A protein factor is essential for in situ reactivation of glycerol-inactivated adenosylcobalamin-dependent diol dehydratase.
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+. beta,gamma-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 alpha-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.
Mori
K
K
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Japan.
Tobimatsu
T
T
Toraya
T
T
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D003001:Cloning, Molecular; D003038:Cobamides / Q000378:metabolism*; D003470:Culture Media; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000378:metabolism*; D004926:Escherichia coli; D007709:Klebsiella / Q000201:enzymology / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D010957:Plasmids; D011408:Propanediol Dehydratase / Q000235:genetics / Q000378:metabolism*
D003038:Cobamides; D003470:Culture Media; D004793:Enzyme Reactivators; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D004247:DNA; D011408:Propanediol Dehydratase; C000913:cobamamide; D008274:Magnesium; C002630:alpha,beta-methyleneadenosine 5'-triphosphate