Prokaryotic pathway

The evolutionary relationships between these two pathways were cemented by the determination of the three-dimensional structure of ThiS, which revealed a structure very similar to that of ubiquitin, despite being only 14% identical in amino acid sequence (Figure 23.10). Thus, a eukaryotic system for protein modification evolved from a preexisting prokaryotic pathway for coenzyme biosynthesis. 

Enzymes of the prokaryotic pathway are localized in plastids, whereas enzymes of the eukaryotic pathway - in the cytosol and ER. In the prokaryotic pathway, FA acyls are directly transferred from AGP to G3P, whereas in the eukaryotic pathway, FA are separated from ACP by acyl-ACP thioesterases and released free FA then are transported in the cytoplasm, where they are converted into acyl-CoA. During the synthesis of membrane and storage lipids acyl groups are used in the ER by acyltransferases of the eukaryotic G3P pathway [66]. In dependence on subcellular localization, these enzymes may differ in their structure, thus forming independent clusters in phylogenetic investigations [67]. 

Introduction Primary Fatty Acids Fatty Acids of Plant Vegetative Parts Biosynthesis Fatty Acid Biosynthesis The Two-Pathway Model of Lipid Biosynthesis The Second 3-Ketoacyl ACP Synthase Isozyme Biosynthesis of Unsaturated Fatty Acids The Prokaryotic Pathway The Eukaryotic Pathway Biosynthesis of Triacylglycerides Degradation of Fatty Acids Unusual Fatty Acids in Plants Fatty Acids from Unusual Starter Units Fatty Acids with Unusual Patterns of Unsaturation Hydroxy Fatty Acids Epoxy Fatty Acids... 

Research since about 1980 indicates that the leaves of higher plants utilize two distinctive pathways for glyceroli-pid biosynthesis. Palmitoleic acid (Cie) (13) and oleic acid (Ci8 i) (3) are synthesized de novo in the chloroplast and may be used directly for the bios)mthesis of chloroplast lipids via the prokaryotic pathway (see below) or exported from the chloroplast as CoA esters that are used in the eukaryotic pathway primarily at sites on the endoplasmic reticulum (Browse and Somerville, 1991). However, in all higher plants, a proportion of the diglycerol moiety of phosphatidylcholine, synthesized by the eukaryotic pathway, again enters the chloroplast where it contributes to the production of thy-lakoid membranes (Browse and Somerville, 1991). 

In many families of angiosperms, phosphatidylglycerol (9) is the only major product of the prokaryotic pathway. Other chloroplast lipids in these plants are synthesized only by the eukaryotic pathway. In some primitive angiosperm families, both pathways contribute to the s)mthesis of mono-galactosyldiacylglycerol (10) and other lipids (Browse and Somerville, 1991). 

Lipids from the prokaryotic pathway of both leaves and seeds contain only oleic and palmitic acids when they are first synthesized in the chloroplast or proplastid (Browse and Somerville, 1991). 

The results of the first characterization steps concerning this new Arabidopsis mutant with reduced 16 3 levels demonstrate that other mutations than actl can (partly) block the prokaryotic pathway of lipid synthesis. We are currently investigating the two hypotheses indicated above in order to identify the biochemical step affected by the mutation in JB19. 

Membrane lipids in 77825-83 contained significantly higher levels of saturates than the host canola variety, A112. Phospholipids, which comprise the majority of membrane lipid in mature seed, contained 23 mol% saturates, with 15 mol% stearate. Glycolipids in mature seed, plastid lipids probably remaining from when the embryo was green, contained 33 mol% saturates with 18 mol% stearate. These lipids may be synthesized via the prokaryotic pathway which incorporates 16 0 at sn-2, and potentially could have stearate at sn-2 as well as sn-1. 

In plants, de novo fatty acid biosynthesis occurs exclusively in the stroma of plastids, whereas, with the exception of plastidial desaturation, modification of fatty acid residues including further desaturation and triacylglrycerol (TAG) assembly are localized in the cytosol/endoplasmic reticulum (ER). The primary fatty acids formed in the plastid (palmitic, stearic, and oleic acid) are used in the plastidic prokaryotic pathway for membrane lipid synthesis or diverted to the cytoplasmic eukaryotic pathway for the synthesis of membrane lipids or storage TAGs (1). Movement of glycerolipids is believed to occur in the reverse direction between the cytosol/ER and the plastids in the highly regulated manner (2). 

S.2 Fatty acyl-CoA transferases. The enzyme systems involved with fatty acyl-CoA utilization in the cytosol appear to be membrane-bound. Consequently, detailed knowledge of their individual structure, specificity and genetic control is generally lacking due to the particular inability to obtain ready isolation and purification of the relevant proteins. Studies, however, support the concept of the operation of the eukaryotic pathway for the production of glycerolipids and polyunsaturated fatty acid (Browse et al., 1990 Stymne et al., 1990). While this pathway may contribute a significant quantity of fatty acid for use in membrane synthesis in the plastid (chloroplast) (Browse et al., 1990), its major importance would seem to lie with the production of unsaturated oils (Frentzen, 1986). On the other hand the occurrence of the prokaryotic pathway in the plastid permits more direct membrane lipid formation in both 16 3 and 18 3 plants (Browse et al., 1990 Somerville and Browse, 1991). Different sets of acyltransferase may be associated with the two pathways (Hills and Murphy, 1991). 

