Molecular cloning, sequencing, and expression of the genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca

Takamasa Tobimatsu, Tetsuya Hara, Munetoh Sakaguchi, Yasuhiro Kishimoto, Yukihisa Wada, Masaki Isoda, Takahiro Sakai, Tetsuo Toraya

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucleotide as a hybridization probe followed by measuring the enzyme activity of each clone. Five clones of Escherichia coli exhibited diol dehydrase activity. At least one of them was shown to express diol dehydrase 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 second 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 (α), 24,113 (β), and 19,173 (γ), respectively. Overexpression of these three genes in E. coli produced more than 50-fold higher level of functional apodiol dehydrase than that in K. oxytoca. The recombinant enzyme was indistinguishable from the wild-type one of K. oxytoca by the criteria of polyacrylamide gel electrophoretic and immunochemical properties. It was thus concluded that these three gene products are the subunits of functional diol dehydrase. Comparisons of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.

Original languageEnglish
Pages (from-to)7142-7148
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number13
DOIs
Publication statusPublished - Mar 31 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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