A’-desaturase system

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

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. 

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.)...

It has been demonstrated that independent reactions and different chemical precursors involved in AFBl and AFB2 syntheses are catalyzed by common enzyme systems (79, 27, 27, 30, 34) AFBl precursors are the preferred substrates for the relevant enzymes (79). A desaturase activity has been demonstrated in cell-free fimgal extracts at the branch (Figure 3) in the AFB1/B2 biosynthetic pathways (18). 

Figure 4.7 Consensus bootstrap tree of full-length amino acid sequences of insect desaturases generated with MacVector 7.0 (Oxford Molecular Limited). Branch points (internal nodes) are retained if they occur in >50 percent of resampling trees (1000 x resampling) all other nodes are collapsed. Names in bold are from the published literature names in parentheses reflect a nomenclature system for insect desaturases (proposed in Knipple ef a/.,...

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. 

In higher plants, animals, protozoa, and fungi, saturated fatty acids are acted upon by desaturases to introduce double bonds, usually of the cis (Z) configuration. The substrates may be fatty acyl-ACP, fatty acyl-CoA molecules, membrane phospholipids, or glycolipids. The A desaturase, isolated from liver or from yeast, converts stearoyl-CoA to oleoyl-CoA (Eq. 21-3). ° This membrane-associated enzyme system... 

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). 

Since the A -desaturase is beUeved to be the rate limiting step in the conversion of linoleic acid to arachidonic acid, efforts have been made to utilize the relatively high content (7%) of y-linolenic acid (18 3w6) in the evening primrose oil to influence the activity of the prostaglandin system. The essential fatty acid potency of y-linolenic acid is 50% greater than that of linoleic acid [424]. However, there is to date no evidence on the possible relationship between dietary intake of this acid and of levels of prostaglandins and prostaglandin metabolites metabolites in vivo. 

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. 

In food, CLA is found in milk fat, the tissue fat of ruminant animals, and products derived from them, although there are exceptions. About 90% of the CLA is represented by the cis-9, trani-11-isomer or rumenic acid (RA) (4). This isomer is produced in the rumen by the action of a linoleic acid isomerase on dietary linoleic acid as a first step in the biohydrogenation process. Further hydrogenation produces trans- - %. or vaccenic acid (VA), the predominant trans monounsaturat-ed fatty acid of milk and animal tissue fat. The major polyunsaturated fatty acids of pasture, a-and y-linolenic acid, cannot be converted to RA, but they do produce VA. A proportion of RA and VA escape further hydrogenation in the rumen and after absorption pass via the circulatory system to adipose tissue and the mammary gland where A -desaturases convert VA to RA. About 70% of RA in milk fat is produced by this pathway (5). 

The current findings of CLA effects on bone fatty acid composition are consistent with previously reported values. For example, CLA reduced 18 ln-9 and total monounsaturated fatty acids in both neutral and polar lipids, and reduced 20 4n-6 in the neutral fraction of liver. This could be due to reduced liver stearoyl-CoA desaturase (A9 desaturase, catalyze the formation of 18 ln-9 from 18 0) expression (16). CLA was reported to decrease the mRNA level of the A9-desaturase enzyme in both liver tissue and hepatocyte cultures (16). More interestingly, the c9,fll CLA isomer, which is implicated as the primary biologically active isomer (21), had no effect on the expression of A9 desaturase compared with the chemically synthesized CLA, which contained as much tl0,cl2 as c9,tll and small amounts of other isomers. This indicates that individual CLA isomers may have their own regulatory functions in a biological system. 

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). 

A transient assay system was developed using carrot suspension cell protoplasts to detect the activity of the putative gene. Carrot suspension cells were chosen as the source for the protoplasts because of an abundance of the 18 2 fatty acid. Introduction of the borage gene resulted in the transient production of GLA in the transformed carrot protoplasts. These results confirmed the identity of the borage A -desaturase gene. 

The fully oxidized and reduced forms of A desaturase have been studied in great detail by various spectroscopic methods, including optical, resonance Raman, Mossbauer, (magnetic) circular dichroism, and extended x-ray absorption fine structure [20,179,352,364]. Specifically, an analysis of the oxidized system by Mossbauer spectroscopy has shown the presence of high-spin Fe ions, anti-ferromagnetically coupled with an exchange coupling constant of —J > 30 cm ... 

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