© © Monoacylglycerol pathway

Fig. 3.6 The biosynthesis of triacylglycerols in mammals. The figure illustrates the three main pathways: the glycerol 3-phosphate (G3P) pathway (reactions 1,4,5, 7), the dihydroxyacetone phosphate (DHAP) pathway (reactions 2, 3, 4, 5, 7) and the monoacylglycerol pathway (reactions 6, 7). The enzymes involved are: (1) glycerol 3-phosphate acyltransferase (GPAT); (2) dihydroxyacetone phosphate acyltransferase; (3) acyl-dihydroxyacetone phosphate reductase; (4) lysophosphatidate acyltransferase (LPAT); (5) phosphatidate phosphohydrolase (PAP); (6) monoacylglycerol acyltransferase; (7) diacylglycerol acyltransferase (DAGAT). A minor route from diacylglycerols proceeds via reaction 8: diacylglycerol transacylase. In some tissues, diacylglycerol can be reconverted into phosphatidic acid by diacylglycerol kinase (9).

drate, the mRNA for p90 was rapidly induced at high levels in liver, muscle and kidney and at relatively low levels in brain. Such induction was up-regulated by insulin and down-regulated by cyclic AMP. This protein has also been detected in fully differentiated adipocytes but not pre-adipocytes.

LPAT, which transfers an acyl group to position 2 of 1-acylglycerol 3-phosphate to form phosphatidic acid, is also found in both mitochondrial and microsomal fractions, but predominantly the latter. It has proved more difficult to purify this activity but two human isoforms, a and p with 46% homology, have been cloned. LPAT-a is present in all tissues (but predominantly skeletal muscle), whereas the p-isoform is found primarily in heart, liver and pancreas. It is localized in the endo-plasmic reticulum. (A third protein with LPAT activity has been identified. This is called endo-philin-I and is involved, not in TAG biosynthesis, but in the formation of synaptic vesicles, during which process arachidonate is transferred to lyso-phosphatidic acid.)

When mitochondria are incubated with glycerol 3-phosphate and acyl-CoA, the major product is phosphatidic acid. However, if a liver fatty acid binding protein is present as a lysophosphatidic acid acceptor, lysophosphatidate accumulates. It has been shown that lysophosphatidate synthesized in the mitochondria is transferred to endo-plasmic reticulum and is there converted into phosphatidic acid. More research is required into the co-operativity between intracellular sites in the process of TAG assembly and the role of fatty acid binding proteins (or 'Z' proteins; Sections 2.2.1 and 5.1.3) in this process.

It should be noted that phosphatidic acid, the product of the esterification of the two hydroxyls of glycerol 3-phosphate, can also be formed by phos-phorylation of diacylglycerols by diacylglycerol kinase in the presence of ATP. The contribution of this reaction to overall biosynthesis of phosphatidic acid is unknown but it is unlikely to be important in TAG biosynthesis. Its importance is more likely to be in the phosphorylation of diacylglycerols as part of the signal transduction pathway involving phosphoinositides, described in Section 7.9.

The next step of the glycerol 3-phosphate pathway (step 5 in Fig. 3.6) is catalysed by phosphati-date phosphohydrolase (PAP). This enzyme is found in both membrane-bound and soluble forms, which may be relevant to the regulation of its activity (Section 3.6.2).

The final step in the pathway (step 7, Fig. 3.6) is catalysed by diacylglycerol acyltransferase (DAGAT), an enzyme unique to TAG biosynthesis. The enzyme has recently been cloned and sequenced but is membrane-bound and insoluble and has long eluded traditional methods of purification. The enzyme accepts a wide variety of acyl groups for transfer to diacylglycerols formed from phosphatidic acid by the action of PAP. Its activity is highest in tissues specialized in TAG biosynthesis: adipose tissue, liver, lactating mammary gland, small intestinal mucosa and adrenal gland. In mammalian cells, DAGAT forms a family with acyl-CoA:cholesterol acyltransferases-1 and -2. Yeast cells contain an acyl-CoA:sterol acyltransfer-ase, which has been well characterized. Because of considerable homology between this yeast enzyme and mammalian DAGAT, it has now been possible to clone human and mouse DAGAT. The concentration of DAGAT mRNA increases eightfold during differentiation of 3T3-L1 adipocytes in culture. However, because enzyme activity increases nearly 60-fold at this time, the enzyme may also be regulated post-transcriptionally.

