H. Kajiura et al., Characterization and mechanism of action of a reactivating factor for adenosylcobalamin-dependent glycerol dehydratase, J BIOL CHEM, 276(39), 2001, pp. 36514-36519
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.