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

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, ␤-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.Fatty acids are synthesized by the enzymatic reactions of acetyl-CoA carboxylase (ACCase) 1 and fatty acid synthase (FAS). FAS enzyme complexes are classified into two groups based on their structural forms and organization. Complexes consisting of multifunctional polypeptides encoded by one or two genes (type I) are present in the cytoplasm of animals and fungi (1, 2), whereas those composed of monofunctional enzymes (type II) are present in most bacteria and plant plastids (3, 4) as well as in mitochondria, as will be detailed below. The incipient reaction of fatty acid synthesis is catalyzed by ACCase to form malonyl-CoA from acetyl-CoA, the initial carbon source. The first FAS activity, malonyl-CoA:ACP transacylase (MCAT), transfers the malonyl group from CoA to acyl carrier protein (ACP) to form the donor substrate malonyl-ACP that provides the C 2 -units for elongation. Repetitive elongation cycles accomplished by FAS start with condensation of the acyl primer substrate with the C 2 -unit to give a ␤-ketoacyl-ACP. The introduced ␤-keto group is then removed by three reactions, a ␤-keto reduction, a ␤-dehydration, and an enoyl reduction. The resulting saturated acyl-ACP serves as a substrate for the next extension. Most frequently seven or eight cycles yield palmitoyl (C 16 )-ACP and stearoyl (C 18 )-ACP, respectively. Additional FAS ac...

Mitochondrial Protein Lipoylation Does Not Exclusively Depend on the mtKAS Pathway of de Novo Fatty Acid Synthesis in Arabidopsis

“…These four lines of evidence, recombination mapping, phenotype analysis of T-DNA insertion lines, complementation analysis, and sequencing, evidently show that the gld1 (mtkas-1) locus is identical with the singlecopy gene encoding the recently predicted (Mekhedov et al, 2000) and identified (Yasuno et al, 2004) Arabidopsis type II mitochondrial fatty acid synthase, mtKAS. Primed with malonyl-ACP, mtKAS catalyzes both the initial condensation step and chain elongation in mitochondrial acyl-ACP biosynthesis and represents the only known source of octanoyl-ACP in plant mitochondria (Yasuno et al, 2004).…”

“…Primed with malonyl-ACP, mtKAS catalyzes both the initial condensation step and chain elongation in mitochondrial acyl-ACP biosynthesis and represents the only known source of octanoyl-ACP in plant mitochondria (Yasuno et al, 2004). Elimination of mitochondrial octanoyl-ACP synthesis thus must exert deleterious effects on H protein and GDC, explaining the heavily disturbed photorespiratory metabolism of all three mtkas mutants.…”

“…Here, we report on the identification of the gld1 locus (now designated mtkas-1) and show that it resides on gene At2g04540 encoding b-ketoacyl-[acyl carrier protein (ACP)] synthase (mtKAS; EC 2.3.1.41), a key enzyme of mitochondrial fatty acid biosynthesis (Wada et al, 1997;Gueguen et al, 2000;Yasuno et al, 2004). One of the major products of mtKAS, octanoyl-ACP, is required for the lipoylation of essential mitochondrial proteins including the H protein of GDC and the dihydrolipoyl acyltransferase (E2) subunits of the pyruvate dehydrogenase (PDH) and a-ketoglutarate dehydrogenase (KGDH) multienzyme complexes.…”

β‐Ketoacyl‐ACP synthase I/II from Plasmodium falciparum (PfFabB/F)—Is it B or F?

“…E. coli CY244 cells were kind gift from Prof. Knowles (Copenhagen University, Denmark). CY244 cells harbor mutations in the alleles coding for elongation condensing enzymes (20). The mutation in fabB allele results in Ala329Val substitution resulting in temperature sensitivity.…”

“…Thus, PfFabB/F seems to be closer to FabF class of enzymes. We decided to elucidate whether PfFabB/F functions like FabB or FabF and for this study E. coli CY244 cells were used (20). E. coli CY244 cells were kind gift from Prof. Knowles (Copenhagen University, Denmark).…”