In this section, much has been said about the specificity of acyltransferases that leads to characteristic distributions of fatty acids on the three positions of the glycerol backbone. Despite this sometimes seemingly tight specificity, numerous examples have been found of TAG that contain unnatural or 'xenobiotic' acyl groups. Xenobiotic carboxylic acids may arise from many sources including herbicides, pesticides and drugs of various kinds. Many are substrates for acyl-CoA syn-thetase and the resulting xenobiotic acyl-CoAs may be incorporated into TAG, cholesteryl esters, phospholipids and other complex lipids. An example of a xenobiotic TAG is the one formed from ibuprofen, a commonly used non-steroidal anti-inflammatory drug. Such compounds are normally stored in adipose tissue where they may have relatively long half-lives since the ester bonds of many xenobiotic acyl groups are poor substrates for lipases. Much needs to be learned of their metabolism and potential toxicity. The dihydroxyacetone phosphate pathway in mammalian tissues is a slight variant to the main glycerol 3-phosphate pathway and provides an important route to ether lipids

In the 1960s the American biochemists Hajra and Agranoff discovered that radio-phosphorus was incorporated from 32P[ATP] into a hitherto unknown lipid, which they identified as acyl dihydroxyacetone phosphate. Further research demonstrated that dihydroxyacetone phosphate could provide the glycerol backbone of TAG without first being converted into glycerol 3-phosphate (Fig. 3.6). The first reaction of this so-called 'dihydroxyacetone phosphate pathway' is the acylation of dihydroxyacetone phosphate at position 1, catalysed by the enzyme dihydroxyacetone phosphate acyltransferase. Although first studied in a micro-somal fraction, the activity has also been demonstrated in mitochondria and in peroxisomes. Peroxisomes (sometimes called microbodies) are subcellular organelles specialized for the oxidation of small molecules by hydrogen peroxide. They also contain many enzymes of lipid metabolism including those of the dihydroxyacetone phosphate pathway. Whereas it is now established that the principal location of dihydroxyacetone phosphate acyltransferase is in peroxisomes, there is still debate about whether the activity in microsomal and mitochondrial fractions is due to contamination by peroxisomal enzymes or to non-specificity of the GPAT in those organelles. Evidence from molecular mass of the purified enzymes and the differential effects of diet on the activities suggests that glycerol 3-phosphate and dihydroxyacetone phosphate acyltransferases may be separate enzymes.

It now seems certain that dihydroxyacetone phosphate acyltransferase has two separate roles. The first is in providing an alternative route to TAG

as illustrated in Fig. 3.6. (The second role, in ether lipid synthesis is described below.)

Dihydroxyacetone phosphate acyltransferase has been purified after detergent treatment of perox-isomal membranes from guinea pig liver and human placenta. The purified protein has a molecular mass of 65-69 kDa. Certain chemical substances, including the hypolipidaemic drug clofibrate, cause peroxisomes to proliferate. Under such conditions, the activity of dihydroxyacetone phosphate acyltransferase increases 2-3-fold.

