A reactivating factor for coenzyme B-12-dependent diol dehydratase

Adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoe s suicide inactivation by glycerol, a physiological substrate. The coenzyme is modified through irreversible cleavage of its cobalt-carbon bond, resul ting in inactivation of the enzyme by tight binding of the modified coenzym e to the active site. Recombinant DdrA and DdrB proteins of K. oxytoca were co-purified to homogeneity from cell-free extracts of Escherichia coli ove rexpressing the ddrAB genes. They existed as a tight complex, i.e. a putati ve reactivating factor, with an apparent molecular weight of 150,000, The f actor consists of equimolar amounts of the two subunits with M-r of 64,000 (A) and 14,000 (B), encoded by the ddrA and ddrB genes, respectively. There fore, its subunit structure is most likely A(2)B(2). The factor not only re activated glycerol-inactivated and O-2-inactivated holoenzymes but also act ivated enzyme-cyanocobalamin complex in the presence of free adenosylcobala min, ATP, and Mg2+. The reactivating factor mediated ATP-dependent exchange of the enzyme-bound cyanocobalamin for free 5-adeninylpentylcobalamin in t he presence of ATP and Mg2+, but the reverse was not the case. Thus, it can be concluded that the inactivated holoenzyme becomes reactivated by exchan ge of the enzyme-bound, adenine-lacking cobalamins for free adenosylcobalam in, an adenine-containing cobalamin.