Characterization, sequencing, and expression of the genes encoding a reactivating factor for glycerol-inactivated adenosylcobalamin-dependent diol dehydratase.
Diol dehydratase undergoes suicide inactivation by glycerol during catalysis involving irreversible cleavage of the Co-C bond of adenosylcobalamin. In permeabilized Klebsiella oxytoca and Klebsiella pneumoniae cells, the glycerol-inactivated holoenzyme or the enzyme-cyanocobalamin complex is rapidly activated by the exchange of the inactivated coenzyme or cyanocobalamin for free adenosylcobalamin in the presence of ATP and Mg2+ (Honda, S., Toraya, T., and Fukui, S. (1980) J. Bacteriol. 143, 1458-1465; Ushio, K., Honda, S., Toraya, T., and Fukui, S. (1982) J. Nutr. Sci. Vitaminol. 28, 225-236). Permeabilized Escherichia coli cells co-expressing the diol dehydratase genes with two open reading frames in the 3'-flanking region were capable of reactivating glycerol-inactivated diol dehydratase as well as activating the enzyme-cyanocobalamin complex in situ in the presence of free adenosylcobalamin, ATP, and Mg2+. These open reading frames, designated as ddrA and ddrB genes, were identified as the genes of a putative reactivating factor for inactivated diol dehydratase. The genes encoded polypeptides consisting of 610 and 125 amino acid residues with predicted molecular weights of 64,266 and 13,620, respectively. Co-expression of the open reading frame in the 5'-flanking region was stimulatory but not obligatory for conferring the reactivating activity upon E. coli. Thus, the product of this gene was considered not an essential component of the reactivating factor.
Mori
K
K
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700, Japan.
Tobimatsu
T
T
Hara
T
T
Toraya
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004274:DNA, Recombinant; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004793:Enzyme Reactivators / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005990:Glycerol / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011408:Propanediol Dehydratase / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004274:DNA, Recombinant; C109737:DdrA protein, Klebsiella oxytoca; C109738:DdrB protein, Klebsiella oxytoca; D004791:Enzyme Inhibitors; D004793:Enzyme Reactivators; D011994:Recombinant Proteins; D011408:Propanediol Dehydratase; D005990:Glycerol