3-Ketoacyl-Acyl Carrier Protein Synthase III from Spinach (Spinacia oleracea) Is Not Similar to Other Condensing Enzymes of Fatty Acid Synthase

Tai, Heeyoung; Jaworski, Jan C.

doi:10.1104/pp.103.4.1361

Cited by 51 publications

(29 citation statements)

References 25 publications

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“…In this study, we report the isolation of the Arabidopsis FAE7 gene by a third genetic approach, heterologous transposon tagging with the maize element Activator (Ac). The FAEl protein predicted from the corresponding cDNA sequence shares homology with chalcone synthase (CHS) and stilbene synthase (STS), two condensing enzymes that also catalyze the addition of malonyl COA molecules to a starter COA ester, and with p-ketoacyl-acyl carrier protein synthase 111 (KASIII) from Escherichia coli (Tsay et al, 1992) and spinach (Tai and Jaworski, 1993). This suggests that the product of the fAE7 gene is in fact a synthase or condensing enzyme.…”

Section: Introductionmentioning

confidence: 89%

Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator.

et al. 1995

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The FATTYAClD ELONGATlONl (FAEl) gene of Arabidopsis is required for the synthesis of very long chain fatty acids in the seed. The product of the FAEl gene is presumed to be a condensing enzyme that extends the chain length of fatty acids from C18 to C20 and C22. We report here the cloning of FAE7 by directed transposon tagging with the maire element Activator (Ac). An unstable fael mutant was isolated in a line carrying Ac linked to the FAEl locus on chromosome 4. Cosegregation and reversion analyses established that the new mutant was tagged by Ac. A DNA fragment flanking Ac was cloned by inverse polymerase chain reaction and used to isolate FAEl genomic clones and a cDNA clone from a library made from immature siliques. The predicted amino acid sequence of the FAEl protein shares homology with those of other condensing enzymes (chalcone synthase, stilbene synthases, and p-ketoacyl-acyl carrier protein synthase lll), supporting the notion that FAE7 is the structural gene for a synthase or condensing enzyme. FAEl is expressed in developing seed, but not in leaves, as expected from the effect of the fael mutation on the fatty acid compositions of those tissues.

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Section: Introductionmentioning

confidence: 89%

Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator.

et al. 1995

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“…Proteins were fractionated by 10% SDS-PAGE and transferred to nitrocellulose (0.45-m membrane). Antisera raised against castor biotin carboxylase (Roesler et al, 1996), avocado stearoyl-ACP desaturase (Shanklin and Somerville, 1991), and spinach 3-ketoacyl-ACP synthase III (Tai and Jaworski, 1993) were prepared as previously described. Anti-biotin antibody was purchased from Sigma.…”

Section: Immunoblot Analysismentioning

confidence: 99%

Expression of Lauroyl–Acyl Carrier Protein Thioesterase in Brassica napus Seeds Induces Pathways for Both Fatty Acid Oxidation and Biosynthesis and Implies a Set Point for Triacylglycerol Accumulation

1998

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Expression of a California bay lauroyl-acyl carrier protein thioesterase (MCTE) in developing seeds of transgenic oilseed rape alters the fatty acid composition of the mature seed, resulting in up to 60 mol% of laurate in triacylglycerols. In this study, we examined the metabolism of lauric acid and 14 C-acetate in developing seeds of oilseed rape that express high levels of MCTE. Lauroyl-CoA oxidase activity but not palmitoyl-CoA oxidase activity was increased several-fold in developing seeds expressing MCTE. In addition, isocitrate lyase and malate synthase activities were sixand 30-fold higher, respectively, in high-laurate developing seeds. Control seeds incorporated 14 C-acetate almost entirely into fatty acids, whereas in seeds expressing MCTE, only 50% of the label was recovered in lipids and the remainder was in a range of water-soluble components, including sucrose and malate. Together, these results indicate that the pathways for ␤ -oxidation and the glyoxylate cycle have been induced in seeds expressing high levels of MCTE. Although a substantial portion of the fatty acid produced in these seeds is recycled to acetyl-CoA and sucrose through the ␤ -oxidation and glyoxylate cycle pathways, total seed oil is not reduced. How is oil content maintained if lauric acid is inefficiently converted to triacylglycerol? The levels of acyl carrier protein and several enzymes of fatty acid synthesis were increased two-to threefold at midstage development in high-laurate seeds. These results indicate that a coordinate induction of the fatty acid synthesis pathway occurs, presumably to compensate for the lauric acid lost through ␤ -oxidation or for a shortage of long-chain fatty acids.

