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-Sepharore and Matrex Gel Red-A, and by gel filtration on Superdex 200. The relative molecular mass (M,) of the native SDH was 61 000 as calculated from its Stokes' radius (r, = 3.5 nm) and sedimentation coefficient (S20,w = 4.235). SDS-PAGE resulted in one single band representing a polypeptide with a M, 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 Dfructose, galactitol to D-tagatose and of L-iditol. The apparent K, values were NAD+, 096 mM; D-glucitol, 6 2 mM; galactitol, 1.5 mM; NADH, 013 mM; Dfructose, 160 mM; and D-tagatose, 13 mM. The pH-optimum of substrate oxidation was 11.0 and that of substrate reduction 69-7.2. 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.

A polyol dehydrogenase was detected in cell extracts of the facultative phototrophic bacterium Rhodobacter sphaeroides strain Si 4 grown on D-glucitol (sorbitol) as the sole carbon source. The enzyme was purified 150-fold to apparent homogeneity by steps involving fractionated (NH&S04 precipitation, chromatography on Q-Sepharose and phenyl-Sepharose, and FPLC on Superose 12. The relative molecular mass ( M E ) of the native polyol dehydrogenase was 47200 as calculated from its Stokes' radius (r, = 2.76 nm) and sedimentation coefficient (szO,,, = 4.15 S). SDS/PAGE resulted in one single band representing a polypeptide with a M , of 52200, indicating that the native protein is a monomer. The isoelectric point of the polyol dehydrogenase was determined to be pH 4.

A gain of function mutant of Rhodobacter sphaeroides Si4, capable of growing on galactitol, was isolated from a chemostat culture. Continuous cultivation was performed for 54 d with a limiting concentration (1 mM) of the substrate D-glucitol and an excess (20 mM) of the non-metabolizable galactitol. The mutant strain, R. sphaeroides D, grew in galactitol minimal medium with a growth rate of 0-11 h-1 (t d = 6-3 h). In crude extracts of R. sphaeroides D, a specific galactitol dehydrogenase (GDH) activity of 380 mU mg-1 was found, while the wild-type strain exhibited GDH activities lower than 50 mU mg-1 when grown on different polyols. Unlike mannitol, sorbitol or ribitol dehydrogenase from the wild-type strain, the new GDH was expressed constitutively. To study whether it was a newly evolved enzyme or an improved side activity of one of the pre-existing polyol dehydrogenases, GDH was purified to apparent homogeneity by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, Q-Sepharose, Matrex Gel Red-A and Mono-Q. The relative molecular mass (M r) of the native GDH was 110000. SDS-PAGE resulted in one single band that represented a polypeptide with a M r of 28000, indicating that the native protein is a tetramer. The isoelectric point of GDH was determined to be pH 4-2. The enzyme was specific for NAD+ but catalysed the oxidation of different sugar alcohols as well as different diols and secondary alcohols. The apparent K m values were: galactitol, 240 mM; D-threitol, 85 mM; 1,2-hexandiol, 0-2 mM; NAD+, 12μM; L-erythrulose, 144 mM; acetoin, 62 mM; dihydroxyacetone, 48 mM; and NADH, 4μM. GDH activity was strictly dependent on the presence of divalent cations. The properties of GDH are different to any of the three polyol dehydrogenases from R. sphaeroides Si4. In addition, comparison of the N-terminal amino acid sequence of the isolated GDH with the N-terminal sequence of the other three polyol dehydrogenases clearly demonstrates that GDH is an additional enzyme, so far unrecognized in the wild-type strain.

The phototrophic bacterium Z t h b k t e r spkroides strain Si4 induced ribitol dehydrogenase (EC 1.1.1 . ! Xi ) when grown on ribitol-or xylitol-containing medium. This ribitol dehydrogenase was purified to apparent homogeneity by ammonium sdphate precipitation, afl[inity chromatography on Procion red, and chromatography on Q-Sepharcwe. For the native enzyme an isoelectric point of pH6.1 and an apparent M, of WOO0 was determined. SDS-PAGE yielded a single peptide band of M, 25000 suggesting a dimeric enzyme structure. The ribitol dehydrogenase was specific for NAD+ but unspecific as to its polyol substrate. In order of decreasing activity ribitol, xylitol, erythritol, D-@UCitOl and D-arabitol were oxidized. The pH optimum of Substrate oxidation was 10, and that of substrate reduction was 6.5. The equilibrium constant of the interconversion of ribitol to D-ribulose was determined to be 0.33 nM at pH 7.0 and 25 OC. The K,-values determined for ribitol, ribulose, xylitol and NAD+ (in the presence of ribitol) were 6.3, 12.5, 77 and 0-077mM, respectively. Because of the favourable K,,, for ribitol, a method for quantitative ribitol determination was elaborated.

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