Characterization and mechanism of action of a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase

Adenosylcobalamin-dependent glycerol dehydratase undergoes mechanism-based inactivation by its physiological substrate glycerol. We identified two gen es (gdrAB) of Klebsiella pneumoniae for a glycerol dehydratase-reactivating factor (Tobimatsu, T., Kajiura, H., Yunoki, M., Azuma, M., and Toraya, T. (1999) J. Bacteriol 181, 4110-4113). Recombinant GdrA and GdrB proteins for med a tight complex of (GdrA)(2)(GdrB)(2), which is a putative reactivating factor. The purified factor reactivated the glycerol-inactivated and O-2-i nactivated glycerol dehydratases as well as activated the enzyme-cyanocobal amin complex in vitro in the presence of ATP, Mg2+, and adenosylcobalamin. The factor mediated the exchange of the enzyme-bound, adenine-lacking cobal amins for free, adenine-containing cobalamins in the presence of ATP and Mg 2+ through intermediate formation of apoenzyme. The factor showed extremely low ATP-hydrolyzing activity and formed a tight complex with apoenzyme in the presence of ADP. Incubation of the enzyme-cyanocobalamin complex with t he reactivating factor in the presence of ADP brought about release of the enzyme-bound cobalamin. The resulting tight inactive complex of apoenzyme w ith the factor dissociated upon incubation with ATP, forming functional apo enzyme and a low affinity form of factor. Thus, it was established that the reactivation of the inactivated holoenzymes takes place in two steps: ADP- dependent cobalamin release and ATP-dependent dissociation of the apoenzyme -factor complex. We propose that the glycerol dehydratase-reactivating fact or is a molecular chaperone that participates in reactivation of the inacti vated enzymes.