ENZYME EVOLUTION IN RHODOBACTER-SPHAEROIDES - SELECTION OF A MUTANT EXPRESSING A NEW GALACTITOL DEHYDROGENASE AND BIOCHEMICAL-CHARACTERIZATION OF THE ENZYME

A gain of function mutant of Rhodobacter sphaeroides Si4, capable of g rowing on galactitol, was isolated from a chemostat culture. continuou s 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-metab olizable galactitol. The mutant strain, R. sphaeroides D, grew in gala ctitol 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 dehydrog enase (GDH) activity of 380 mU mg(-1) was found, while the wild-type s train exhibited GDH activities lower than 50 mU mg(-1) when grown on d ifferent polyols. Unlike mannitol, sorbitol or ribitol dehydrogenase f rom the wild-type strain, the new GDH was expressed constitutively. To study whether it was a newly evolved enzyme or an improved side activ ity of one of the pre-existing polyol dehydrogenases, GDH was purified to apparent homogeneity by ammonium sulfate precipitation and chromat ography on Phenyl-Sepharose, Q-Sepharose, Matrex Gel Red-A and Mono-Q. The relative molecular mass (M(r)) of the native GDH was 110 000. SDS -PAGE resulted in one single band that represented a polypeptide with a M(r) of 28 000, indicating that the native protein is a tetramer. Th e isoelectric point of GDH was determined to be ph 4.2. The enzyme was specific for NAD(+) but catalysed the oxidation of different sugar al cohols 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(+), 12 mu M; L-erythrulose, 144 mM; acetoin, 62 mM; dihy droxyacetone, 48 mM; and NADH, 4 mu M. GDH activity was strictly depen dent on the presence of divalent cations. The properties of CDH are di fferent 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 polyo l dehydrogenases clearly demonstrates that GDH is an additional enzyme , so far unrecognized in the wild-type strain.