The next step in the pathway is the reduction of the keto group in 1-acyl-dihydroxyacetone phosphate to form 1-acylglycerol 3-phosphate, linking once more into the main triacylglycerol biosyn-thetic pathway. This enzyme is located on the cytosolic side of the peroxisomal membrane and is notable in that it requires NADPH rather than NADH. NADPH is normally associated with reactions of reductive synthesis such as fatty acid biosynthesis. There is some evidence to support the view that the activity of the dihydroxyacetone phosphate pathway is enhanced under conditions of increased fatty acid synthesis and relatively reduced in conditions of starvation or when the animal is fed a relatively high fat diet (particularly unsaturated fat). Evidence from Amiya Hajra's laboratory in Ann Arbor, published in early 2000, suggests that the DHAP pathway may indeed make a significant contribution to TAG accumulation when 3T3-L1 preadipocytes differentiate in culture. However, we do not yet have a clear picture of the quantitative significance of the dihydroxyacetone phosphate pathway in overall triacylglycerol assembly.

Peroxisomes do not contain the enzymes catalysing the final steps of acylglycerol biosynthesis. The end-products of peroxisomal biosynthesis, acyl or alkyl (see below) dihydroxyacetone phosphate or 1-O-acyl- or 1-O-alkylglycerol 3-phos-phate must be exported to the endoplasmic reticulum before acylation at position 2, depho-sphorylation to the diacylglycerol or its ether analogue and the final acylation at position 3 by diacylglycerol acyltransferase can occur. The details of the transport mechanism from perox-isomal to microsomal membranes are unclear, but there is some evidence for the participation of a fatty acid binding protein.

It is now generally agreed that the other and most important role for dihydroxyacetone phosphate acyltransferase is to catalyse the first step in the biosynthesis of ether lipids, a reaction that occurs on the luminal side of peroxisomal membranes. The peroxisome is now regarded as the principal site of biosynthesis of the alkyl (ether) lipids. The mechanism of formation of the alkyl linkage at position 1 of dihydroxyacetone phosphate is described in more detail in Section 7.1.9 because the alkyl phospholipids are more widespread and of greater physiological importance than the neutral alkyl acylglycerols. However, as described in Section 3.2.1, the neutral alkyl lipids are found in significant quantities in the liver oils of sharks. The vital importance of ether lipids is illustrated by a number of serious neurological diseases resulting from lack of peroxisomes or peroxisomal enzymes. Thus, one piece of research, in which human cDNA for dihydroxyacetone phosphate acyltransferase was cloned, showed that absence of the enzyme causes severe neurological impairment and skeletal deformities, but no alteration in overall TAG biosynthesis. Formation of triacylglycerols in plants involves the co-operation of different subcellular compartments

The primary pathway for the biosynthesis of TAG in plants that use lipids as their major energy store, is the glycerol 3-phosphate ('Kennedy7) pathway. However, there are sufficient differences from animals in terms of subcellular location and sources of substrates to merit a separate discussion of plant TAG biosynthesis.

To study the different enzymes in the pathway, seeds or fruits need to be harvested at the time when the rate of lipid accumulation is most rapid, since there are distinct phases of development as illustrated in Fig. 3.7. In phase 1, cell division is rapid but there is little deposition of storage material, whether it be protein, lipid or carbohydrate. In phase 2, there is a fast accumulation of storage material. Moreover, if the TAG in the lipid stores contain unusual fatty acids, the special enzymes needed for synthesis are active only at this stage. Finally, in phase 3, desiccation takes place with little further metabolism.

Fatty acid biosynthesis de novo is concentrated in the plastid of the plant cell (Section 2.2.3). By a

Starvation Lipid Protein Phase Three

Fig. 3.7 The accumulation of lipid during seed development.

Period of seed development

Fig. 3.7 The accumulation of lipid during seed development.

combination of acetyl-CoA carboxylase and fatty acid synthase, palmitoyl-acyl carrier protein (ACP) is produced. This is chain-lengthened by a specific condensing enzyme to stearoyl-ACP, which is desaturated to oleoyl-ACP by a A9-desaturase (Section Under most conditions, palmitate and oleate are the main products of biosynthesis in plastids (chloroplasts in leaves). These acyl groups can be transferred to glycerol 3-phosphate within the organelle or hydrolysed, converted into CoA-esters and exported outside the plastid (Fig. 3.8). If fatty acids are esterified to glycerol 3-phosphate within the plastid, then 16C acids (mainly palmi tate) are attached at position 2, whereas 18C acids (mainly oleate) are attached at position 1.

