Identification and molecular characterization of the beta-ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid synthase.
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, beta-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 C(4)-C(18) fatty acids exhibiting a bimodal distribution with peaks at C(8) and C(14)-C(16). 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.
Yasuno
Rie
R
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.
von Wettstein-Knowles
Penny
P
Wada
Hajime
H
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D015153:Blotting, Western; D002569:Cerulenin / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D018744:DNA, Plant / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis; D017353:Gene Deletion; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014162:Transfection
D018076:DNA, Complementary; D018744:DNA, Plant; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002569:Cerulenin; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D064429:Fatty Acid Synthases