Diol Dehydratase-Reactivase Is Essential for Recycling of Coenzyme B12 in Diol Dehydratase

Holoenzymes of adenosylcobalamin-dependent diol and glycerol dehydratases undergo mechanism-based inactivation by glycerol and O2 inactivation in the absence of substrate, which accompanies irreversible cleavage of the coenzyme Co–C bond. The inactivated holodiol dehydratase and the inactive enzyme·cyanocobalamin complex were (re)activated by incubation with NADH, ATP, and Mg2+ (or Mn2+) in crude extracts of Klebsiella oxytoca, suggesting the presence of a reactivating system in the extract. The reducing system with NADH could be replaced by FMNH2. When inactivated holoenzyme or the enzyme·cyanocobalamin complex, a model of inactivated holoenzyme, was incubated with purified recombinant diol dehydratase-reactivase (DD-R) and an ATP:cob(I)alamin adenosyltransferase in the presence of FMNH2, ATP, and Mg2+, diol dehydratase activity was restored. Among the three adenosyltransferases (PduO, EutT, and CobA) of this bacterium, PduO and CobA were much more efficient for the reactivation than EutT, although PduO ...