Ii

10-methytene stearate CH3(CH2)7C—CH2(CH2)7CO—phospholipid

10-methy! stearate CH3(CH2)7CHCH2(CH2)7CO—phospholipid

Fig. 2.14 Production of tuberculostearic acid in Mycobacterium phlei.

chain. However, mid-chain hydroxylations are also found - a good example being the formation of ricinoleic acid (D-12-hydroxyoleic acid; Table 2.4). This acid accounts for about 90% of the triacylgly-cerol fatty acids of castor oil and about 40% of those of ergot oil, the lipid produced by the parasitic fungus, Claviceps purpurea. In developing castor seed, ricinoleic acid is synthesized by hydroxyla-tion of oleate while attached to position 2 of phos-phatidylcholine. The A12-hydroxylase accepts electrons from either NADPH or NADH via a cytochrome b5 protein and uses molecular oxygen. The gene for the enzyme shows many similarities to the fatty acid desaturases and deduced protein structures suggest a common evolutionary origin and reaction mechanism. In contrast to the method of hydroxylation in castor seed, the pathway in Claviceps involves hydration of linoleic acid under anaerobic conditions. Thus the hydroxyl group in this case comes from water and not from molecular oxygen.

a-Oxidation systems producing a-hydroxy (2-hydroxy) fatty acids have been demonstrated in micro-organisms, plants and animals. In plants and animals these hydroxy fatty acids appear to be preferentially esterified in sphingolipids. a-Oxida-tion is described more fully in Section 2.3.2.

ro-Oxidation involves a typical mixed-function oxidase. The major hydroxy fatty acids of plants have an ro-OH and an in-chain OH group (e.g. 10,16-dihydroxypalmitic acid). Their synthesis seems to involve ro-hydroxylation with NADPH and O2 as cofactors, followed by in-chain hydro-xylation with the same substrates. If the precursor is oleic acid then the double bond is converted to an epoxide, which is then hydrated to yield 9,10-hydroxy groups. These conversions involve CoA esters. ro-Oxidation is discussed further in Section 2.3.3.

2.2.5 The biosynthesis of unsaturated fatty acids is mainly by oxidative desaturation

2.2.5.1 Monounsaturated fatty acids

Unsaturated fatty acids can either be produced anaerobically or in the presence of oxygen, which acts as an essential cofactor. The anaerobic mechanism is rather rare but is used by E. coli (and other members of the Eubacteriales) as part of its FAS complex (Section 2.2.3.2). By far the most widespread pathway is by an oxidative mechanism, discovered by Bloch's team, in which a double bond is introduced directly into the preformed saturated long-chain fatty acid with O2 and a reduced compound (such as NADH) as cofactors. This pathway is almost universal and is used by bacteria, yeasts, algae, higher plants, protozoa and animals. Apparently, the two pathways are mutually exclusive because no organism has yet been discovered that contains both the aerobic and anaerobic mechanisms of desaturation. Most of the monounsaturated acids produced have a A9 double bond. Exceptions are A7 bonds in some algae, A5 and A10 monoenoic acids in Bacilli and a A6 acid (petroselenic) in some plants.

The pathway was first demonstrated in yeast.

Cell-free preparations could catalyse the conversion of palmitic into palmitoleic acid (hexadec-9-enoic acid, 9-16:1) only if both a particulate fraction (microsomes) and the supernatant fraction were present. The membrane fraction alone could perform the dehydrogenation provided that the substrate was the acyl-CoA thiolester. The supernatant contained the acid:CoA ligase to activate the fatty acid. More recently another protein fraction in the soluble cytoplasm has been found to stimulate desaturation. This is probably the fatty acid binding protein, which regulates the availability of fatty acid or fatty acyl-CoA for lipid metabolizing enzymes. Bloch found that the cofactors for the desaturation were NADH or NADPH and molecular oxygen, which suggested to him a mechanism similar to many mixed-function oxygenase reactions.

It has been particularly difficult and slow to obtain a detailed understanding of the biochemistry of the desaturase enzymes. Not only are they usually located in membranes, but the substrates are micellar (Section 2.1.8) at concentrations that are suitable for studies in vitro. It was only when methods for solubilizing membranes with detergents were developed that the stearoyl-CoA desaturase enzyme was purified and a better understanding of the enzymic complex emerged. Work by Sato's group in Japan and Holloway in the USA has identified three component proteins of the complex: a flavoprotein, NADH-cytochrome b5 reductase; a haem-containing protein, cytochrome b5; and the desaturase itself which, because of its inhibition by low concentrations of cyanide, is sometimes referred to as the cyanide-sensitive factor (CSF). Strittmatter's group in the USA has purified this latter protein (and identified its gene), which is a single polypeptide chain of 53 kDa containing one non-haem iron atom per molecule of enzyme. The iron can be reduced in the absence of stearoyl-CoA, by NADH and the electron transport proteins, and when it is removed, enzymic activity is lost.