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“…This is mainly due to the complexity of the membrane fractions used as the enzyme source and the presence of a high level of background activities. Currently, there is only one report of an extensively purified membrane-bound KCS, jojoba KCS [13], and its characterization was limited to the substrate specificity.Despite their membrane-bound nature, some domains of the elongase condensing enzymes have limited homology to two soluble condensing enzymes: plant chalcone synthases [16] and 3-ketoacyl-ACP synthase III (KAS III) from plants [17] and Escherichia coli [18]. The reaction mechanism of the soluble condensing enzymes has been extensively studied, and recently the crystal structures of chalcone synthase [19] and all isoforms of KASs from E. coli [20][21][22][23] have been published.…”

mentioning

confidence: 99%

“…Despite their membrane-bound nature, some domains of the elongase condensing enzymes have limited homology to two soluble condensing enzymes: plant chalcone synthases [16] and 3-ketoacyl-ACP synthase III (KAS III) from plants [17] and Escherichia coli [18]. The reaction mechanism of the soluble condensing enzymes has been extensively studied, and recently the crystal structures of chalcone synthase [19] and all isoforms of KASs from E. coli [20][21][22][23] have been published.…”

mentioning

confidence: 99%

Engineering and mechanistic studies of theArabidopsisFAE1 β‐ketoacyl‐CoA synthase, FAE1 KCS

Ghanevati

Jaworski

2002

European Journal of Biochemistry

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The Arabidopsis FAE1 b-ketoacyl-CoA synthase (FAE1 KCS) catalyzes the condensation of malonyl-CoA with longchain acyl-CoAs. Sequence analysis of FAE1 KCS predicted that this condensing enzyme is anchored to a membrane by two adjacent N-terminal membrane-spanning domains. In order to characterize the FAE1 KCS and analyze its mechanism, FAE1 KCS and its mutants were engineered with a His 6 -tag at their N-terminus, and expressed in Saccharomyces cerevisiae. The membrane-bound enzyme was then solubilized and purified to near homogeneity on a metal affinity column. Wild-type recombinant FAE1 KCS was active with several acyl-CoA substrates, with highest activity towards saturated and monounsaturated C16 and C18. In the absence of an acyl-CoA substrate, FAE1 KCS was unable to carry out decarboxylation of [3][4][5][6][7][8][9][10][11][12][13][14] C]malonyl-CoA, indicating that it requires binding of the acyl-CoA for decarboxylation activity. Site-directed mutagenesis was carried out on the FAE1 KCS to assess if this condensing enzyme was mechanistically related to the well characterized soluble condensing enzymes of fatty acid and flavonoid syntheses. A C223A mutant enzyme lacking the acylation site was unable to carry out decarboxylation of malonyl-CoA even when 18:1-CoA was present. Mutational analyses of the conserved Asn424 and His391 residues indicated the importance of these residues for FAE1-KCS activity. The results presented here provide the initial analysis of the reaction mechanism for a membrane-bound condensing enzyme from any source and provide evidence for a mechanism similar to the soluble condensing enzymes.Keywords: very long chain fatty acids; fatty acid elongation; condensation mechanism.Fatty acids with greater than 18 carbon atoms (very long chain fatty acids, VLCFA) are precursors of many biologically important compounds such as sphingolipids [1,2], waxes [3], and triacylglycerols in many seed oils [4]. Biosynthesis of VLCFA in plants and animals, is dependent on the activity of a membrane-bound fatty acid elongation system which consists of four component reactions similar to fatty acid synthase. The first reaction of elongation involves condensation of malonyl-CoA with a long chain acyl substrate producing a b-ketoacyl-CoA. Subsequent reactions are reduction to b-hydroxyacyl-CoA, dehydration to an enoyl-CoA, followed by a second reduction to form the elongated acyl-CoA [5]. In both animals and plants, the initial condensation reaction is believed to be the ratelimiting step [6,7].In Arabidopsis, FAE1 codes for a b-ketoacyl-CoA synthase (FAE1 KCS) which is expressed exclusively in the seed and catalyzes the initial condensation step in the elongation pathway [8]. Based on fatty acid profiles of transgenic plants and yeast, it has been reported that FAE1 KCS has a substrate preference for C18:1, producing eicosenoic (C20:1) acid as the major product and erucic acid (C22:1) as a minor product [7].A prominent feature of b-ketoacyl-CoA synthases involved in VLCFA biosynthesis is their membrane-bound...

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3-Ketoacyl-Acyl Carrier Protein Synthase III from Spinach (Spinacia oleracea) Is Not Similar to Other Condensing Enzymes of Fatty Acid Synthase

Cited by 51 publications

References 25 publications

Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator.

Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator.

Expression of Lauroyl–Acyl Carrier Protein Thioesterase in Brassica napus Seeds Induces Pathways for Both Fatty Acid Oxidation and Biosynthesis and Implies a Set Point for Triacylglycerol Accumulation

Engineering and mechanistic studies of theArabidopsisFAE1 β‐ketoacyl‐CoA synthase, FAE1 KCS

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