Nitrile pathway involving acyl-CoA synthetase: overall metabolic gene organization and purification and characterization of the enzyme.
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.
Hashimoto
Yoshiteru
Y
Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
Hosaka
Hideaki
H
Oinuma
Ken-Ichi
K
Goda
Masahiko
M
Higashibata
Hiroki
H
Kobayashi
Michihiko
M
D001483:Base Sequence; D003062:Codon / Q000235:genetics; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D009570:Nitriles / Q000378:metabolism*; D016366:Open Reading Frames; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D003062:Codon; D017931:DNA Primers; D009570:Nitriles; D011994:Recombinant Proteins; D003066:Coenzyme A Ligases