Although the desaturation reaction has all the characteristics of a mixed-function oxygenation, nobody has ever successfully demonstrated an hydroxylation as an intermediate step in double bond formation. In spite of our lack of knowledge of the mechanism, certain details have emerged concerning the stereochemistry of the dehydrogena-tion. Schroepfer and Bloch in the USA and James and coworkers in the UK have demonstrated that the D-9 and D-10 (as) hydrogen atoms are removed by animal, plant and bacterial systems. Experiments with deuterium-labelled stearate substrates showed isotope effects at both the 9 and 10 positions, which are consistent with the concerted removal of hydrogens rather than a mechanism involving a hydroxylated intermediate followed by dehydration.

The three essential components of the animal A9-desaturase are thought to be arranged in the endoplasmic reticulum in a manner shown in Fig. 2.15. The cytochrome b5 component is a small haem-containing protein (16-17 kDa), which has a major hydrophilic region and a hydrophobic anchor of about 40 amino acids at the carboxyl terminus. The cyanide-sensitive desaturase component is largely within the membrane with only its active centre exposed to the cytosol. Most animal A9-desaturases work well with saturated acyl-CoAs in the range 14C-18C. Although it was originally thought (from substrate selectivity studies) that liver contained two isoforms, isolation of genes coding for the desaturase suggests that there is only one (SCD-1) in liver. However, other tissues, such as lung and kidney express two genes (SCD-1 and SCD-2), while brain expresses only the SCD-2 gene. In some classes of tumours an increased level of desaturase mRNA has been found.

In yeast, the A9-desaturase is expressed by the OLE-1 gene. In deficient mutants, the rat liver A9-desaturase gene can effectively substitute for the missing activity.

A general scheme for aerobic fatty acid desaturation is shown in Fig. 2.16 and this applies to the animal A9-desaturase. However, although the animal enzyme clearly uses NADH and cytochrome b5 the A9-desaturase of other organisms may not. Interestingly, the enzyme from plants is soluble. Moreover, it uses stearoyl-ACP as substrate instead of stearoyl-CoA and reduced ferredoxin as a source of reductant (a sensible choice of substrate for a desaturase that is located in the chloroplast).

The availability of the plant A9-desaturase as a

NADH: Cytochrome Cytochrome Desaturase b5 Reductase b5 (Cyanide Sensitive Factor)

Fig. 2.15 Diagrammatic representation of the animal A9-fatty acid desaturase complex. Adapted from H.W. Cook (1996) with kind permission of the author and Elsevier Science. Note that the nature of the hydrogen transfer from NADH (which is depicted in the diagram as H+) has not been proven. In some A5 or A6 polyunsaturated fatty acid desaturases, the cytochrome b5 component is part of the same protein as the desaturase.

Fatty Acid Desaturation Nadh

Fig. 2.16 A generalized scheme for aerobic fatty acid desaturation: *e.g. NADH, NADPH, reduced ferredoxin; **e.g. cytochrome b5; ***e.g. acyl-ACP (stearoyl-ACP A9-desaturase in plants); acyl-CoA (stearoyl-CoA A9-desaturase in animals); oleoyl-phosphatidylcholine (A12-desaturase in yeast or plants); linoleoyl-monogalactosyldiacylglycerol (A15-desaturase in plant chloroplasts).

Fig. 2.16 A generalized scheme for aerobic fatty acid desaturation: *e.g. NADH, NADPH, reduced ferredoxin; **e.g. cytochrome b5; ***e.g. acyl-ACP (stearoyl-ACP A9-desaturase in plants); acyl-CoA (stearoyl-CoA A9-desaturase in animals); oleoyl-phosphatidylcholine (A12-desaturase in yeast or plants); linoleoyl-monogalactosyldiacylglycerol (A15-desaturase in plant chloroplasts).

soluble protein allowed it to be purified in Stumpf's laboratory and, later, with the availability of a gene coding for the enzyme it has been possible to obtain a lot of important information about its reaction mechanism using point mutations. Much of this work has come from Shanklin's laboratory at Brookhaven and he and his coworkers have succeeded in obtaining structural information by X-ray studies down to about 3 A resolution. Once sequence information was available from a number of desaturases, it was clear that they belonged to a group of proteins with di-iron centres. This di-iron cluster is also found in the reaction centre of enzymes like methane monooxygenase and, interestingly, in the oleate hydroxylase that gives rise to ricinoleate (Section 2.2.4). A computer-generated model of the plant A9-desaturase is shown in Fig. 2.17.

