Abstract
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.
Highlights
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
Isolation of a cDNA Encoding mtKAS—The T1O3.5 sequence in GenBankTM was previously suggested to code for a ketoacyl-[acyl carrier protein] (ACP) synthase (KAS) participating in mitochondrial fatty acid synthesis in A. thaliana [43]
With the pfabB and pfabF transformant extracts, as expected, predominantly long chain acyl-acyl carrier protein] (ACP), both saturated and unsaturated, were produced (Fig. 4B, lanes 3 and 4). These results demonstrate that mtKAS can catalyze many of the condensation reactions in fatty acid synthesis, and that it is compatible with all relative components of the E. coli FAS
Summary
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. 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. MtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required. The -ketoacyl-ACP synthase (KAS) components of the type II FAS enzyme complexes present in bacteria and plant plastids carry out the condensation steps in fatty acid synthesis using a Claisen reaction consisting of three parts: (i) transfer of ACP bound acyl primer substrate to the active site cysteine of KAS, (ii) decarboxylation of malonyl-ACP to form the acetylACP carbanion, and (iii) condensation of the carbanion with the carbonyl carbon of the acyl primer substrate. KAS IV enzymes in some seeds, such as those from Cuphea sp., show a marked preference for medium (C6–C14) acyl chains [8]
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