The adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca undergoes suicidal inactivation by glycerol during catalysis involving irreversible dissociation of the Co-C bond of the coenzyme. The glycerol-inactivated holoenzyme in permeabilized cells (in situ) of E. coli harboring a plasmid containing the diol dehydratase genes and their flanking regions was rapidly reactivated in the presence of free AdoCbl, ATP, and Mg²⁺. β, γ-Methylene ATP was not able to replace ATP. Inactive complexes of the enzyme with aqCbl, CN-Cbl, And PeCbl were activated in situ in the presence of AdoCbl, ATP, and Mg²⁺, but the complex with AdePeCbl was not. These results suggest that the inactivated holoenzyme is reactivated in situ in the presence of ATP and Mg²⁺ by exchange of the inactivated coenzyme lacking the adenine moiety for free intact AdoCbl. The in situ reactivation was also observed when an analog lacking the α-ribose moiety of the nucleotide loop was used as coenzyme. The results with a recombinant E. coli strains carrying a deletion mutant plasmid demonstrate that certain protein(s) encoded by the 3'-flanking region of the diol dehydratase genes are essential for the in situ reactivation of inactivated diol dehydratase.

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