The lipids which contain either saturated or unsaturated fatty acids at the n-2 position of their glycerol moieties are derived from phosphatidic acid synthesized within the chloroplast. The biosynthesis of lipids containing such a configuration is referred to as the prokaryotic pathway. The desaturation of palmitic acid to hexadecatrienoic acid is only observed in the prokaryotic pathway of the so-called plants. 

Arabidopsis is a typical 16 3-plant in which both the prokaryotic and eukaryotic pathways [4] contribute to the production of chloroplast lipids. We have investigated the pattern of lipid metabolism in wild type Arabidopsis and calculated the fluxes of carbon involved [5]. An abbreviated version of this analysis is shown in Fig. la. For every 1000 fatty acid molecules synthesized in the chloroplast 390 enter the prokaryotic pathway in the chloroplast envelope while 610 are exported as CoA esters to enter the eukaryotic pathway. Of these 340 are reimported into the chloroplast. Overall, almost equal amounts of chloroplast lipids are produced by each pathway. However, the quantities of individual lipids synthesized by the two routes are very different. All the chloroplast phosphatidylglycerol (PG) and over 70% of the monogalactosyldiacylglycerol (MGD) is derived from the prokaryotic pathway while digalactosyldiacylglycerol is synthesized mainly on the eukaryotic pathway [5]. In this paper we have outlined how four of the Arabidopsis mutants have changed the way we view the operation of the two pathways involved in leaf membrane lipid synthesis. More detailed information on each mutant can be found elsewhere [1-3, 5,6 and in preparation]. 

Recently [3] we described a mutant (actl) which was almost completely deficient in activity of the chloroplast glycerol-3-phosphate acyltransferase the first enzyme of the prokaryotic pathway. Analysis of the fatty acid composition of leaf lipids and C-labelling experiments indicated that PG was essentially the only lipid produced by the prokaryotic pathway in this mutant. However, this blockage in the prokaryotic pathway is compensated for by increased flux through the eukaryotic pathway and furthermore the proportions of the different chloroplast lipids made by the eukaryotic pathway are altered so that there is only a small alteration in lipid composition in the actl mutant compared with wild type [3]. Thus in the actl mutant (Fig. lb) 950 of every 1000 fatty acids enter the eukaryotic pathway and 650 of these are subsequently returned to the chloroplast. The mutant is essentially an 18 3-plant and clearly demonstrates the existence of controls which regulate reactions in the chloroplast and endoplasmic reticulum so as to provide the complement of lipids required for correct membrane synthesis in leaf cells. 

The fade mutant is deficient in a chloroplast w-6 desaturase which is responsible for desaturation of 18 1 and 16 1 fatty acids on the prokaryotic pathway. One effect of this mutation is a 37% decrease in the amount of prokaryotic MGD species compared with the wild type. This decrease is compensated for by increased synthesis of eukaryotic MGD so that there is only a small change in the total amount of leaf... 

MGD[6]. In Fig. Id this is shown by a decreased flux through the prokaryotic pathway of only 280 fatty acids compared with 390 in the wild type and an increased flux through the eukaryotic pathway. The extrachloroplast lipids-phosphatidylcholine (PC), and phosphatidylethanolamine (PE)- of the fade mutant also show increased levels of 18 1. We did not expect this result since the mutant contains normal activity of the endoplasmic reticulum 18 1 desaturase. While the eukaryotic pathway very probably delivers some 18 1-containing lipids to the chloroplast, any 18 1 reexported to the... 

When leaves were Incubated with [ C] 12 0, the C16 and C18 FA at both positions of eukaryotic and prokaryotic MGDG were labeled indicating access to the FA elongation system. In WT, declining radioactivity In 18 2/16 2 And 18 3/16 2 coincided with a rise in 18 3/16 3 showing progressive desaturation In the prokaryotic pathway (Fig. 2). In JBl, the 18/16C species were labeled more slowly and radioactivity accumulated in 18 2/16 2, Indicating Inhibition of these chloroplast desaturase(s). 

As a prelude to detailed genetic studies of lipid metabolism in Arabidopsis we have characterized the lipid composition and the pattern of lipid metabolism of the wild-type in some detail (Browse et al. 1986c). These studies established that Arabidopsis is a 16 3 plant in which approximately 38% of newly synthesized fatty acids enter the prokaryotic pathway of lipid biosynthesis (Roughan and Slack, 1982). Of the 62% which is exported as acyl-CoA species to enter the eukaryotic pathway, 56% (34% of the total) is ultimately reimported into the... 

The distinguishing characteristic of a mutant vd.th a lesion at the fadP locus is a substantial decrease in the amount of both 16 3 and 18 3 fatty acids in extracts of whole leaves and a corresponding increase in the amount of 16 2 and 18 2, respectively (Browse et al., 1986c). Thus, it was inferred that the fadP locus controls the activity of a desaturase which converts both 16- and 18-carbon dienoic to trienoic acyl groups. Analysis of the molecular species of MGP and PGP in the mutant and the wild-type indicated that the mutation specifically affects the desaturation of fatty acids in the prokaryotic pathway of lipid synthesis in the chloroplast and that both the sn-1 and sn-2 position of MGP was affected (Norman and St. John, 1986). 

Figure 2 A proposed model for the acyl carrier protein directed apportionment of oleic acid between the eucaryotlc and prokaryotic pathways of fatty acid metabolism.

---