-R/NADP A1-R in complex with NADP r m s d root-mean-square deviation PDB Protein Data Bank MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight SDR short-chain dehydrogenase/reductase |
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