POLYOL METABOLISM OF RHODOBACTER-SPHAEROIDES - BIOCHEMICAL-CHARACTERIZATION OF A SHORT-CHAIN SORBITOL DEHYDROGENASE

A sorbitol dehydrogenase (5DH; L-iditol: NAD(+) 2-oxidoreductase; EC 1 .1.1.14) was isolated from the phototrophic bacterium Rhodobacter spha eroides 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 precipi tation, 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,S-w = 4.2 3S). SDS-PAGE resulted in one single band representing a polypeptide w ith a M(r) of 29 000, indicating that the native protein is a dimer. T he isoelectric point of SDH was determined to be ph 4.8. The enzyme wa s specific for NAD(+) and catalysed the oxidation of o-glucitol (sorbi tol) to D-fructose, galactitol to D-tagatose and of L-iditol. The appa rent 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 reduc tion 6.0-7.2. It was demonstrated that SDH is expressed in the wild-ty pe strain R. sphaeroides Si4 together with MDH during growth on D-gluc itol. Forty-four amino acids of the SDH N terminus were sequenced. Thi s sequence exhibited 45-55% identity to the N-terminal sequence of 10 enzymes belonging to the short-chain alcohol dehydrogenase family.