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J. Biol. Chem., Vol. 279, Issue 9, 8242-8251, February 27, 2004

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Identification and Molecular Characterization of the -Ketoacyl-[Acyl Carrier Protein] Synthase Component of the Arabidopsis Mitochondrial Fatty Acid Synthase^*

Rie Yasuno, Penny von Wettstein-Knowles¶, and Hajime Wada||

From the Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan and the Department of Genetics, Molecular Biology Institute, University of Copenhagen, Oester Farimagsgade 2A, DK-1353 Copenhagen K, Denmark

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, -ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C₄-C₁₈ fatty acids exhibiting a bimodal distribution with peaks at C₈ and C₁₄-C₁₆. Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.

Received for publication, August 12, 2003 , and in revised form, November 26, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB073746.

^* This work was supported by grants-in-aid from the Japan Society for the Promotion of Science, the Program for Promotion of Basic Research Activities for Innovative Biosciences from the Bio-oriented Technology Research Advancement Institution, the Novo Nordisk Foundation, and the Danish Natural Science Research Council. 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 online version of this article (available at http://www.jbc.org) contains Supplemental Fig. 1.

^¶ To whom correspondence may be addressed. Fax: 45-3532-2113; E-mail: knowles{at}biobase.dk.

^|| To whom correspondence may be addressed. Tel./Fax: +81-3-5454-6656; E-mail: hwada{at}bio.c.u-tokyo.ac.jp.

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