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 cat alysis involving irreversible cleavage of the Go-G bond of adenosylcob alamin. In permeabilized Klebsiella oxytoca and Klebsiella pneumoniae cells, the glycerol-inactivated holoenzyme or the enzyme-cyanocobalami n complex is rapidly activated by the exchange of the inactivated coen zyme or cyanocobalamin for free adenosylcobalamin in the presence of A TP 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 coi l cells co-expressing the diol dehydratase genes with two open reading frames in the 3'-flanking region were capable; of reactivating glycer ol-inactivated diol dehydratase as well as activating the enzyme-cyano cobalamin complex in situ in the presence of free adenosylcobalamin, A TP, and Mg2+. These open reading frames, designated as ddrA and ddrB g enes, were identified as the genes of a putative reactivating factor f or inactivated diol dehydratase. The genes encoded polypeptides consis ting of 610 and 125 amino acid residues with predicted molecular weigh ts of 64,266 and 13,620, respectively. Go-expression of the open readi ng frame in the 5'-flanking region was stimulatory but not obligatory for conferring the reactivating activity upon E. coli. Thus, the produ ct of this gene was considered not an essential component of the react ivating factor.