Identification of a reactivating factor for adenosylcobalamin-dependent ethanolamine ammonia lyase.
The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5' end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.
Mori
Koichi
K
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700-8530, Japan.
Bando
Reiko
R
Hieda
Naoki
N
Toraya
Tetsuo
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003038:Cobamides / Q000378:metabolism*; D004789:Enzyme Activation; D004793:Enzyme Reactivators / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004981:Ethanolamine Ammonia-Lyase / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D020035:Holoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D009876:Operon; D011994:Recombinant Proteins / Q000378:metabolism
D003038:Cobamides; D004793:Enzyme Reactivators; D029968:Escherichia coli Proteins; C492797:EutA protein, E coli; D020035:Holoenzymes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004981:Ethanolamine Ammonia-Lyase; C000913:cobamamide