A9-desaturases

Figure 13.25 Three-dimensional structures of diiron proteins. The iron-binding subunits of (a) haemery-thrin, (b) bacterioferritin, (c) rubryerythrin (the FeS centre is on the top), (d) ribonucleotide reductase R2 subunit, (e) stearoyl-acyl carrier protein A9 desaturase, (f) methane monooxygenase hydroxylase a-subunit. (From Nordlund and Eklund, 1995. Copyright 1995, with permission from Elsevier.)...

Figure 13.26 Dioxygen-utilizing carboxylate-bridged diiron centres (a) Oxidized (top) and reduced (bottom) MMOH (b) oxidized (top) and Mnn-reconstituted ToMOH (bottom) (c) oxidized (top) and reduced (bottom) RNR-R2 (d) oxidized (top) and reduced (bottom) rubryerythrin (e) reduced stearoyl-acyl carrier protein A9 desaturase (f) reduced bacterioferritin (g) methaemerythrin. Fel is on the left and Fe2 on the right. (Reprinted with permission from Sazinsky and Lippard, 2006. Copyright (2006) American Chemical Society.)...

In the diiron active sites of RNR-R2, A9 desaturase, bacterioferritin and rubrerythrin the flanking carboxyl ligands on the opposite side of the diiron centre are all quite different,... 

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,97 or glycolipids.98 The A9 desaturase, isolated from liver or from yeast, converts stearoyl-CoA to oleoyl-CoA (Eq. 21-3).99-102 This membrane-associated enzyme system... 

In plants a similar enzyme catalyzes formation of the first double bond in a fatty acyl group converting stearoyl-ACP into oleoyl-ACP in the chloroplasts.72 753/105 108 The soluble A9 stearoyl-ACP desaturase has a diiron-oxo active site (Fig. 16-20, B, C).i°9 no Electrons are donated from light-generated reduced ferredoxin (see Chapter 23). In addition to the A9 desaturase both plants and cyanobacteria usually desaturate C18 acids also at the A12 and A15 positions and C16 acids at the A7, A20, and A13 (co3) positions.iii ii2 Desaturation of oleate occurs primari-... 

Enzyme complexes occur in the endoplasmic reticulum of animal cells that desaturate at A5 if there is a double bond at the A8 position, or at A6 if there is a double bond at the A9 position. These enzymes are different from each other and from the A9-desaturase discussed in the previous section, but the A5 and A6 desaturases do appear to utilize the same cytochrome b5 reductase and cytochrome b5 mentioned previously. Also present in the endoplasmic reticulum are enzymes that elongate saturated and unsaturated fatty acids by two carbons. As in the biosynthesis of palmitic acid, the fatty acid elongation system uses malonyl-CoA as a donor of the two-carbon unit. A combination of the desaturation and elongation enzymes allows for the biosynthesis of arachidonic acid and docosahexaenoic acid in the mammalian liver. As an example, the pathway by which linoleic acid is converted to arachidonic acid is shown in figure 18.17. Interestingly, cats are unable to synthesize arachidonic acid from linoleic acid. This may be why cats are carnivores and depend on other animals to make arachidonic acid for them. Also note that the elongation system in the endoplasmic reticulum is important for the conversion of palmitoyl-CoA to stearoyl-CoA. 

Synthesis in mammalian tissues of arachidonic acid from linoleic acid. The A5 and A6 desaturases are separate enzymes and are also different from the A9 desaturase (fig. 18.16). The mechanisms, however, seem to be the same, involving cytochrome b5 and cytochrome reductase. The enzymes for elongation of unsaturated fatty acid such as 18 3 to 20 3 occur on the endoplasmic reticulum. 

R = (P03) ) [36] (c) a,fi-dehydrogenation of fatty acids by soluble stearoyl-ACP A9-desaturase (A9D) (R = holo-acyl carrier protein) [37],... 

Malvaceae). Malvalic acid is produced from sterculic acid by chain shortening from the carboxyl end (Figure 3.14). Sterculic acid is an inhibitor of the A9-desaturase which converts stearic acid into oleic acid and is potentially harmful to humans in that it can alter membrane permeability and inhibit reproduction. 

The next example is used to demonstrate how different pathways could produce the same pheromone component. Helicoverpa zea and Helicoverpa assulta are closely related species that use aldehydes as the major pheromone. Helicoverpa zea uses a blend of components with Z11-16 Aid as the major component, and minor components include 16 Ald, Z9-16 Aid, and Z7-16 Aid (Klun et al., 1980). H. assulta uses Z9-16 Ald as the major component and Z11-16 Aid as a minor component (Cork et al., 1992 Sugie et al., 1991). The biosynthesis of Zll-16 Aid occurs by Al 1 desaturation of 16 CoA to produce Z1 l-16 CoA, which is reduced to the aldehyde. This probably occurs in both species, but Z9-16 Ald could be produced by the action of a A9 desaturase using 16 CoA as a substrate or by the Al 1 desaturation of 18 CoA to produce Zll-18 CoA that is then chain shortened to Z9-16 CoA. To determine between these two pathways, deuterium-labeled precursors were applied topically to the glands in dimethyl sulfoxide and females injected with PBAN 1 h later the glands were extracted and analyzed for incorporation using GC/MS (Choi et al., 2002). Figure 3.4 shows the data and biosynthetic pathways. 

Molecular cloning and functional expression of a A9 desaturase-encoding cDNA from T. ni fat body... 

An expression construct consisting of the open reading frame of the TnFB A9Ds cDNA inserted into the yeast desaturase expression vector YEpOLEX was used to transform the olel strain of S. cerevisiae as described above. Many transformant colonies were obtained on medium lacking unsaturated fatty acids, indicating complementation of the olel mutation by the encoded T. ni desaturase. GC/MS analysis of the fatty acid methyl esters obtained from the transformants showed that the TnFBA9Ds cDNA encoded a A9 desaturase that produced oleic acid (Z9-18 Acid) and palmitoleic acid (Z9-16 Acid) (Liu et al., 1999). Quantitation of these unsaturated fatty acids under standard conditions as described above revealed about three times more of the former than the latter (Rosenfield et al., 2001). 

Northern blot and RT-PCR analyses showed that the A9 desaturase-encoding RNA was present in T. ni adult and larval fat body, as well as in pheromone-gland and muscle tissue. Interestingly, no A9 desaturase-encoding cDNA was... 

The two additional full-length desaturase-encoding cDNAs obtained from the H. zea pheromone gland were designated HzPGDs2 and HzFBDs (Rosenfield et al., 2001). The HzPGDs2 cDNA encoded a 353 amino acid protein that had only 64 percent identity to the T. ni A9 desaturase. Its corresponding RNA was... 

Figure 4.3 Mass quantities of unsaturated fatty acids extracted from ole1 strains of S. cerevisiae transformed with plasmids encoding the wildtype yeast desaturase (OLE1), two moth A9 desaturases (TnFBd9Ds and HzPGDs2), and two moth A11 desaturases (TnPGd11Ds and HzPGDsI). Growth conditions, extraction procedures, and quantitation were standardized as in Figure 4.2. (Reproduced with permission from Rosenfield etai., 2001. 2001 by Insect Biochemistry and Molecular Biology.)...

A second desaturase-encoding cDNA was isolated from this species and found to encode a 352 amino acid protein that is orthologous to the H. zea A9 desaturase HzPGDs2 based on its 83 percent sequence identity to the latter vs only 63-64 percent identity to the TnFBDs and HZFBDs desaturases. When this cDNA, designated Pocto-Z9, was functionally expressed in the olel yeast mutant it... 

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