Oleoyl-CoA elongase catalyzes four successive reactions: condensation of malonyl-CoA to oleoyl-CoA, reduction, dehydration, and another reduction. Evidence supporting this mechanism and the multienzymatic nature of the elongation complex are reported. A particulate membrane fraction from rapeseed is able to elongate intermediates (R,S) 3-hydroxy-20:0-CoA and (E) 2,3-20:1-CoA to very long chain fatty acids in the presence of malonyl-CoA. Studies of the 3-ketoacyl-CoA synthase activities showed that maximal activity could be measured by using 15 to 30 microM 18:1-CoA and 30 microM malonyl-CoA, and that 18:0-CoA and 18:1-CoA were the best substrates. Comparison of the condensation and the overall elongation activities indicated that condensation is the rate-limiting step of the elongation process. The 3-hydroxyacyl-CoA dehydratase activity was maximal in the presence of 75 microM Triton X-100 and 25 microg of proteins. Finally, the acyl-CoA elongase complex was solubilized and purified. During the purification process, the 3-hydroxyacyl-CoA dehydratase copurified with the elongase complex, strongly suggesting that this enzyme belongs to the elongase complex. The apparent molecular mass of 700 kDa determined for the elongase complex, and the fact that four different polypeptide bands were detected after sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fraction, further suggest that the acyl-CoA elongase is a multienzymatic complex.

The elongation of different substrates was studied using several subcellular fractions from Brassica napus rapeseed. In the presence of malonyl-CoA, NADH and NADPH, very-long-chain fatty acid (VLCFA) synthesis was observed from either oleoyl-CoA (acyl-CoA elongation) or endogenous primers (ATP-dependent elongation). No activity was detected using oleic acid as precursor. Acyl-CoA and ATP-dependent elongation activities were mainly associated with the 15 000 g/25 min membrane fraction. Reverse-phase TLC analysis showed that the proportions of fatty acids synthesized by these activities were different. Acyl-CoA elongation increased up to 60 mm oleoyl-CoA, and ATP-dependent elongation was maximum at 1 mm ATP. Both activities increased with malonyl-CoA concentration (up to 200 mm). Under all conditions tested, acyl-CoA elongation was higher than ATP-dependent elongation, and, in the presence of both ATP and oleoyl-CoA, the elongation activity was always lower. ATP strongly inhibited acyl-CoA elongation, whereas ATP-dependent elongation was slightly stimulated by low oleoyl-CoA concentrations (up to 15 mm) and decreased in the presence of higher concentrations. CoA (up to 150 mm) had no effect on the ATP-dependent elongation, whereas it inhibited the acyl-CoA elongation. These marked differences strongly support the presence in maturing rapeseed of two different elongating activities differently modulated by ATP and oleoyl-CoA.Keywords: acyl-CoA elongase; acyl-CoAs; ATP-dependent elongation; erucic acid; plant lipids.Very-long-chain fatty acids (VLCFAs, fatty acids with at least 20 carbon atoms) are widely distributed in nature. In plants, they are primarily found in epicuticular waxes (mostly saturated VLCFAs) and in seed storage oils [mostly monounsaturated VLCFAs, generally belonging to the cis (n-9) series]. The enzymes responsible for their synthesis, referred to as elongases, are membrane-bound proteins that utilize malonyl-CoA as the C2-unit donor and primers having at least 18 carbon atoms [1,2].Genetic [3,4] and biochemical [5,6] studies have suggested the existence of several elongases. Several studies using leek epidermis microsomes [7±9] have hypothesized the existence of at least two different elongating activities. In the presence of labeled malonyl-CoA, VLCFAs could be synthesized from either acyl-CoAs (acyl-CoA elongation) or endogenous acyl components of the microsomal lipids; in the latter case, there is an absolute requirement for ATP (ATP-dependent elongation). In the case of acyl-CoA elongation, Lessire et al. [9] demonstrated that successive additions of C2 units to stearoyl-CoA (C 18 : 0 -CoA) were responsible for the sequential synthesis of arachidonoyl-CoA (C 20 : 0 -CoA), behenoyl-CoA (C 22 : 0 -CoA) and lignoceroyl-CoA (C 24 : 0 -CoA). On the other hand, ATP-dependent elongation involves a one-step condensation from an endogenous primer (Cn) with malonylCoA to a Cn + 2 [2,7]. Using linear sucrose-gradient centrifugation, Moreau et al.[10] studied the intracellular localization of these el...

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