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.