HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 280, Issue 10, 8660-8667, March 11, 2005

This Article Full Text Full Text (PDF) All Versions of this Article: 280/10/8660    most recent M405686200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hashimoto, Y. Articles by Kobayashi, M. Search for Related Content PubMed PubMed Citation Articles by Hashimoto, Y. Articles by Kobayashi, M. Social Bookmarking                      What's this?

Nitrile Pathway Involving Acyl-CoA Synthetase

OVERALL METABOLIC GENE ORGANIZATION AND PURIFICATION AND CHARACTERIZATION OF THE ENZYME^*

Yoshiteru Hashimoto, Hideaki Hosaka, Ken-Ichi Oinuma, Masahiko Goda, Hiroki Higashibata, and Michihiko Kobayashi

From the 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

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 K_m 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 (k_cat/K_m) 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" (aldoximenitrileamideacidacyl-CoA) comprising these enzymes.

Received for publication, May 21, 2004 , and in revised form, December 27, 2004.

^* This work was supported in part by the 21st Century COE Program (www.tara.tsukuba.ac.jp/~coe21/) of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan; a grant-in-aid for scientific research from MEXT; the Industrial Technology Research Grant Program in 2002 of the New Energy and Industrial Technology Development Organization (NEDO) of Japan; the National Project on Protein Structural and Functional Analyses; and Research Grant (A) of the University Research Projects. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB125061.

Both authors contributed equally to this work.

To whom correspondence should be addressed. Fax: 81-29-853-4605 (Institute).

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				T. Sakashita, Y. Hashimoto, K.-I. Oinuma, and M. Kobayashi Transcriptional Regulation of the Nitrile Hydratase Gene Cluster in Pseudomonas chlororaphis B23 J. Bacteriol., June 15, 2008; 190(12): 4210 - 4217. [Abstract] [Full Text] [PDF]

				T. Abe, Y. Hashimoto, H. Hosaka, K. Tomita-Yokotani, and M. Kobayashi Discovery of Amide (Peptide) Bond Synthetic Activity in Acyl-CoA Synthetase J. Biol. Chem., April 25, 2008; 283(17): 11312 - 11321. [Abstract] [Full Text] [PDF]