A Plastidial Lysophosphatidic Acid Acyltransferase from Oilseed Rape1

Abstract

The biosynthesis of phosphatidic acid, a key intermediate in the biosynthesis of lipids, is controlled by lysophosphatidic acid (LPA, or 1-acyl-glycerol-3-P) acyltransferase (LPAAT, EC 2.3.1.51). We have isolated a cDNA encoding a novel LPAAT by functional complementation of the Escherichia coli mutant plsC with an immature embryo cDNA library of oilseed rape (Brassica napus). Transformation of the acyltransferase-deficient E. coli strain JC201 with the cDNA sequence BAT2 alleviated the temperature-sensitive phenotype of the plsC mutant and conferred a palmitoyl-coenzyme A-preferring acyltransferase activity to membrane fractions. The BAT2 cDNA encoded a protein of 351 amino acids with a predicted molecular mass of 38 kD and an isoelectric point of 9.7. Chloroplast-import experiments showed processing of a BAT2 precursor protein to a mature protein of approximately 32 kD, which was localized in the membrane fraction. BAT2 is encoded by a minimum of two genes that may be expressed ubiquitously. These data are consistent with the identity of BAT2 as the plastidial enzyme of the prokaryotic glycerol-3-P pathway that uses a palmitoyl-ACP to produce phosphatidic acid with a prokaryotictype acyl composition. The homologies between the deduced protein sequence of BAT2 with prokaryotic and eukaryotic microsomal LAP acytransferases suggest that seed microsomal forms may have evolved from the plastidial enzyme.Phosphatidic acid is a key intermediate in the biosynthesis of phospho-and glycerolipids, essential components of all cellular membranes and of triacylglycerols. In most plant species, palmitoyl-ACP and oleoyl-ACP are the predominant products of de novo fatty acid biosynthesis in the chloroplast (Ohlrogge et al., 1993). These fatty acids may enter the prokaryotic pathway of lipid biosynthesis by transfer of the acyl group from ACP to glycerol-3-P, which is mediated by a stromal glycerol-3-P acyltransferase to form LPA or to position sn-2 of glycerol-3-P mediated by LPAAT (EC 2.3.1.51) to form phosphatidic acid. The phosphatidic acid produced by the prokaryotic pathway of the chloroplast is then used to produce phosphatidylglycerol (for review, see Ohlrogge and Browse, 1995) or is dephosphorylated to diacylglycerol, from which the glycodiacylglycerols characteristic of the thylakoid membrane are derived (for review, see Douce and Joyard, 1990).Alternatively, the elongation of fatty acids may be terminated by the action of an acyl-ACP thioesterase, which hydrolyzes the acyl-ACP to release a free fatty acid, which then leaves the plastid. The fatty acid in the form of an acyl-CoA thioester may participate in the synthesis of glycerolipids via the eukaryotic pathway located at the ER. The phosphatidic acid of the cytoplasmic pathway is used to produce the phospholipids characteristic of extrachloroplastic lipids. The phosphatidylcholine produced by the eukaryotic pathway is a substrate for desaturation, after which the diacylglycerol moiety may be returned to the chloroplast. The contribution of each pathway to the syn...

Proteomics of the Chloroplast Envelope Membranes from Arabidopsis thaliana

“…75). Indeed, we identified 2-lysophosphatidate acyltransferase (76), an enzyme catalyzing the transfer of 16:0 (from 16:0-ACP) to the sn-2 position of lysophosphatidic acid, leading to the synthesis of phosphatidic acid, the precursor for chloroplast glycerolipids. MGD1, one of the three Arabidopsis monogalactosyldiacylglycerol (MGDG) synthases, the last committed step in MGDG biosynthesis, was identified in our previous study on spinach (7).…”

The Significance of C16 Fatty Acids in the sn-2 Positions of Glycerolipids in the Photosynthetic Growth of Synechocystis sp. PCC6803

“…Evolution of Higher-Plant LPAAT Bourgis et al (1999) suggested that the alignment of the deduced amino acid sequences has the same orthologous relationship in sll1848 and plastidial LPAAT from Brassica napus (BAT2). This idea is confirmed by the observation that both LPAAT1/ATS2 from Arabidopsis and sll1848 from Synechocystis sp PCC6803 show a preference for 16:0 ACP (and 16:0 CoA in vitro).…”

Metabolic Interactions between the Lands Cycle and the Kennedy Pathway of Glycerolipid Synthesis in Arabidopsis Developing Seeds

“…A fascinating aspect of glycerolipid biosynthesis is that there often exists an asymmetric distribution of fatty acid (FA) molecules between the sn-1 and sn-2 positions: Saturated and monounsaturated FAs are normally found at sn-1, whereas polyunsaturated fatty acids (PUFAs) are enriched at sn-2. Pioneering work on plant acyltransferases has shown that GPATs and LPAATs are not only stereo-specific with regard to acylation of the glycerol backbone but also selective in terms of acyl-CoA donors (Griffiths et al, 1985;Ohlrogge and Browse, 1995;Oo and Huang, 1989;Bourgis et al, 1999;Kim and Huang, 2004;Kim et al, 2005). Therefore, the substrate specificity of the acyltransferases of the Kennedy pathway is one of the determining factors of the composition of glycerolipid molecules.…”