Enzyme evolution in Rhodobacter sphaeroides: selection of a mutant expressing a new galactitol dehydrogenase and biochemical characterization of the enzyme

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Abstract
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.