The biosynthesis of TAG for energy storage purposes takes place in the endoplasmic reticulum, not in the plastid. Therefore, during the relatively short period of oil accumulation (a few days only) mechanisms must be in place to export acyl groups from plastid to endoplasmic reticulum membranes. The first requirement is a thiolesterase to hydrolyse ACP derivatives to free fatty acids. During oil accumulation, the activity of this enzyme needs to be much higher than that of plastid GPAT and PAP (thought to be rate limiting for TAG biosynthesis)

Fig. 3.8 The 'plant pathway' for triacylglycerol biosynthesis. lyso-Pc, 1-acyl phosphatidylcholine; PDAT, phospholipid: diacylglycerol acyltransferase. This newly discovered enzyme probably plays an important role in channelling unusual fatty acids out of membrane phosphoglycerides into TAG stores.

as there is no need for a rapid production of dia-cylglycerols in plastids when the carbon flux is mainly to endoplasmic reticulum.

After non-esterified fatty acids are attached to CoA in the plastid envelope they move rapidly to the endoplasmic reticulum (Fig. 3.8). It is not known how this transport takes place, although acyl-CoA-binding transport proteins have been purified from a number of plant tissues. It is clear, however, that acyl-CoAs (rather than acyl-ACPs as in the plastid) serve as substrates for acyl-transferases in the endoplasmic reticulum.

GPAT, the enzyme that transfers an acyl group to position 1, generally has broad specificity and accepts both saturated and unsaturated fatty acids. The acyl group at position 1 should therefore broadly represent the distribution of acyl groups in the acyl-CoA pool. In some species that produce unusual seed-specific fatty acids (e.g. Cuphea) GPAT may exhibit particularly high specificity for that fatty acid. 'Unusual' fatty acids are almost invariably concentrated in TAG, little being ester-ified in membrane phospholipids. There must, therefore, be mechanisms for channelling 'unusual' acyl groups away from membrane lipids (where they might adversely influence membrane function) into storage lipids. One possible mechanism is that there are isoforms, specific to storage lipids, of each of the Kennedy pathway enzymes in the endoplasmic reticulum, although this remains to be demonstrated unequivocally. Microsomal fractions isolated from Cuphea, a plant whose seed oil contains high levels of medium-chain fatty acids, incorporate those fatty acids into TAG, actively excluding them from membrane lipids. Even seeds that do not normally synthesize 'unusual' fatty acids have a mechanism for excluding such fatty acids from their membrane lipids.

The enzyme LPAT, which catalyses the acylation at position 2, usually has a stricter specificity for unsaturated fatty acids. Thus, the action of GPAT and LPAT results in a fatty acid distribution in diacylglycerols produced in the endoplasmic reti-culum that is the reverse of that in diacylglycerols produced in plastids (Fig. 3.8). For example, if a microsomal fraction of developing safflower seeds is incubated with 16:0-CoA alone, lysophos-

phatidate accumulates, with little formation of phosphatidic acid. Incubation with 18:1-CoA or 18:2-CoA, which are accepted efficiently by both GPAT and LPAT, results in the formation of phosphatidic acid. Likewise, accumulation of lysophosphatidate is also seen when a microsomal fraction from developing rapeseed is incubated with erucoyl-CoA. In rapeseed oil, erucic acid is exclusively located at positions 1 and 3. As erucic acid is a valuable industrial commodity (for use in lubricating oils) there is currently much interest in either modifying the substrate specificity of LPAT or introducing into rapeseed a gene for the enzyme from another plant species that does not discriminate against erucic acid.

