Fatty Acid Desaturases

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In most plant tissues, over 75% of the fatty acids are unsaturated. Two types of desaturases have been identified, one soluble and the other membrane bound, that have different consensus motifs. Database searching for these motifs reveals that these enzymes belong to two distinct multifunctional classes, each of which includes desaturases, acetylenases, hydrolases and epoxygenases that act on fatty acids or other substrates (Lee et al., 1998; Shanklin and Cahoon, 1998). Free fatty acids are not thought to be desaturated in vivo; rather they are esterified to either acyl carrier protein (ACP) for the soluble plastid desaturase, to coenzyme-A (CoA) or to phospholipids for integral membrane desaturases.

2.3.1 A -9 Desaturases

The first double bond in unsaturated FAs in plants is introduced by the soluble enzyme stearoyl-ACP desaturase. This fatty acid desaturase is unusual in that only a few other known desaturases are soluble. Soluble A -9 stearoyl-ACP desaturases are found in all plant cells and are essential for the biosynthesis of unsaturated membrane lipids (Shanklin and Somerville, 1991; Cahoon et al., 1994, 1997; Kaup et al., 2002). Desaturases that convert saturated fatty acids to mono-unsaturated fatty acids share several common characteristics. They perform stereospecific A -9 desaturation of an 18:0/16:0 substrate with the removal of the 9-A and 10-A hydrogens (Bloomfield and Bloch, 1960; Mudd and Stumpf, 1961). Protein crystallographic studies on the purified desaturase from castor bean have shown that it contains a diiron cluster (Fox et al., 1993). The protein is active as a homodimer and consists of a single domain of 11 helices. This diiron centre is the active site of the desaturase (Lindqvist et al., 1996).

Expression and regulation of A -9 desaturase in plants have been extensively studied (Fawcett et al., 1994; Slocombe et al., 1994). The expression of the promoter of the Brassica napus stearoyl desaturase gene in tobacco was found to be temporally regulated in developing seed tissues. However, the promoter is also particularly active in other oleogenic tissues such as tapetum and pollen grains, raising the interesting question of whether seed-expressed lipid synthesis genes are regulated by separate tissue-specific determinants or by a single factor common to all oleogenic tissues (Slocombe et al., 1994). In Saccharomyces cerevisiae, addition of saturated fatty acids induces A -9 fatty acid desaturase mRNA (Ole1 mRNA) by 1.6-fold, whereas a large family of unsaturated fatty acids represses Ole1 transcription by 60-fold. A 111 bp fatty acid regulation region (FAR) approximately 580 bp upstream of the start codon that is essential for the transcription activation and unsaturated fatty acid repression was identified (Quittnat et al., 2004). In addition to the transcriptional regulation, unsaturated fatty acids mediate changes in the half-life of the Ole1 mRNA (Gonzalez and Martin,1996).

Currently, industries that manufacture shortening, margarine, and confectionery products use considerable amounts of stearate (18:0) produced mainly from partially hydrogenated plant oils (Facciotti et al., 1999). Hydrogenation not only adds extra cost, but also generates significant amounts of trans-fatty acids which have been associated with an elevated risk of heart disease (Katan et al., 1995; Nelson, 1998; Facciotti et al., 1999). Industries manufacturing shortenings and confectionery products could benefit from oil crops that accumulate high levels of stearate. However, stearate (18:0) does not naturally accumulate to abundant levels in most cultivated oil crops including soybeans, and the production of a high-stearate phenotype has only had modest success so far through conventional breeding and mutagenesis techniques (Facciotti et al., 1999). Although stearic acid (18:0) is one of the major saturated fatty acids in most seed oils, its percentages vary among the different oilseed crops from 1.0% in rape seed oil to 3.6% in sesame and corn seed oils and 4.0% in soybean oil, with a range from 2.2% to 7.2% for the soybean genotypes available in the world germplasm collection (Hymowitz et al., 1972; Downey and McGregor, 1975; Rahman et al., 1997). The fatty acid composition of soybeans has been improved by using selective breeding techniques utilising natural variants or induced mutagenesis (Ladd and Knowles, 1970; Graef et al., 1985a,b). Hammond and Fehr (1983) were able to increase the amount of stearate (C18:0) produced in the soybean oil to levels up to about 28.1% of the total fatty acid content using mutagenesis. Rahman et al. (2003) reported a novel soybean germplasm with high stearic levels. This novel soybean was obtained as a consequence of the combination of the loci of high palmitic and stearic acids to determine the effects of altered contents of these two fatty acids on other fatty acids. As a result, two lines (M25 and HPS) with a five-fold increase in stearic acid (from 34 to 181 and 171 g kg-1) were developed. This increase in stearic acid was also found to be associated with a change in oleic and linoleic acid contents. Furthermore, these authors reported that when the palmitic and stearic acid levels in the oil of HPS were combined, this line had a saturated fatty acid content of >380 g kg-1. Thus, such oil might have the potential to increase the utility and also to improve the quality of soybean oil for specific purposes (Rahman et al., 2003; Clemente and Cahoon, 2009). Aghoram et al. (2006) showed that the fap2 locus which mediates an elevated seed palmitate phenotype in soybeans is due to a point mutation resulting in a premature stop codon.