An unusual monounsaturated fatty acid, specifically linked to phosphatidylglycerol and found in chloroplasts, is trans-3-hexadecenoic acid (Table 2.2). Palmitic acid is its precursor and oxygen is required as cofactor. When radiolabelled trans-3-hexadecenoic acid is incubated with chloroplast preparations, it is not specifically esterified in phosphatidylglycerols but is either randomly esterified in all chloroplast lipids or reduced to palmitic acid. These results could be explained if the direct precursor of trans-3-hexadecenoic acid were palmitoyl-phosphatidylglycerol and not

Fig. 2.17 Models of the plant A9-stearoyl-ACP desaturase: (A) schematic of secondary structural elements; (B) structural elements; (C) overlay of the desaturase with methane mono-oxygenase; (D) cartoon of functional regions of the desaturase. Reproduced from Fig. 1 of J. Shanklin et al. (1997) with kind permission of the author and Kluwer Academic Publishers.

Fig. 2.17 Models of the plant A9-stearoyl-ACP desaturase: (A) schematic of secondary structural elements; (B) structural elements; (C) overlay of the desaturase with methane mono-oxygenase; (D) cartoon of functional regions of the desaturase. Reproduced from Fig. 1 of J. Shanklin et al. (1997) with kind permission of the author and Kluwer Academic Publishers.

palmitoyl-S-CoA or palmitoyl-S-ACP. This is an example of a complex lipid acting as a desaturase substrate and often such substrates seem to be important for polyunsaturated fatty acid production.

2.2.5.2 Polyunsaturated fatty acids

Although most bacteria are incapable of producing polyunsaturated fatty acids, other organisms, including many cyanobacteria and all eukaryotes, can. These acids usually contain methylene-inter-rupted double bonds, i.e. they are separated by a single methylene group. Animal enzymes normally introduce new double bonds between an existing double bond and the carboxyl group (Fig. 2.18); plants normally introduce the new double bond between the existing double bond and the terminal methyl group (Fig. 2.19).

We shall describe polydesaturations in plants first. The reason for this is that one of the most abundant polyunsaturated acids produced by plants, linoleic acid (c¿s,c¿s-9,12-18:2) cannot be made by animals [although it is always dangerous in biochemistry to make dogmatic statements and, indeed, some protozoa and a few species of insects are capable of forming linoleic acid] yet this acid is necessary to maintain animals in a healthy condition. For this reason linoleic acid must be supplied in the diet from plant sources, and in order to discuss adequately the metabolism of polyunsaturated fatty acids in animals, it is first necessary to understand their formation in plants. Acids of the linoleic family are known as essential fatty acids and will be discussed in later sections.

The precursor for polyunsaturated fatty acid formation in plants and algae is oleate. The next double bond is normally introduced at the 12,13 position (A12 desaturase) to form linoleate followed by desaturation at the 15,16 position (A 15 desaturase) to form a-linolenic acid (all c¿s-9,12,15-18:3) as summarized in Fig. 2.19. With the exception of some cyanobacteria, a-linolenic acid is the most common fatty acid found in plants and fresh-water algae. In marine algae, highly unsaturated 20C acids are predominant, the principal of which

(arachidonic and eicosapentaenoic) are made by the pathways shown in Fig. 2.19.

The possibility that desaturation could occur on fatty acyl chains esterified in complex lipids was first suggested by experiments with the phyto-flagellate, Euglena gracilis. This organism can live as a plant or animal and can synthesize both plant and animal types of polyunsaturated fatty acids. The animal type of fatty acids accumulate in the phos-pholipids and the plant types in the galactolipids. It proved impossible to demonstrate plant-type desaturations in vitro when acyl-CoA or acyl-ACP thiolesters were incubated with isolated cell fractions. Either the desaturase enzymes were labile during the fractionation of the plant cells, or the substrates needed to be incorporated into the appropriate lipids before desaturation could take place. The next series of experiments was done with Chlorella vulgaris, a green alga that produces a very simple pattern of plant-type lipids. When cultures of the alga were labelled with 14C-oleic acid as a precursor of the 18C polyunsaturated fatty acids, labelled linoleic and linolenic acids were produced and the label was only located in the phosphati-dylcholine fraction. Next synthetic 14C-labelled oleoyl-phosphatidylcholine was tested as a substrate for desaturation: the only product formed was linoleoyl-phosphatidylcholine. An important loophole that had to be closed was the possibility that during the incubation, labelled oleic acid might be released, activated to the CoA or ACP thiolester, desaturated as a thiolester and then re-esterified to the same complex lipid very rapidly. Appropriate control experiments eliminated this possibility.

Desaturations involving lipid-bound fatty acids have also been shown to occur in the mould Neu-rospora crassa, various yeasts such as Candida utilis and Candida lipolytica, higher plants and several animal tissues.

One of the best studied systems has been that in the leaves of higher plants. It will be recalled that synthesis of fatty acids de novo in plants occurs predominantly in the plastids. Fatty acid synthase forms palmitoyl-ACP, which is elongated to stear-oyl-ACP and then desaturated to oleoyl-ACP. The latter can then be hydrolysed and re-esterified by the chloroplast envelope to oleoyl-CoA. Although

De novo Synthesis

Acetate +

malonate

Lipids Lipids / /

Lipids

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