Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca

Recombinant adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca overexpressed in Escherichia coli was 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 of K. 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 apparent M(r) of 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 α₂γ₂ complex, respectively. Apparent K(m) for 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 sub- units of diol dehydratase were overexpressed individually or in various combinations in Escherichia coli. The α and γ subunits mutually required each other for correct folding forming the soluble, active α₂γ₂ complex (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.