Vegetable oils rich in mono-unsaturated fatty acids (MUFA) are not only important in human nutrition but can also be used as renewable sources of industrial chemicals (Cahoon et al., 1997). Palmitoleic acid, cis-9-hexadecenoic acid, (16:1 A 9) has the nutritional and industrial chemical advantages of the much more common longer chain MUFA, oleic acid (18:1 A 9), but with much better cold flow properties. Sea buckthorn (Hippophae rhamnoides) and cat's claw, Macfadyena unguis-cati (formerly Doxantha unguis-cati L.), have high levels of 16:1A 9 accumulating 40% and 80%, respectively (Chisholm and Hopkins, 1965; Cahoon et al., 1998; Yang and Kallio, 2001). Macadamia ((Macadamia integrifolia) nut oil is the main commercially available source of palmitoleic acid. Its oil is unique among edible sources in that mono-unsaturated fatty acids are the predominant component (about 80%) and a considerable portion (17-21%) of this is palmitoleic acid (a component not present in substantial amounts in olive oil) (Curb et al., 2000). Grayburn and Hildebrand (1995) and Wang et al. (1996) reported large increases in palmitoleic acid (16:1 A -9) after expressing a mammalian or yeast A -9 desaturase gene in both tobacco and tomato. Since soybeans are an important oil source that is high in linoleic and saturated fatty acids (mostly linoleic and palmitic acid: about 55% and 15%), conversion of all or part of these saturated fatty acids into palmitoleic acid would be a great benefit for health, while converting much of the remaining PUFAs (polyunsaturated fatty acids) into palmitoleic acid could have industrial value. Liu et al. (1996) reported converting about half of the palmitic acid of soybean somatic embryos into palmitoleic acid with good expression of a A 9-CoA desaturase. The transformed embryos had 16:1 levels from 0% to over 10% of total fatty acids, while the levels of 16:0 dropped from 25% to approximately 5% of total fatty acids.

A number of studies have demonstrated apparently beneficial effects of diets based on high MUFA content primarily derived from olive oil (Kris-Etherton et al., 1988; Spiller et al., 1992; Hegsted et al., 1993; Curb et al., 2000). The health implications of palmitoleic acid were first addressed by Yamori et al. (1986) and Abraham et al. (1989). Curb et al. (2000) compared the effects of (i) a typical American diet high in saturated fat - "37% energy from fat", (ii) the American Heart Association 'step 1' diet - "30% energy from fat" (half the saturated fatty acids, normal amounts of MUFAs and PUFAs, and high levels of carbohydrates), and (iii) a macadamia nut-based mono-unsaturated fat diet (37% energy from fat). When compared with the typical diet, the 'step 1' and macadamia nut diets both had potentially beneficial effects on cholesterol and LDL cholesterol levels. These results are consistent with previously reported lipid-altering benefits of MUFA-rich diets, particularly those involving macadamia nut oil (Ako and Okuda, 1995; Griel et al., 2008). Palmitoleic acid has also been reported to protect rats from stroke (Yamori et al., 1986), apparently by increasing cell membrane fluidity, clearing lipids from the blood, and altering the activity of important cell membrane transport systems particularly through inhibition of the Na+,K(+)-ATPase activity within a narrow range (Swarts et al., 1990). In men and women, elevated blood/tissue levels of palmitoleic acid were found to be correlated with protection from ventricular arrhythmias (Abraham et al., 1989) and negatively correlated with markers of artherosclerosis (Theret et al., 1993). Palmitoleic acid was also found to reportedly inhibit mutagenesis in animals (Hayatsu et al., 1988) and to be negatively correlated with breast cancer incidence in women (Simonsen et al., 1998).

Many vegetable oils are partially hydrogenated to increase the stability of cooking oils, and hydrogenated further for use as margarines and shortenings. To reduce or eliminate the need for hydrogenation of vegetable oils used for margarines and shortenings, a goal of plant geneticists has been to develop high stearate oils. As described in the section above on high stearate oils, many groups have been successful in achieving this goal with a variety of vegetable oils using different approaches, including genetic engineering of soybeans to a 53% stearic acid content of oil (Knutzon et al., 1992; Kridl, 2002; Martinez-Force et al., 2002). There have been several strategies to increase stearic acid levels in oilseed crops, and the increases achieved have usually been at the expense of oleic (18:1) and linoleic (18:2) acids. Among other strategies, both anti-sense suppression and co-suppression to reduce or knock out the activity of stearoyl-ACP desaturase, which is responsible for converting stearoyl-ACP (saturated) to oleoyl-ACP (unsaturated) (Budziszewski et al., 1996), have been used routinely. Also the stearoyl-ACP thioesterase is another possible metabolic target. Up-regulation of this enzyme by sense-oriented reintroduction of the stearoyl-ACP thioesterase has been found to result in an increase of free stearate release. Kridl (2002)

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