MOLECULAR-CLONING, SEQUENCING, AND EXPRESSION OF THE GENES ENCODING ADENOSYLCOBALAMIN-DEPENDENT DIOL DEHYDRASE OF KLEBSIELLA-OXYTOCA

The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of K lebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucle otide as a hybridization probe followed by measuring the enzyme activi ty of each clone. Five clones of Escherichia coli exhibited diol dehyd rase activity. At least one of them was shown to express diol dehydras e genes under control of their own promoter, Sequence analysis of the DNA fragments found in common in the inserts of these five clones and the flanking regions revealed four open reading frames separated by 10 -18 base pairs. The sequential three open reading frames from the seco nd to the fourth (pddA, pddB, and pddC genes) encoded polypeptides of 554, 224, and 173 amino acid residues with predicted molecular weights of 60,348 (alpha), 24,113 (beta), and 19,173 (gamma), respectively, O verexpression of these three genes in E. coli produced more than 50 fo ld higher level of functional apodiol dehydrase than that in K. oxytoc a. The recombinant enzyme was indistinguishable from the wild-type one of R. oxytoca by the criteria of polyacrylamide gel electrophoretic a nd immunochemical properties. It was thus concluded that these three g ene products are the subunits of functional diol dehydrase. Comparison s of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.