A PAP located in the endoplasmic reticulum then acts to generate diacylglycerols. PAP may regulate carbon flux through the TAG biosynthesis pathway. As in mammalian cells, it exists in two separate forms, one associated with the endoplasmic reticulum membranes, the other in the cytoplasm. The amount of the enzyme attached to membranes is influenced by the local concentration of non-esterified fatty acids, thereby allowing feed-forward control over carbon flux to TAG.

The diacylglycerol products of PAP can be further acylated at position 3 with acyl-CoA, catalysed by the enzyme DAGAT to complete the Kennedy pathway. This enzyme usually has less specificity than the acyltransferases that esterify positions 1 and 2. Thus, in many plants, the fatty acids that accumulate at the sn-3 position depend upon the composition of the acyl-CoA pool. Nevertheless, in a few tissues, the substrate specificity of the DAGAT may also have an important role in determining the nature of the final stored lipid. From measurements of enzyme activities in vitro and because diacylglycerol accumulates during lipid deposition, it is often considered that DAGAT may have a regulatory role in the rate of TAG biosynthesis. It may also be involved in channelling 'unusual' fatty acids away from membrane phos-pholipids and into triacylglycerols as discussed above. Thus, the DAGAT of castor bean, when presented with a mixture of di-ricinoleoyl and di-oleoyl species in vitro, specifically selects the di-ricinoleoyl species.

During active oil accumulation, carbon from diacylglycerols is preferentially channelled into TAG biosynthesis. However, at other phases of the seed's life cycle, the utilization of diacylglycerols for the biosynthesis of membrane phospholipids, such as phosphatidylcholine, catalysed by the enzyme CDPcholine:diacylglycerol choline phosphotransferase, is more important. In some plants, the latter enzyme may also play a role in TAG biosynthesis, however, as illustrated in Fig. 3.8. The reaction catalysed by this enzyme is approximately in equilibrium and can, therefore, allow the rapid exchange of diacylglycerols between their pool and that of the newly synthesized phosphatidylcholine. Since phosphatidylcholine is the substrate for oleate (and linoleate) desaturation in seeds, the reversible nature of the choline phosphotransferase allows the diacylglycerol pool to become enriched in polyunsaturated fatty acids. This process has been studied in particular detail in safflower (which has an oil rich in polyunsaturated fatty acids, comprising 75% linoleate). It has also been shown that the acyl-CoA pool can also be utilized through the activity of an acyl-CoA:lysophosphatidylcho-line acyltransferase. In contrast, for seeds where less unsaturated oils accumulate (e.g. avocado) the subsidiary flux of diacylglycerols through phos-phatidylcholines and their consequent desaturation, is much less important.

In some plants, diacylglycerol molecules may have yet another fate. In their studies with developing safflower seed microsomal fractions, Stobart and Stymne made a puzzling observation. Apparently TAG could be formed from diacylglycerols in the absence of acyl-CoA. The most likely explanation is a transacylase reaction in which two molecules of diacylglycerol can form one of monoacylglycerol and one of triacylglycerol (Fig. 3.8). It is further proposed that the mono-acylglycerols thus formed are rapidly converted first into diacylglycerols and then into TAG. Details of the transacylation mechanism are still to be elucidated and its significance is a matter of speculation. It may be involved in 'retailoring' molecular species of TAG or of regulating the size of the diacylglycerol pool and protecting phospholipid biosynthesis at a period of rapid oil accumulation.

From this discussion it will be apparent that the plastid and endoplasmic reticulum compartments of the plant cell have to integrate their metabolism during lipid storage (Fig. 3.8). Questions remaining to be answered include the mechanism by which acyl groups are exported from plastid to endo-plasmic reticulum, how specific fatty acids are channelled into storage TAG and excluded from membrane phospholipids (Fig. 3.8), how switching between phospholipid and TAG biosynthesis is regulated during seed development and what, if any, role the transacylation of diacylglycerols plays in regulating these processes.

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