Isolation and function of spinach leaf β-ketoacyl-[acyl-carrier-protein] synthases

Abstract

Crude spinach leaf extract readily forms the stearoyl derivative ofacyl-carrier-protein (ACP) when acetyl-ACP and malonyl-ACP are incubated together. Palmitoyl-ACP is also elongated by malonyl-ACP to stearoyl-ACP. When P-ketoacyl-ACP synthase {3-oxoacyl- [ACP] synthase; acyl-[ACP]:malonyl- [ACP] C-acyltransferase (decarboxylating), EC 2.3.1.41} is purified with decanoyl-ACP as the assay substrate, palmitoyl-ACP elongation activity is lost. When palmitoyl-ACP is the assay substrate, another protein is isolated that specifically elongates pal-.mitoyl-ACP to P-ketostearoyl-ACP but has no. activity towards decanoyl-ACP. The first protein is designated -ketoacyl-ACP synthase I and participates in the conversion of acetyl-ACP to palmitoyl-ACP, whereas the second-protein is designated (3-ketoacyl-ACP synthase II, and its substrate specificity is highly restricted to myristoyl-ACP and palmitoyl-ACP. The purification ofsynthase II is described, and its activity is compared to synthase L Reconstitution experiments with the highly purified nonassociated enzymes in fatty acid synthesis plus synthases I and II clearly demonstrate the roles of these two proteins in fatty acid synthesis. Vma than did synthase I with palmitoyl-ACP. The E. coli synthase I is a dimer with -a molecular weight of 80,000, whereas synthase II is' a dimer with a molecular weight of 85,000 (2). It was proposed that synthase II played a role in modulating fatty acid synthesis and, hence, fatty acid composition when E. coli' were grown at different temperatures (2).In 1974, Jaworski et al. (3) reported on the conversion ofpalmitoyl-ACP to stearoyl-ACP by an extract ofmaturing safflower seeds. The system appeared to differ from the de novo fatty acid synthesis (FAS) enzyme system (with acetyl-ACP as the primer substrate) in that (i) it was inactivated at 370C, whereas the de novo system that formed palmitoyl-ACP remained fully active, and (ii) the elongation system was relatively insensitive to cerulenin, whereas the de novo-system was highly sensitive. These and other data suggested a de novo system responsible for the conversion of C2 to C16 fatty acids, and a restrictive elongation system that specifically converted C16 to C18 fatty acids.Because the plant FAS (PFAS) enzyme system in the spinach chloroplast is of a nonassociated type (4), the opportunity to separate the de novo and the elongation systems was quite feasible. Therefore, this investigation reports on (i) the isolation oftwo distinct synthases I and II from spinach leafextracts with unusual substrate specificities and (ii) reconstitution experiments with highly purified enzymes of the PFAS system to which synthases I and II alone and together were added to determine their role in. FAS. While this work. was in progress, Schfiz et at (5) reported on the isolation of a &k-etoacyl-ACP synthase for suspension cells of parsley, which appears to be identical to synthase I of spinach leaf. MATERIALS AND METHODSPurification ofSpinach f-Ketoacyl-ACP Synthases I and II.Methods for preparin...

Quantitative trait loci (QTLs) analysis of palm oil fatty acid composition in an interspecific pseudo-backcross from Elaeis oleifera (H.B.K.) Cortés and oil palm (Elaeis guineensis Jacq.)

“…Also, the gene KAS I seems to be a good candidate underlined by the putative QTL for C14:0 on LG 15. KAS II (chain lengthening of C16:0 to C18:0) mapped at that same locus could also support the accuracy of this QTL, as it has been demonstrated that KAS II enzyme can use C10:0 to C14:0 as substrates, although substrate C16:0 is preferred (Shimakata and Stumpf 1982). Other gene SNP markers were mapped outside QTLs.…”

A Mutant of Arabidopsis Deficient in the Elongation of Palmitic Acid

“…From our initial observations, we inferred that the mutant is partially deficient in activity of KASII. The choice of KASII as the putative target of the mutation rather than 3-ketoacyl-ACP reductase, 3-hydroxyacyl-ACP dehydratase, or enoyl-ACP reductase was based on the observation that these three additional enzymes of the fatty acid synthesis cycle probably also act in the synthesis of 16:O-ACP, whereas KASII is thought to be specific for the further elongation of 16:O-ACP (Shimikata and Stumpf, 1982). The defect in KASII in the mutant was confirmed by direct assay of the enzyme.…”

The Critical Requirement for Linolenic Acid Is Pollen Development, Not Photosynthesis, in an Arabidopsis Mutant.

“…of polyunsaturated fatty acids (Roughan et al., 1980;Browse and Somerville, 1991). 60th pathways are initiated by the synthesis of 16:O-acyl carrier protein (ACP) and 18:l-ACP by the combined action of a type II fatty acid synthase (Shimakata and Stumpf, 1982) and a soluble stearoyl-ACP desaturase (McKeon and Stumpf, 1982;Shanklin and Somerville, 1991) located in the chloroplasts or other plastids. The prokaryotic pathway (Roughan et al, 1980) located in the chloroplast inner envelope uses 18:l-ACP and 16:O-ACP for the sequential acylation of glyceral-3-phosphate and synthesis of glycerolipid components for the chloroplast membranes (Frentzen, 1993;Joyard et al, 1993).…”