Molecular Cloning, Sequencing, and Expression of the Genes Encoding Adenosylcobalamin-dependent Diol Dehydrase of Klebsiella oxytoca

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 (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.

A Reactivating Factor for Coenzyme B12-dependent Diol Dehydratase

“…The 6.6-kilobase HindIII-BglII fragment containing the tac promoter sequence and the ddrA and ddrB genes was excised from pCXV(6/5b) and ligated to the 5-kilobase HindIII-BglII fragment of pUSI2ENd(DD) to construct pUSI2ENd(6/5b). pUSI2ENd(DD) was a derivative of pUSI2E(DD) (23), an expression plasmid for the diol dehydratase genes. The unnecessary NdeI site on the vector region of pUSI2E(DD) was eliminated in pUSI2ENd(DD).…”

Construction and Characterization of Hybrid Dehydratases between Adenosylcobalamin-Dependent Diol and Glycerol Dehydratases

“…One unit is defined as the amount of enzyme activity that catalyzes the formation of 1 mol of propionaldehyde per min under the standard assay conditions using 1,2-propanediol as substrate. Since the solubilities of diol dehydratase ( 6,16 ) and the ␣ G ␤ D ␥ D hybrid enzyme are low, homogenates, crude extracts, and purified preparations of enzymes to be assayed were diluted in 50 m M potassium phosphate buffer (pH 8.0) containing 2% substrate and 0.1-1% Brij35. Apparent Km of the hybrid enzyme for glycerol was determined by the 1-min MBTH assay method ( 11 ).…”

“…In contrast, glycerol dehydratase is highly soluble and exists in a cytosolic fraction. Predicted amino acid sequences of the subunit are well conserved between the two dehydratases except that the ␤ and ␥ subunits of diol dehydratase have extra amino acid residues in their N-terminal regions (16)(17)(18). Although hydropathy profiles show that these N-terminal regions are neither hydrophobic nor hydrophilic, removal of the regions from the ␤ and ␥ subunits of diol dehydratase by limited proteolysis with trypsin (23,34) or by gene engineering techniques (23) makes the enzyme highly soluble.…”

“…1) ( 18 ). The N-terminal 32 and 37 amino acid residues of the ␤ and ␥ subunits, respectively, of diol dehydratase are lacking in the corresponding subunits of glycerol dehydratase (16)(17)(18). These regions of ␤ and ␥ subunits of diol dehydratase lower the solubility of diol dehydratase ( 23 ).…”

“…[13][14][15]). In order to reveal their similarities and differences on a molecular level, we cloned, sequenced, and expressed the diol dehydratase genes of Klebsiella oxytoca ( 16 ) and K lebsiella pneumoniae ( 17 ) and the glycerol dehydratase genes of K. pneumoniae ( 18 ). The glycerol dehydratase genes of Citrobacter freundii ( 19 ) and Clostridium pasteurianum ( 20 ) and the diol dehydratase genes of Salmonella typhimurium ( 21 ) and Lactobacillus collinoides ( 22 ) have also been cloned by other investigators.…”