Acetyl coenzyme A synthetase (ADP forming) from the hyperthermophilic Archaeon pyrococcus furiosus: identification, cloning, separate expression of the encoding genes, acdAI and acdBI, in Escherichia coli, and in vitro reconstitution of the active heterotetrameric enzyme from its recombinant subunits.
Acetyl-coenzyme A (acetyl-CoA) synthetase (ADP forming) represents a novel enzyme in archaea of acetate formation and energy conservation (acetyl-CoA + ADP + P(i) --> acetate + ATP + CoA). Two isoforms of the enzyme have been purified from the hyperthermophile Pyrococcus furiosus. Isoform I is a heterotetramer (alpha(2)beta(2)) with an apparent molecular mass of 145 kDa, composed of two subunits, alpha and beta, with apparent molecular masses of 47 and 25 kDa, respectively. By using N-terminal amino acid sequences of both subunits, the encoding genes, designated acdAI and acdBI, were identified in the genome of P. furiosus. The genes were separately overexpressed in Escherichia coli, and the recombinant subunits were reconstituted in vitro to the active heterotetrameric enzyme. The purified recombinant enzyme showed molecular and catalytical properties very similar to those shown by acetyl-CoA synthetase (ADP forming) purified from P. furiosus.
Musfeldt
M
M
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany.
Selig
M
M
Schönheit
P
P
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli; D019766:Genes, Archaeal; D006358:Hot Temperature; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003066:Coenzyme A Ligases; C101958:acetate-CoA ligase (ADP-forming)