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 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 (td = 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 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. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Schneider
Karl-Heinz
KH
Lehrstuhl für Angewandte Mikrobiologie, Universitat des Saarlandes,Postfach 15 11 50, D-66041 Saarbrücken,Germany.
Jakel
Gregor
G
Lehrstuhl für Angewandte Mikrobiologie, Universitat des Saarlandes,Postfach 15 11 50, D-66041 Saarbrücken,Germany.
Hoffmann
Ralf
R
Fachrichtung 12.4 Biochemie, Universitat des Saarlandes,Postfach 15 11 50, D-66041 Saarbrücken,Germany.
Giffhorn
Friedrich
F
Lehrstuhl für Angewandte Mikrobiologie, Universitat des Saarlandes,Postfach 15 11 50, D-66041 Saarbrücken,Germany.
D000438:Alcohols / Q000378:metabolism; D000595:Amino Acid Sequence; D005075:Biological Evolution; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D004376:Galactitol / Q000378:metabolism*; D007526:Isoelectric Point; D007700:Kinetics; D008293:Malates / Q000378:metabolism; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000378:metabolism; D009154:Mutation; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D016415:Sequence Alignment; D013401:Sugar Alcohol Dehydrogenases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013402:Sugar Alcohols / Q000378:metabolism; D013997:Time Factors
D000438:Alcohols; D008293:Malates; D009005:Monosaccharides; D013402:Sugar Alcohols; D004376:Galactitol; D013401:Sugar Alcohol Dehydrogenases; C095946:galactitol 2-dehydrogenase