Polyol metabolism of Rhodobacter sphaeroides: biochemical characterization of a short-chain sorbitol dehydrogenase.
A sorbitol dehydrogenase (SDH; L-iditol:NAD+ 2-oxidoreductase; EC 1.1.1.14) 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 61000 as calculated from its Stokes' radius (rs = 3.5 nm) and sedimentation coefficient (S20,w = 4.23S). SDS-PAGE resulted in one single band representing a polypeptide with a M(r) of 29,000, 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 Km 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.
Schauder
Stephan
S
Schneider
Karl-Heinz
KH
Giffhorn
Friedrich
F
D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007064:L-Iditol 2-Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008354:Mannitol Dehydrogenases / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009005:Monosaccharides / Q000378:metabolism; D016254:Mutagenesis, Insertional; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics; D013402:Sugar Alcohols / Q000378:metabolism*
D009005:Monosaccharides; D013402:Sugar Alcohols; D000429:Alcohol Oxidoreductases; D008354:Mannitol Dehydrogenases; D013401:Sugar Alcohol Dehydrogenases; D007064:L-Iditol 2-Dehydrogenase