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 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 ( ) of the native SDH was 61 000 as calculated from its Stokes’ radius (r = 3.5 nm) and sedimentation coefficient ( ,w = 4.235). SDS-PAGE resulted in one single band representing a polypeptide with a 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 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 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.


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