A6-Desaturase

Assay supernatant for A12-A6-desaturase activites according to Galle et al. (31). 

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. 

Mutant Mut49 was considered to be defective in A6 desaturase because of a high 18 2co6 level and low levels of 18 3o>6, DHGA and A A in its mycelia. In addition to the accumulation of 18 2 6, two nonmethylene-interrupted PUFAs, i.e., 5,11,14-cis-eicosatrienoic acid (20 3A5) and 5,11,14,17-cis-eicosatetraenoic acid (20 4A5), were found [158], These PUFAs are considered to be converted from 18 2co6 or 18 3ca3, respectively, by three subsequent reactions, i.e., A6 desaturation, elongation and A5 desaturation (Fig. 15). 

The HUFAs (AA, EPA, and DHA), required by mammals, are synthesized by the microsomal elongases and A5 and A6 desaturases (8). The substrates for synthesis, LA and LN, are obtained primarily from vegetable oils in the diet. Elongation and desaturation of LA produces AA, whereas elongation and desaturation of LN produces EPA and DHA. Synthesis of DHA, which is concentrated in the brain, proceeds through the elongation and desaturation pathways to the product C24 6 (1)3, which is then the substrate for one cycle of peroxisomal fS-oxidation to yield C22 6 (u3 (8). 

An alternative route to EPA and DHA can come from elongation and further desaturation of stearidonic acid (18 4A ). Certain plants, including blackberry, borage, and evening primrose, contain up to 25% of y-linolenic acid (18 3A ) in their seed oil, with considerably smaller amounts of stearidonic acid. The y-linolenate arises from the action of a A6-desaturase on linoleate. Small amounts of co-3-desaturase present in these seeds account for the stearidonate produced. When A6-desaturase from borage was introduced into soy, plants producing up to 29% y-linolenate (precursor to arachidonate), with up to 4% stearidonate in oil, resulted (32). Further desaturation to stearidonate could be promoted with high expression of an co-3-desaturase. 

Marzo, J., Alava, M. A Pineiro, A., and Naval, J. (1996). Biosynthesis of docohexaenoic acid in human cells Evidence that two different A6-desaturase activities may exist. Biochim. Biophys. Acia 13D1, 263-272. 

Ivanetich, K. M., Bradshaw, J. J., and Ziman, M. R. (1996). A6-Desaturase Improved methodology and analysis of the kinetics in a multi-enzyme system. Biochem. Biophys. Acta 1292,120-132. 

Several plants A6-desaturase genes have been cloned from Anemone leveillei, borago [211], three Echium species, Marchantia polymorpha. Primula farcinosa, P. vialii and P. juliae, as well as from fungi Mortierella alpine, M. isabelina, Mucor rouxii [205] and Pythium irregulare, from the diatom Phaeodactylum tricornutum [203] and from mosses the Ceratodom purpureus and Physcomitrella patens [212], although not all of them have been expressed in plants. 

Analogously, A6-desaturase of B. officinalis has been overexpressed in tobacco plants and the determination of the FA distribution in the total lipid pool was determined [208]. A6-... 

Likewise a A5-desaturase from humans has been characterized [231]. In a first attempt, the gene was isolated and then cloned and expressed in Chinese hamster ovary cells. Its structural characteristics and nutritional regulation were compared with other A5 and A6-desaturases. Concerning function analysis, the transgenic hamster cells were able to convert DGLA into AA. Furthermore, the structure was similar to other known desaturases, sharing a 61% identity with... 

In another attempt, a A5-desaturase from C. elegans and a A6-desaturase from B. officinalis were coexpressed with CEELOl, an elongase from C. elegans in S. cerevisiae [239] to reconstitute the PUFA biosynthetic pathway. Expression of the A6-desaturase and CEELOl was confirmed in the presence of LA and ALA, whilst the A5-desaturase and the elongase were expressed only when LA was... 

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