ABSTRACT
Acyl-CoA synthetase, which is one of the acid-thiol ligases (EC 6.2.1), plays key roles in metabolic and regulatory processes. This enzyme forms a carbon-sulfur bond in the presence of ATP and Mg(2+), yielding acyl-CoA thioesters from the corresponding free acids and CoA. This enzyme belongs to the superfamily of adenylate-forming enzymes, whose three-dimensional structures are analogous to one another. We here discovered a new reaction while studying the short-chain acyl-CoA synthetase that we recently reported (Hashimoto, Y., Hosaka, H., Oinuma, K., Goda, M., Higashibata, H., and Kobayashi, M. (2005) J. Biol. Chem. 280, 8660-8667). When l-cysteine was used as a substrate instead of CoA, N-acyl-l-cysteine was surprisingly detected as a reaction product. This finding demonstrated that the enzyme formed a carbon-nitrogen bond (EC 6.3.1 acid-ammonia (or amide) ligase (amide synthase); EC 6.3.2 acid-amino acid ligase (peptide synthase)) comprising the amino group of the cysteine and the carboxyl group of the acid. N-Acyl-d-cysteine, N-acyl-dl-homocysteine, and N-acyl-l-cysteine methyl ester were also synthesized from the corresponding cysteine analog substrates by the enzyme. Furthermore, this unexpected enzyme activity was also observed for acetyl-CoA synthetase and firefly luciferase, indicating the generality of the new reaction in the superfamily of adenylate-forming enzymes.
Subject(s)
Acetate-CoA Ligase/physiology , Amides/chemistry , Peptides/chemistry , Acetate-CoA Ligase/metabolism , Biochemistry/methods , Butyrates/chemistry , Carbon/chemistry , Cysteine/chemistry , Luciferases/metabolism , Magnesium/chemistry , Mass Spectrometry/methods , Models, Chemical , Nitrogen/chemistry , Pseudomonas/enzymology , Saccharomyces cerevisiae/enzymology , Sulfur/chemistryABSTRACT
Two open reading frames (nhpS and acsA) were identified immediately downstream of the previously described Pseudomonas chlororaphis B23 nitrile hydratase (NHase) gene cluster (encoding aldoxime dehydratase, amidase, the two NHase subunits, and an uncharacterized protein). The amino acid sequence deduced from acsA shows similarity to that of acyl-CoA synthetase (AcsA). The acsA gene product expressed in Escherichia coli showed acyl-CoA synthetase activity toward butyric acid and CoA as substrates, with butyryl-CoA being synthesized. From the E. coli transformant, AcsA was purified to homogeneity and characterized. The quality of the recombinant protein was verified by the NH2-terminal amino acid sequence and the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The apparent Km values for butyric acid, CoA, and ATP were 0.32 +/- 0.04, 0.37 +/- 0.02, and 0.22 +/- 0.02 mm, respectively. AcsA was shown to be a short-chain acyl-CoA synthetase, according to the catalytic efficiencies (kcat/Km) for various acids. The substrate specificity of AcsA was similar to those of aldoxime dehydratase, NHase, and amidase, the genes of which coexist in the same orientation in the gene cluster. P. chlororaphis B23 grew when cultured in a medium containing butyraldoxime as the sole carbon and nitrogen source. The activities of aldoxime dehydratase, NHase, and amidase were detected together with that of acyl-CoA synthetase under the culture conditions used. Moreover, on culture in a medium containing butyric acid as the sole carbon source, acyl-CoA synthetase activity was also detected. Together with the adjacent locations of the aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase genes, these findings suggest that the four enzymes are sequentially correlated with one another in vivo to utilize butyraldoxime as a carbon and nitrogen source. This is the first report of an overall "nitrile pathway" (aldoxime-->nitrile-->amide-->acid-->acyl-CoA) comprising these enzymes.
