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 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 (alpha), 24,113 (beta), and 19,173 (gamma), 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.
Tobimatsu
T
T
Department of Biotechnology, Faculty of Engineering, Okayama University, Japan.
Hara
T
T
Sakaguchi
M
M
Kishimoto
Y
Y
Wada
Y
Y
Isoda
M
M
Sakai
T
T
Toraya
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002474:Cell-Free System; D003001:Cloning, Molecular; D003038:Cobamides / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D007709:Klebsiella / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010957:Plasmids; D011408:Propanediol Dehydratase / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D003038:Cobamides; D011994:Recombinant Proteins; D011408:Propanediol Dehydratase; C000913:cobamamide