Desaturase system

MONOUNSATURATED FATTY ACIDS ARE SYNTHESIZED BY A A DESATURASE SYSTEM... 

Figure 23-3. Biosynthesis of the co9, co6,and co3 families of polyunsaturated fatty acids. Each step is catalyzed by the microsomal chain elongation or desaturase system 1,elongase 2,A desaturase 3,A desaturase 4,A desaturase. ( .Inhibition.)...

The association of proton movement with electron transport is not reflected in the fatty acyl desaturase system universal to endomembranes. In these enzymes the dehydrogenase, NADH cyt b5 reductase and the cytochrome b5 (a single heme cytochrome) are associated exclusively with the cytosolic side of the membrane by acyl groups and have no transmembrane segment. The cytochrome b5 oxidase associated with the desaturation of fatty acyl CoA may be transmembranous but has not been associated with proton movement. It is an iron-containing protein. The other type of endomembrane cytochromes are the P-450 group of cytochrome bs... 

Fatty acids are the building blocks of TAG. More than 90 percent of fatty acids have an even number of carbon atoms, and are in aliphatic chains ranging from 4 to 22 carbons in length. The major fatty acid synthesis pathway is production of stearic acid (18 carbons) after which separate desaturase systems introduce 1, 2, or 3 unsaturated (double) bonds. Additional enzymes become active in elongating the chain as needed. Shorter fatty acids also are produced. Trace amounts of odd-number carbon fatty acids are found in most fats, and also have been synthesized for research purposes. Microorganisms frequently produce odd-number carbon fatty acids, with heptadecenoic (17 carbon) acid a major component of Candida tropicalis yeast fat. Up to 8 percent C17 fatty acids have been found in milk and meat fats of ruminants (cattle, sheep, goats) and are of rumen microbe origin. 

Acyl-CoA molecules are desaturated in ER membrane in the presence of NADH and 02. All components of the desaturase system are integral membrane proteins that are apparently randomly distributed on the cytoplasmic surface of the ER. The association of cytochrome b5 reductase (a flavoprotein), cytochrome b5, and oxygen-dependent desaturases constitutes an electron transport system. This system efficiently introduces double bonds into long-chain fatty acids (Figure 12.15). Both the flavoprotein and cytochrome b5 (found in a ratio of approximately 1 30) have hydrophobic peptides that anchor the proteins into the microsomal membrane. Animals typically have A9, A6, and A5 desaturases that use electrons supplied by NADH via the electron transport system to activate the oxygen needed to create the double bond. Plants contain additional desaturases for the A12 and A15 positions. 

The r-alkyl desaturase system, a microsomal mixed-function oxidase responsible for the biosynthesis of ethanolamine plasmalogens from alkyl lipids (Fig. 6), was initially characterized in the early 1970s (F. Snyder, 1971 A. Paltauf, 1973). The reverse of this reaction (i.e., conversion of an alk-l -enyl moiety to an alkyl chain via a reductase) has not been observed. The alkyl desaturase is a unique activity since it can specifically and stereospecifically abstract hydrogen atoms from C-1 and C-2 of the 0-alkyl chain of an intact phospholipid molecule to form the cis double bond of the O-alk-l -enyl moiety. Only intact l-alkyl-2-acyl-in-glycero-3-phosphoethanolamine is known to serve as a substrate for the alkyl desaturase. As with other reactions in complex ether phospholipid synthesis, the molecular identity of the responsible enzyme is unknown. 

Investigation of the desaturase systems has been hampered by the enzymes being tenaciously bonded to membranes and by their substrates typically being micellar at convenient concentrations in buffers. Unlike those unsaturated fatty acids that are components of seed fats and waxes of the plant cuticle, the alkynes—or at best the alkynic... 

The animal and plant desaturase systems diverge at this point. Animal systems desaturate oleate only between the Cq double bond and the carboxyl group, while plants desaturate between the C q carbon and the u-methyl. The plant desaturase systems giving rise to linoleic acid and a-linolenic acid are not yet well characterized. This is because the desaturases are membrane-bound and have not been isolated. Current indications are that phosphatidyl choline with oleic acid at the 2-position is the substrate for the 18 1->18 2 reaction (2). There is evidence (10) that the 18 2- 18 3 reaction takes place in the chloroplast and that the substrate is monogalactosyldiacylglyceride (HGDG). 

C.hj from the microsomal fraction of bird and mammal livers is thought to deliver electrons to the fatty acid desaturase system of the endoplasmic reticulum. The heme group is noncovalently bound to the protein via histidine and does not react with O . It also protects the C.b molecule from denaturation and proteolytic attack. C.b solubilized by detergent treatment is an oligomer (A/, 120,000) of several monomers (M, 16,000, 126 amino acids), while C.65 solubilized by protease or lipase treatment has only 82 to 98 amino acids, depending on the species. The primary sequences of these fragments are known. They have a-helix and 25% -structure. 

