Abstract
Adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicide inactivation by glycerol, a physiological substrate. The coenzyme is modified through irreversible cleavage of its cobalt-carbon bond, resulting in inactivation of the enzyme by tight binding of the modified coenzyme to the active site. Recombinant DdrA and DdrB proteins of K. oxytoca were co-purified to homogeneity from cell-free extracts of Escherichia coli overexpressing the ddrAB genes. They existed as a tight complex, i.e. a putative reactivating factor, with an apparent molecular weight of 150,000. The factor consists of equimolar amounts of the two subunits with Mr of 64,000 (A) and 14,000 (B), encoded by the ddrA and ddrB genes, respectively. Therefore, its subunit structure is most likely A2B2. The factor not only reactivated glycerol-inactivated and O2-inactivated holoenzymes but also activated enzyme-cyanocobalamin complex in the presence of free adenosylcobalamin, ATP, and Mg2+. The reactivating factor mediated ATP-dependent exchange of the enzyme-bound cyanocobalamin for free 5-adeninylpentylcobalamin in the presence of ATP and Mg2+, but the reverse was not the case. Thus, it can be concluded that the inactivated holoenzyme becomes reactivated by exchange of the enzyme-bound, adenine-lacking cobalamins for free adenosylcobalamin, an adenine-containing cobalamin.
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