Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase

• Published in: Microbiology (Society for General Microbiology) Vol. 141; no. 8; pp. 1857 - 1863
• Main Authors: Schauder, Stephan, Schneider, Karl-Heinz, Giffhorn, Friedrich
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.08.1995; Society for General Microbiology
• Subjects: Alcohol Oxidoreductases - genetics; Amino Acid Sequence; Bacteriology; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Chromatography; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; L-Iditol 2-Dehydrogenase - chemistry; L-Iditol 2-Dehydrogenase - genetics; L-Iditol 2-Dehydrogenase - isolation & purification; Mannitol Dehydrogenases - genetics; Metabolism. Enzymes; Microbiology; Mission oriented research; Molecular Sequence Data; Molecular Weight; Monosaccharides - metabolism; Mutagenesis, Insertional; Physiology and metabolism; Rhodobacter sphaeroides; Rhodobacter sphaeroides - enzymology; Rhodobacter sphaeroides - genetics; Sequence Alignment; Sugar Alcohol Dehydrogenases - genetics; Sugar Alcohols - metabolism; Purification; Enzyme; Rhodospirillaceae; Metabolism; Rhodospirillales; Rhodobacter sphaeroides; Bacteria; L-Iditol 2-dehydrogenase; Oxidoreductases; Polyol; Rhodospirillineae; Physicochemical properties; Structural analysis
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/13500872-141-8-1857

Lehrstuhl für Angewandte Mikrobiologie, Universitat des Saarlandes, Postfach 15 11 50, D-66041 Saarbrücken, Germany Author for correspondence: Friedrich Giffhorn. Tel: +49 681 302 3444. Fax: +49 681 302 4360. ABSTRACT A sorbitol dehydrogenase (SDH; L-iditol: NAD + 2-oxidoreductase; EC1.1.1.14) was isolated from the phototrophic bacterium Rhodobacter sphaeroides strain M22, a transposon mutant of R. sphaeroides Si4 with the transposon inserted in the mannitol dehydrogenase (MDH) gene. SDH was purified 470-fold to apparent homogeneity by ammonium sulfate precipitation, chromatography on Phenyl-Sepharose, Q-Sepharose and Matrex Gel Red-A, and by gel filtration on Superdex 200. The relative molecular mass ( M r ) of the native SDH was 61 000 as calculated from its Stokes’ radius (r s = 3.5 nm) and sedimentation coefficient ( S 20 ,w = 4.235). SDS-PAGE resulted in one single band representing a polypeptide with a M r of 29000, indicating that the native protein is a dimer. The isoelectric point of SDH was determined to be pH 4-8. The enzyme was specific for NAD + and catalysed the oxidation of D-glucitol (sorbitol) to D-fructose, galactitol to D-tagatose and of L-iditol. The apparent K m values were NAD + , 0-06 mM; D-glucitol, 6-2 mM; galactitol, 1-5 mM; NADH, 0-13 mM; D-fructose, 160 mM; and D-tagatose, 13 mM. The pH-optimum of substrate oxidation was 11-0 and that of substrate reduction 6-0-7-2. It was demonstrated that SDH is expressed in the wild-type strain R. sphaeroides Si4 together with MDH during growth on D-glucitol. Forty-four amino acids of the SDH N terminus were sequenced. This sequence exhibited 45-55% identity to the N-terminal sequence of 10 enzymes belonging to the short-chain alcohol dehydrogenase family. Keywords: Rhodobacter sphaeroides, polyol metabolism, sorbitol dehydrogenase