Plant desaturase systems appear to be soluble, may contain ferredoxin in place of cytochrome 65, and act upon fatty acyl-ACP or upon acyl groups already incorporated into membrane lipids (e.g. [l-oleoyl]-dia-cylgalactosylglycerol desaturated to [l-linoleoyl]-dia-cylgalactosyl ycerol and then to [l-linolenoyl]-diacyl-galactosyl ycerol by spinach chloroplast desaturase). 

Fig. 5. Fatty acid biosynthesis. Desaturase system of rat (iver endoplasmic reticulum.

The fatty acid desaturase system was one of the first microsomal enzymes for which a lipid requirement was established. Using Fleischer s procedure of aqueous acetone extraction (Lester Fleischer, 1961) we were able to reduce the desaturase activity of hen liver microsomes to very low levels and restore activity completely with a mixture of lipids isolated from the microsomes (Holloway Peluffo, 1964 Jones at al., 1969). Shortly after this observation cytochrome bs was implicated in the desaturation process (Oshino et al., 1966). Progress in the isolation and purification of the stearyl CoA desaturase has been slow and it is only in the last two years that the complete resolution and reconstitution of the stearyl CoA desaturase has been achieved by Strittmatter and co-workers (Strittmatter et al., 1974). 

Our approach from the beginning was to use detergents to resolve the desaturase system. Although we never pushed these techniques to the point of obtaining homogeneous desaturase protein, we were able to use this approach to remove cytochrome bs from the majority of the... 

Most evidence suggests that oleoyl phosphatidylcholine is the true substrate of the complex oleate-desaturase system present in the endoplasmic reticulum of higher plants [ 3-6]. 

It clearly appears that in each tissue analyzed several PC molecular species can be utilized as substrates by the microsomal oleate-desaturase system. The main species are the same in all tissues but the quantitative... 

The major unsaturated fatty acids synthesized by N-limited R.gracilis are oleic, linoleic and a-linolenic acids, indicating the presence of A9, A12 and A15 desaturase enzymes [1]. In this report, we discuss experiments In which the activities of these desaturase systems have been altered by temperature and sterculate treatment. 

Throughout these studies, mechanisms involved in the control of membrane fluidity have been shown to play a major role in the regulation of the degree of unsaturation exhibited by the major acyl lipid classes (see [7]). As both 15 and 30 C cultures possessed very active A9 desaturase systems, treatment of cultures with sterculate proved to be the only method by which the synthesis of unsaturated triacylglycerols could be significantly controlled. 

In the sequence of carotene desaturation are two targets for herbicide interaction the step from phytoene to -carotene and the step from -carotene to lycopene. It is assumed that two individual desaturase systems work in sequence. 

The effects of methyl sterculate on R gracilis were also studied via the use of (l- C]acetate and ammonium [ 1- C]stearate as possible precursors for oleic acid synthesis (Table 2). When labelled stearate was the precursor molecule, methyl sterculate was able to decrease the conversion of stearate to oleate from approximately 40% to 4%, whilst In samples In which (1- x]acetate was the precursor, very little Inhibition was observed. Such evidence Implies that the conversion of acetate to oleate Is by a route which Is not susceptible to sterculate Inhibition. Similar observations have been reported for both plant and mammalian" desaturase systems. 

In work performed by Jeffcoat and Pollard, the Inhibitory form of stercullc acid In mammalian tissue was reported to be sterculoyl-CoA. If this Is also true for R. gracilis, we may tentatively propose that the alternative route of oleic acid synthesis In the presence of methyl sterculate Is via a A9 desaturase system that Is capable of desaturatlng stearate when attached to a complex lipid. Further desaturation of phospholipid substrate to llnoleate and then llnolenate would explain the lack of sterculate Inhibition of the latter s S3rnthesls. These possibilities are being Investigated. 

In safflower, a high stearic acid strain, similar to that found In soybeans, was identified within the world germplasm collection. As with the soya mutant the level of stearic acid formed was found to be conditioned by two alleles at a single locus, with low stearic acid content being almost completely dominant over the high stearic acid characteristic (Ladd and Knowles, 1971 Hammond and Fehr, 1984). In safflower it was found that the level of stearic acid produced was inherited independently of the oleic-linolelc acid desaturase system (Ladd and Knowles, 1971). Although the level of stearic found in the safflower strain is less than that in the high stearic soybean mutant, similar processes appear to be in operation. 

The A -desaturase system consists of three major proteins (1) NADH-cytochrome b reductase, (2) cytochrome bj, and (3) a terminal desaturase component (Fig. 3.3). Under most circumstances, electron transport greatly exceeds the activity of the rate-limiting desaturase component. The A -desaturase contains one atom of a nonheme. 

FIGURE 3.3 An outline of the oxidation-reduction sequence in the A -desaturase system. 

The phospholipid requirement for this system has recently been studied by Enoch et al. (1976), who concluded that the hydrocarbon is necessary both as an initial attachment site for the stearoyl-CoA prior to binding to the enzyme and as an anchor for the hydrophobic regions of the desaturase system components. The study by Enoch et al. supports the view that the fluidity of the hydrocarbon chains in the membrane allows for lateral diffusion of the three enzymes of the system. In... 

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