Acp

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Structural core

1Cys

1Ser

8His

Ketoreductase

Thioesterase

1670GXGXXG

1Ser

2Ser

1886GXGXXG

- Total 2500 residues

Fig. 2.11 Arrangement of the partial reactions of one monomer of the animal fatty acid synthase. Information taken from Smith (1994). The key active-site residues for each domain are indicated.

condensing enzyme on the other half of the dimer (Fig. 2.12). Other experimental evidence demonstrated that translocation of acetyl and malonyl moieties from CoA esters to the 4'-phospho-pantetheine of the ACP domain also needs the dimeric form of the enzyme.

However, Smith and colleagues have produced a number of modified FASs in which the activity of one of the functional domains is specifically compromised by mutations. Heterodimers are then formed from subunits containing different single mutations to observe whether they are capable of fatty acid synthesis. Unexpectedly, Smith and colleagues found that the dehydrase and ACP domains could interact in the same subunit, even though they were separated by more than 1000 residues (see Fig. 2.11). Moreover, both the substrate translocation and condensation reactions could use the ACP of either subunit. These results led to the proposal that the two halves of the dimer are not simply arranged in an extended conformation but are folded in a manner to allow functional contacts within subunits in addition to between subunits. This allows greater flexibility for the interaction of domains (see Fig. 2.12). Based on results with cross-linking agents, it has been estimated that up to 35% of the resting, wild-type FAS adopts a conformation where the condensing enzyme active site cysteine is juxtaposed with the phosphopantetheine residues of the ACP domain of the same subunit.

2.2.3.3 Termination

The typical end-product of animal FAS enzymes is unesterified palmitic acid. The cleavage of this acid from the complex is catalysed by a thioesterase which, as discussed above, is an integral part of the enzyme. Several factors combine to achieve this normal specificity. First, the transferase that catalyses the loading of substrate moieties from CoA-esters to the enzyme-bound thiolester group has a high specificity for acetyl and butyryl groups. Thus, once the acyl chain has grown longer than 4C it cannot readily escape the FAS. Second, the condensation reactions are much faster for medium-chain acyl substrates thereby ensuring that, once elongation has started, it rapidly proceeds to long chain lengths. By contrast, chain lengths of 16C or above are not easily elongated. This is shown most convincingly in experiments where the thioesterase activity has been removed. Under these conditions, palmitate is still the main product. Moreover, chains of 16C or more tend to transfer to the 4'-phosphopantetheine of the ACP domain rather than to the condensing enzyme domain, perhaps because of steric hindrance or hydrophobicity in a similar fashion to what occurs in the yeast FAS (Fig. 2.9). This means that palmi-tate will dwell on the mammalian FAS until it is cleaved by the thioesterase. In addition, the thioesterase itself shows a strong preference for palmitate.

Acp Biochemistry

Fig. 2.12 The conventional and alternative models for the action of animal fatty acid synthase. The two-dimensional cartoon shows a simple caricature of the seven domains on each of the two identical peptides present in the dimer. For clarity a long non-catalytic domain between DH and ER (see Fig. 2.11) and extending approximately between residues 970 and 1630, has been omitted. In the conventional mechanism, the ACP of one-half of the dimer provides the 'primer' acyl chain to KS of the other half of the dimer for condensation. In the alternative mechanism, an altered three-dimensional configuration of the FAS protein allows the ACP to interact with the KS domain on the same half of the dimer. Under these circumstances, all the reactions involved in two-carbon addition take place on one-half of the dimer rather than through co-operation between catalytic domains located on both subunits. Redrawn from Joshi, A.K., Witkowski, A. and Smith, S. (1998) Biochemistry, 37, 2515-2523 with kind permission of the authors and the American Chemical Society.

Fig. 2.12 The conventional and alternative models for the action of animal fatty acid synthase. The two-dimensional cartoon shows a simple caricature of the seven domains on each of the two identical peptides present in the dimer. For clarity a long non-catalytic domain between DH and ER (see Fig. 2.11) and extending approximately between residues 970 and 1630, has been omitted. In the conventional mechanism, the ACP of one-half of the dimer provides the 'primer' acyl chain to KS of the other half of the dimer for condensation. In the alternative mechanism, an altered three-dimensional configuration of the FAS protein allows the ACP to interact with the KS domain on the same half of the dimer. Under these circumstances, all the reactions involved in two-carbon addition take place on one-half of the dimer rather than through co-operation between catalytic domains located on both subunits. Redrawn from Joshi, A.K., Witkowski, A. and Smith, S. (1998) Biochemistry, 37, 2515-2523 with kind permission of the authors and the American Chemical Society.

However, in some mammalian tissues, 4C-14C products are released. Moreover, a variety of organisms has been found that produce different types of fatty acid end-products. Several factors have been found to influence chain termination.

