Phosphatidylcholine desaturation

Figure 21-3 Major pathways of synthesis of fatty acids and glycerolipids in the green plant Arabidopsis. The major site of fatty acid synthesis is chloroplasts. Most is exported to the cytosol as oleic acid (18 1). After conversion to its coenzyme A derivative it is converted to phosphatidic acid (PA), diacylglycerol (DAG), and the phospholipids phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE). Desaturation also occurs, and some linoleic and linolenic acids are returned to the chloroplasts. See text also. From Sommerville and Browse.106 See also Figs. 21-4 and 21-5. Other abbreviations monogalactosyldiacylglycerol (MGD), digalactosyldiacylglycerol (DGD), sulfolipid (SL), glycerol 3-phosphate (G3P), lysophosphatidic acid (LPA), acyl carrier protein (ACP), cytidine diphosphate-DAG (CDP-DAG).

Mutant T4 is considered to be defective in A9 desaturation [155]. Its mycelial fatty acids included 38% stearic acid (18 0) mainly as free fatty acids, the level being only 5% for the wild type. Since the fatty acid composition of phosphatidylethanolamine and phosphatidylcholine at sn-2 position included more than 75% of PUFA, the accumulation of free fatty acids was supposed to be due to the lack of PUFAs. 

Triricinolein (RRR) constitutes about 70% of TAG in castor oil (Lin et al., 2003). Recently, a new tetraacylglycerol, (12-ricinoleoylricinoleoyl)diricinoleo ylglycerol (RRRR), in castor oil was identified and its content was about 0.5% (Lin et al., 2006). These two AG contain ricinoleate only without other FA. The biosynthetic pathway of RRR and RRRR in the castor bean has been established and the key enzymatic steps driving ricinoleate into RRR and RRRR have been identified (Lin et al., 1998, 2000, 2002, 2006) as shown in Figure 25.1. Oleate of oleoyl-CoA is incorporated into 2-oleoyl-phosphatidylcholine (2-oleoyl-PC) by lysophosphatidylcholine-acyltransfer-ase and then hydroxylated to 2-ricinoleoyl-PC by 12-oleoyl-hydroxylase in castor microsomes (Fig. 25.1) (Lin et al., 1998). 2-Oleoyl-PC is also desaturated to 2-linoleoyl-PC and then to 2-linolenoyl-PC (Lin et al, 1998) by desaturases. 

The most abundant membranes in nature are the thylakoids inside chloroplasts of green plants. A surprising amount of lipid traffic is involved in the assembly of these membranes. Almost all the acyl chains that form the core of the photosynthetic membranes are first produced by fatty acid synthase in the chloroplast. In most plants these acyl chains are then exported to the ER where they become esterified to glycerol, desaturated while they are part of phosphatidylcholine and then are returned to the plastid. The exact mechanisms for the export and return of acyl chains are still uncertain although much has been learned (Chapter 17) [10]. The export from plastids across the chloroplast envelope membranes is known to involve a fatty acid intermediate, and probably is a channeled or facilitated process rather than free diffusion because only a tiny pool of free fatty acid is ever detected (A. Koo, 2004). An acyl-CoA synthetase on the envelope membrane is believed to quickly convert the exported fatty acid to a thioester form that is then a substrate for acyltransferases. Transfer of acyl groups to the ER may occur via diffusion of the acyl-CoAs however, recent evidence suggests this initial acyl transfer reaction involves acylation of lyso-phosphatidylcholine and may occur at the chloroplast envelope. 

After the desaturation of 18 1 groups of phosphatidylcholine to 18 2 and 18 3, a major proportion of the leaf cell acyl chains returns to the chloroplast and becomes part of the glycolipid core of the photosynthetic membranes. Most research suggests that two acyl... 

Demandre, C., Tremolieres, A., Justin, A hi., and Mazliak, P. (1986) Oleate Desaturation in Six Phosphatidylcholine Molecular Species from Potato Tuber Microsomes, Biochim. Bio-phys. Acta 877,38(U386. 

In plants the further desaturation of linoleate to a-linolenate uses diacylgalactosylglycerol as a substrate in leaf tissue (Jones and Harwood, 1980). Because this lipid is located in chloroplasts whereas the previous desaturation takes place mainly outside the chloroplasts (on phosphatidylcholine which is the major lipid of the endoplasmic reticulum), the complete desaturation sequence requires the co-operation of subcellular organelles. This process has been discussed by Harwood (1980) and evidence for it has also been obtained by Ohnishi and Yamada (1982). The overall synthesis of a-linolenate in photosynthetic plant cells is summarized in Fig. 11.5... 

