Lipids Desaturase

Polyunsaturated fatty acid synthesis is catalyzed by acyl-lipid-desaturases, also named front-end desaturases due to their action mechanism, which proceeds via introduction of double bonds into preformed acyl chains by oxygen and electron-donor dependent desaturation, between the carboxyl group and the pre-existing unsaturation which acts as substrate. For many microsomal desaturases, the electron donors are cytochrome b5, and a small hemoprotein that operates in numerous redox reactions in plants, involving NADH-dependent acyl-group desaturation [200]. 

According to its solubility these enzymes can be classified into two non evolutively related groups the soluble acyl carrier protein (AGP) desaturases and the membrane-bound desaturases, which includes the acyl-lipid desaturases and the acyl-CoA desaturases. The soluble AGP desaturases introduce double bonds into fatty acids esterified to AGP, and are found in the stroma of plant plastids (Shanklin and Gaboon, 1998) and some bacteria, as Mycobacterium and Streptomyces (Phetsuksiri et al., 2003). The acyl-lipid desaturases, that... 

Some acyl-lipid desaturases recognize specific polar head groups, as well as the in-position of the glycerol backbone to which the fatty acid is esterifed (Murata and Wada, 1995). However, most acyl-lipid desaturases are insensitive to head groups and to in-positions. 

The effects of the unsaturation of fatty acids on membrane fluidity has also been extensively studied in two strains of cyanobacteria, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 (Los and Murata, 2004). The genes for four specific acyl-lipid desaturases, designated desA, desB, desC and desD, have been cloned from Synechocystis. These desaturases introduce double bonds at the A12 (Sakamoto and Bryant, 1997), ( 3 (Sakamoto et al., 1994a), A9 (Sakamoto and Bryant, 1997 Sakamoto et al., 1994b), and A6 (Reddy et al.,... 

Altabe, S.G., Aguilar, P., Caballero, G.M. and de Mendoza, D. The Bacillus subtilis acyl lipid desaturase is a deltas desaturase. J Bacterial, 185 (2003) 3228-3231. 

Cybulski, L.E., Albanesi, D., Mansilla, M.C., Altabe, S., Aguilar, P.S. and de Mendoza, D. Mechanism of membrane fluidity optimization isothermal control of the Bacillus subtilis acyl-lipid desaturase. Mol Microbiol, 45 (2002) 1379-1388. 

Higashi, S. and Murata, N. An in vivo study of substrate specificities of acyl-lipid desaturases and acyltransferases in lipid synthesis in Synechocystis PCC6803. Plant Physiol, 102 (1993) 1275-1278. 

Murata, N. and Wada, H. Acyl-lipid desaturases and their importance in the tolerance and acclimatization to cold of cyanobacteria. Biochem J, 308 ( Pt 1) (1995) 1-8. 

Mustardy, L., Los, D.A., Gombos, Z. and Murata, N. Immunocytochemical localization of acyl-lipid desaturases in cyanobacterial cells evidence that both thylakoid membranes and cytoplasmic membranes are sites of lipid desaturation. ProcNatl AcadSci U SA,92 (1996) 10524-10527. 

Homung E, Pemstich C, Feussner 1. (2002) Formation of conjugated deltal ldeltal3-double bonds by deltal2-hnoleic acid (l,4)-acyl-lipid-desaturase in pomegranate seeds. Eur J Biochem 269 4852-4859. 

Demin, A. N., Deryabin, A. N., Sinkevich, M. S., and Trunova, T. I. Introduction of gene desA. 12-acyl-lipid desaturase of cyanobacterium enhange the resistance of potapo plants to oxidative stress caused by hypothermia. Plant Physiol. (Rus), 55 710-720 (2000). 