(1) In rat mammary gland, where the milk tri-acylglycerols contain large quantities of 8:0 and 10:0 acids, Smith found a second thioes-terase responsible for the release of medium-chain acids. A similar thioesterase II has also been found in rabbit mammary gland. The Californian bay tree produces medium-chain fatty acids in its seed storage lipids. A specific medium-chain thioesterase has been isolated from this species and, by genetic manipulation of oilseed rape, been used to produce laurate (12:0)-enriched oils, which have industrial utility.

(2) In contrast, goat mammary gland, which also produced short- and medium-chain fatty acids, did not contain thioesterase II. In this case a transacylase was found in the FAS and, when incubated in combination with microsomes and appropriate cofactors, rapid transfer of medium-chain fatty acids into triacylglycerols is seen.

(3) The uropygial gland FAS from a number of birds produces medium branch-chain fatty acids as products. These products are released by a specific hydrolase, which is absent from those birds whose end-products are long branched-chain fatty acids.

(4) For E. coli early experiments showed that the specificity of the 3-ketoacyl-ACP synthase was such that palmitoyl- and vaccenoyl-ACP could not act as primers. However, additional evidence from Cronan's laboratory suggests chain elongation can continue in cells if they are starved of glycerol 3-phosphate so that the fatty acid products are not transferred into membrane phospholipids.

(5) In Mycobacterium smegmatis termination involves transacylation of 16C-24C fatty acids to CoA. This transacylation is stimulated by polysaccharides, which seem to act by increasing the diffusion of the acyl-CoA esters from FAS rather than promoting acyl transfer from ACP to CoA.

There may be other mechanisms for controlling the chain length of the fatty acids produced by the various FAS complexes. Certainly, there are plenty of other theories that have been proposed and evidence has been obtained, in some cases, in vitro. Moreover, there are numerous cases where unusual distributions of fatty acid products are found but about which we know very little of the mechanism of termination.

2.2.3.4 Elongation

Although, as discussed above, the major product of FAS is often palmitate, many tissues contain longer chain fatty acids in their (membrane) lipids. For example, in the myelin of nervous tissues, fatty acids of 18C or greater make up two-thirds of the total, while in many sphingolipids fatty acids of 24C are common. In plants, the surface waxes contain mainly very long chain products in the 28C-34C range.

The formation of these very long chain fatty acids is catalysed by the Type III synthases, which are commonly termed elongases because they chain lengthen preformed fatty acids (either produced endogenously or originating from the diet). Most eukaryotic cells have the capacity to carry out elongation reactions.

In liver, brain and many mammalian tissues there are two elongation systems located in the mitochondria and endoplasmic reticulum, respectively. The mitochondrial system, discovered by Wakil in rat liver, occurs by the addition of 2C units from acetyl-CoA and not malonyl-CoA. Monoenoic acyl-CoAs are generally preferred to saturated substrates. In tissues such as liver or brain, both NADPH and NADH are needed, whereas heart or skeletal muscles required only NADH. The German biochemist, Seubert, showed the virtual reversal of P-oxidation (Section 2.3.1) for mitochondrial elongation. However, the enzyme FAD-dependent acyl-CoA dehydrogenase in P-oxidation is replaced by the thermodynamically more favourable enoyl-CoA reductase. The enoyl-CoA reductase isolated from liver mitochondria is different from that of the endoplasmic reticulum and kinetic studies suggest that its activity largely controls the speed of overall mitochondrial elongation.

The principal reactions for the elongation of longer chain fatty acids are found in the membranes of the endoplasmic reticulum that are isolated as the microsomal fraction by ultracentrifugation of tissue homogenates. The reactions involve acyl-CoAs as primers, malonyl-CoA as the donor of 2C units and NADPH as the reducing coenzyme. An example of the microsomal elongation system is in the nervous system, where large amounts of 22C and 24C saturated fatty acids are constituents of myelin sphingolipids (Chapters 6 and 7). Before myelination begins, the activity of stearoyl-CoA elongase is hardly measurable, but it rises rapidly during myelination. The mutant 'quaking mouse' is deficient in myelination and has proved to be a useful model for studies of the elongation process. The rate of elongation of 18:0-CoA to 20:0-CoA is normal and that of 16:0-CoA to 18:0-CoA is rather lower than normal in this mutant. The elongation of 20:0-CoA, however, is very much reduced, suggesting that there are at least three elongases in this tissue.

It is likely that the separate elongases referred to above (in brain) differ in the nature of their condensing enzyme rather than for the other three components (3-ketoacyl-CoA reductase, 3-hydro-xyacyl-CoA dehydrase, enoyl-CoA reductase). Enzyme studies have shown more than one condensing enzyme in several tissues. All of the enzymes involved in elongation have a cytosolic orientation on the endoplasmic reticulum and, although NADPH or NADH are used by the reductases, the flow of electrons appears to be indirect. Involvement of cytochrome b5 or of cytochrome P450 has been proposed.

