Heterologous Expression, Purification, and Properties of Diol Dehydratase, an Adenosylcobalamin-Dependent Enzyme ofKlebsiella oxytoca

Recombinant adenosylcobalamin-dependent diol dehydratase ofKlebsiella oxytocaoverexpressed inEscherichia coliwas purified to homogeneity. The enzyme has a low solubility and was extracted from the crude membrane fraction with 1% Brij 35 in a high recovery. Subsequent chromatography on DEAE–cellulose resulted in 4.9-fold purification of the enzyme in an overall yield of 65%. The enzyme thus obtained showed specific activity comparable to that of the wild-type enzyme ofK. oxytoca.The apparent molecular weight determined by nondenaturing gel electrophoresis on a gradient gel was 220,000. The enzyme consists of equimolar amounts of the three subunits with apparentMrof 60,000 (α), 30,000 (β), and 19,000 (γ). Therefore, the subunit structure of the enzyme is most likely α2β2γ2. The recombinant enzyme was also separated into components F and S upon DEAE–cellulose chromatography in the absence of substrate. Components F and S were identified as the β subunit and α2γ2complex, respectively. ApparentKmfor adenosylcobalamin, 1,2-propanediol, glycerol, and 1,2-ethanediol were 0.83 μm, 0.08 mm, 0.73 mm, and 0.56 mm, respectively. The three genes encoding the subunits of diol dehydratase were overexpressed individually or in various combinations inEscherichia coli.The α and γ subunits mutually required each other for correct folding forming the soluble, active α2γ2complex (component S). Expression of the β subunit in a soluble, active form (component F) was promoted by coexpression with both the α and γ subunits, probably by coexistence with component S. These lines of evidence indicate that each subunit mutually affects the folding of the others in this heterooligomer enzyme.