The problem becomes even more confusing when one considers the evidence for the final desaturation in plants, namely, the conversion of linoleic acid to a-linolenic acid. Based mostly on in vivo rates of appearance of [ Cjlinolenate in complex lipids when the substrate is [ jacetate, [ Cjoleate, or [ K ]linoleate, a number of workers concluded that the substrate was /3-linoleoyl phosphatidylcholine and the product /3-linolenoyl phosphatidylcholine (Roughan, 1970, 1975 Willemot and Verret, 1973 Slack and Roughan, 1975 Slack et al., 1976, 1977). Other workers have shown (Heinz and Harwood, 1977 Siebertz and Heinz, 1977 Bolton and Harwood, 1978 Hawke and Stumpf, 1980) that, while j3-linoleoyl phosphatidylcholine accumulates under in vivo conditions, /3-linolenoyl phosphatidylcholine cannot be detected in contrast, /3-linolenoyl monogalactosyldiglyc-eride appears to be the primary product. 

Fig. 11. Various hypotheses proposed by which higher plants may attain high levels of unsaturated fatty acids in their chloroplast membrane galactolipids. (a) Phosphatidylcholine acts as a carrier molecule involved in the desaturation, (b) Desaturation of fatty acids occurs after formation of the galactolipid molecule, (c) Desaturation occurs before formation of the galactolipid molecule. In the first hypothesis, all the desaturases involved are confined in the chloroplast in the second hypothesis, the conversion of 18 1 to 18 2 is maximal in microsomes," whereas desaturation of 18 2 to 18 3 is highest in chloroplast membranes, (d) Deacylation-reacylation mechanism in which X can be a CoA-thioester, a polar lipid, etc. D, Desaturases T, acyl-ACP thioesterase e.r., endoplasmic reticulum.

Fatty acid synthase (FAS) constitutes a multisubunit complex in the plastid where it catalyzes ordered synthesis of fatty acids, initiated from acetyl CoA and malonyl CoA [146, 147]. Stepwise FAS activity generates the products 16 0-acyl carrier protein (AGP) and 18 0-ACP. Most of the 18 0-ACP is desaturated by a soluble stearoyl-ACP desaturase, yielding 18 1 D9-ACP [148]. Acyl-ACP thioesterases release AGP from 16 0-AGP and 18 0-AGP the deacylated fatty acids exit the plastid and are then esterified with coenzyme A (GoA) to form respective acyl-GoAs [148]. These acyl moieties are then esterified to phosphatidylcholine (PQ and then undergo desaturation by D12- and D15-desaturases to yield the essential fatty acids, linoleic acid, and a-linolenic acid [148-150], All higher plants have the enzymes for synthesizing the G18 PUFAs linoleic acid and a-linolenic acid. The primary genes involved in PUFA biosynthesis have been reviewed [151],... 

All these studies suggested that the lipid must be playing some kind of structure role in the desaturation process and this supposition has been amply supported by subsequent work from several groups. In common with several other groups (Dufourcq et al., 1975 Faucon et al., 1976 Rogers Strittmatter, 1975 Robinson Tanford, 1975), we have chosen to examine the cytochrome -phosphatidylcholine vesicle system. We would like to use this system both to understand the desaturase and also to examine lipid-protein interaction in general. Two specific aspects of our studies I would like to discuss were initiated to characterize the mechanism of binding of cytochrome bs to lipid vesicles. 

Serghini-Caid, H., Demandre, C., Justin, A-M. and Mazliak, P. (1988) Oleoyl-phosphatidylcholine molecular species desaturated in pea leaf microsomes - possible substrates of oleate-desaturase in other green leaves. Plant Sci. 54, 93-101. 

I8j C. DEMANDRE, A. TREMOLIERES, A.M. JUSTIN and P. MAZLIAK - Oleate desaturation in six phosphatidylcholine molecular species from potato tuber microsomes. Biochim. Biophys. Acta, 71 380-386 (1986). 

The biosynthetic pathways leading to alpha-Itnolenic acid in plants are poorely understood. This Is partly due to the lability of the a 15 desaturase(s) (catalysing the conversion of 18 2 to 18 3) which has prohibited any detailed in-vitro study of the enzyme(s). The present concept is that linolenic acid in plants is synthesised from linoleate while this acid is esterifled in a complex lipid. In the leaf system the major lipid undergoing a I 5 desaturation is believed to be monogalactosyldiacylglycerol [4] and in oil seeds, phosphatidylcholine [5,6). 

The desaturases from hen liver (converting 18 0 to 18 1) and from safflower (converting 18 1 to 18 2) have been shown to operate on Co A derivatives and not on the phosphatidylcholines. Safflower desaturase, however, contains an active acyltransferase, so that both oleic and linoleic CoA esters are readily converted to phosphatidylcholines. From a large amount of data on the desaturation step in the production of polyunsaturated acids by animals, Brenner describes a hypothetical model of a 6-olefinase. 

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