All desaturases in cyanobacteria and higher plants, except A9 acyl-ACP desaturase in higher plants, are of the acyl-lipid and membrane-bound type (9). Synechocystis sp. PCC 6803 contains four desaturases which introduce double bonds at their respective positions, namely at the A6, A9, A12, and co3 positions of Cis fatty acids. They are known as A6, A9, A12 and o)3 acyl-lipid desaturases, respectively (9). Arabidopsis thaliana contains A12 and oC acyl-lipid desaturases in chloroplasts. The objective of the present study was to localize these desaturases in cyanobacterial cells and higher-plant chloroplasts by immunocytochemistry to identify the sites of desaturation of fatty acids. 

Sakamoto, T., Wada, H., Nishida, I., Ohmori, M. and Murata, N. A9 acyl-lipid desaturases of cyanobacteria. J. Biol. Chem. 269 (1994), 25576-25580. 

Avelange-Macherel M-H, Macherel D, Wada H, Murata N. 1995. Site-directed mutagenesis of histidine residues in the Delta-12 acyl-lipid desaturase of Synechocys-tis. FEBS Lett 361 111-114. 

The acyl-lipid desaturases can be further classified into subgroups according to their electron donors. One subgroup, present in the endoplasmic reticulum of plant cells, uses cytochrome Jbs as the electron donor [6,7]. The other, present in chloroplastic and cyanobacterial thylakoid membranes, uses ferredoxin as the electron donor [8,9], A unique characteristic of the desaturases is that they recognize exact positions within long various carbon chains at which double bonds are specifically introduced. 

In the present communication, we summarize our recent attempts aimed at the physiological, biochemical and molecular-biological characterization of acyl-lipid desaturases in cyanobacteria. 

To study desaturation reactions in terms of the positions within the carbon chain at which the double bonds are introduced by the desaturases, we induced Synechocystis sp. PCC 6803 to synthesize odd-numbered fatty acids by feeding cells with heptanoic acid (C7). The most prevalent unsaturated fatty acids of membrane lipids synthesized in these cells were 17 4(6,9,12,14), 18 4(6,9,12,15) and 19 4 (6,9,12,16). These results demonstrate that double bonds appeared at positions 6, 9, and 12, counted from the carboxyl terminus, regardless of the chain length of the fatty acid. However, the fourth double bond was located at position 3, counted from the methyl terminus. From these physiological results, we designated the four desaturases A6, A9, A12 and 0)3 acyl-lipid desaturases, respectively [11]. 

The desC gene has also been isolated from Anabaena variabilis. The deduced amino acid sequences of the A9 acyl-lipid desaturases of cyanobacteria are similar to those of the A9 acyl-CoA desaturases from the rat the mouse and yeast, with similarity scores of about 25%. Moreover, histidine residues, which are potential ligands of Fe atoms, are well conserved among the A9 acyl-lipid and the A9 acyl-CoA desaturases. The amino acid sequence of the 0)3 desaturase, deduced from the desB gene [13], resembles those of the o)3 desaturases from higher plants, with similarity scores of 45-50%. 

The desC genes from Synechocystis sp. PCC 6803 and A. variahilis were introduced into pET-3a and overexpressed in E. coli under the control of bacteriophage T7 RNA polymerase. The cells were supplied with 18 0, which is present at very low levels in E. coli cells under normal growth conditions. The externally supplied 18 0 was esterified at both the sn-1 and sn-2 positions of the glycerol moiety of phosphatidylglycerol and phosphatidyl-ethanolamine. The overexpressed A9 acyl-lipid desaturase of Synechocystis sp. PCC 6803 and of A. variahilis was active in converting 18 0 to 18 1(9) at the sn-1 position but not at the sn-2 position, and it did not desaturate 16 0 at the sn-1 or the sn-2 position. These observations lead us to conclude that the A9 acyl-lipid desaturases from Synechocystis sp. PCC 6803 and A. variahilis are specific to 18 0 and the sn-1 position but are nonspecific with respect to the head group. 

Sakamoto T, Wada H, Nishida 1, Ohmori M, Murata N. A9 acyl-lipid desaturases of cyanobacteria molecular cloning and substrate specificities to fatty acids, sn-positions and polar head groups. J Biol Chem 1994 in press. 

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