One of the most important functions of elongation is in the transformation of dietary essential fatty acids to the higher polyunsaturated fatty acids. The starting point is linoleoyl-CoA, which is first desaturated to a trienoic acid. This is followed by a sequence of alternate elongations and desa-turations that are described in further detail in Sections 2.2.5.3 and 4.2.3.2. Indeed, it is noticeable that, in the liver elongation system, y-linolenic acid is the best substrate.

The end-product of the Type II FAS of plants is palmitoyl-ACP and this serves as the substrate for an elongation system (palmitate elongase). This is soluble and appears to differ from the Type II FAS forming palmitate only in having a specific condensing enzyme, 3-ketoacyl-ACP synthetase II. Because the palmitate elongase is able to chain-lengthen preformed palmitate it can be regarded as an elongase but, since it usually functions as part of the de novo system for fatty acid production, it can also be considered part of the Type II FAS by analogy with the two different condensing enzymes present in E. coli.

Very long chain fatty acids in plants are made by membrane-bound enzyme systems utilizing mal-onyl-CoA as the source of 2C units in similar fashion to the animal elongases. Acyl-CoAs have been shown to be the substrates in some of these systems and various elongases have been demonstrated which have different chain-length specificities. Moreover, the individual partial reactions involved have been demonstrated and some purifications achieved. Genes coding for the condensing enzymes have, for example, recently been identified. The production of very long chain (>18C) fatty acids is required for the formation of the surface-covering layers, cutin and suberin (Section 6.6.1), as well as for seed oil production in commercially important crops such as rape and jojoba (Sections 3.4.1.3 and 3.7.1).

2.2.3.5 Branched-chain fatty acids

The formation of branched-chain fatty acids by the Type I FAS of the sebaceous (uropygial) glands of waterfowl has already been mentioned (Section 2.2.3.2 and Table 2.8). These acids arise because of the use of methylmalonyl-CoA rather than mal-onyl-CoA, which is rapidly destroyed by a very active malonyl-CoA decarboxylase. The utilization of methylmalonyl-CoA results in the formation of products such as 2,4,6,8-tetramethyl decanoic acid or 2,4,6,8-tetramethylundecanoic acid as major products when acetyl-CoA or propionyl-CoA, respectively, are used as primers.

A high proportion of odd chain and of various polymethyl-branched fatty acids occurs in the adipose tissue triacylglycerols of sheep and goats when they are fed diets based on cereals such as barley. Cereal starch is fermented by bacteria in the rumen to form propionate, and when the animal's capacity to metabolize propionate via methylma-lonyl-CoA to succinate is overloaded, propionyl-and methylmalonyl-CoA accumulate. Garton and his colleagues showed that methylmalonyl-CoA can take the place of malonyl-CoA in fatty acid synthesis and that with acetyl- or propionyl-CoA as primers, a whole range of mono-, di- and tri-methyl branched fatty acids can be produced.

The major fatty acids in most Gram-positive and some Gram-negative genera are branched-chain iso or anteiso fatty acids. The Type II FAS enzymes present in these bacteria make use of primers different from the usual acetyl-CoA. For example, Micrococcus lysodeikticus is rich in 15C acids of both the iso type, 13-methyl-C14 or anteiso type, 12-methyl-C14. These have been shown to originate from leucine and isoleucine, respectively (Fig. 2.13). Thus, isobutyryl-CoA is used as the primer for iso-branched fatty acids and 2-methylvaleryl-CoA for anteiso products. These branched fatty acids are used to increase membrane fluidity in those bacteria that only have low levels of unsaturated acids under most growth conditions.

Another common branched-chain fatty acid is 10-methylstearic acid, tuberculostearic acid, a major component of the fatty acids of Mycobacterium phlei. In this case, the methyl group originates from the methyl donor S-adenosyl methionine, while the acceptor is oleate esterified in a phospholipid. This is an example, therefore, of fatty acid modification taking place while the acid is in an O-ester rather than the S-esters of CoA or ACP. The formation of tuberculostearic acid takes place in two steps, the intermediate being 10-methylenestearic acid, which is then reduced to 10-methylstearic acid (Fig. 2.14).

2.2.4 The biosynthesis of hydroxy fatty acids results in hydroxyl groups in different positions along the fatty chain

Hydroxy fatty acids are formed as intermediates during various metabolic pathways (e.g. fatty acid synthesis, (3-oxidation) and also because of specific hydroxylation reactions. Usually the hydroxyl group is introduced close to one end of the acyl

Acp Biochemistry

Fig. 2.13 Production of an anteiso branched-chain fatty acid in bacteria.

D(+)-12-methyl tetradecanoic

Fig. 2.13 Production of an anteiso branched-chain fatty acid in bacteria.

O/eate CH3{CH2)7CH=CH(CH2)7CO—phospholipid + adenosyl—S(CH2)2CHCOO~

| (S- Adenosylmethionine)

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