Molecular identification of unsaturated uronate reductase prerequisite for alginate metabolism in Sphingomonas sp. A1.
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.
Takase
Ryuichi
R
Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Ochiai
Akihito
A
Mikami
Bunzo
B
Hashimoto
Wataru
W
Murata
Kousaku
K
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
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