Specificities of reactivating factors for adenosylcobalamin-dependent dioldehydratase and glycerol dehydratase

Adenosylcobalamin-dependent glycerol and diol dehydratases undergo inactiva tion by the physiological substrate glycerol during catalysis. In the perme abilized cells of Klebsiella pneumoniae, Klebsiella oxytoca, and recombinan t Escherichia coli, glycerol-inactivated glycerol dehydratase and diol dehy dratase are reactivated by their respective reactivating factors in the pre sence of ATP, Mg2+, and adenosylcobalamin. Both of the reactivating factors consist of two subunits. To examine the specificities of the reactivating factors, their genes or their hybrid genes were co-expressed with dehydrata se genes in E. coli cells in various combinations. The reactivating factor of K. oxytoca for diol dehydratase efficiently cross-reactivated the inacti vated glycerol dehydratase, whereas the reactivating factor of K. pneumonia e for glycerol dehydratase hardly cross-reactivated the inactivated diol de hydratase. Both of the two hybrid reactivating factors rapidly reactivated the inactivated glycerol dehydratase. In contrast, the hybrid reactivating factor containing the large subunit of the glycerol dehydratase reactivatin g factor hardly reactivated the inactivated diol dehydratase. These results indicate that the glycerol dehydratase reactivating factor is much more sp ecific for the dehydratase partner than the diol dehydratase reactivating f actor and that a large subunit of the reactivating factors principally dete rmines the specificity for a dehydratase.