Purification of the acyl‐CoA elongase complex from developing rapeseed and characterization of the 3‐ketoacyl‐CoA synthase and the 3‐hydroxyacyl‐CoA dehydratase

Abstract

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 VLCFA elongase gene family in Arabidopsis thaliana: phylogenetic analysis, 3D modelling and expression profiling

“…These KCS enzymes have different substrate specificities suggesting that multiple fatty acid elongases exist in plants. These results are consistent with the findings that at least three fatty acid elongase-containing preparations with different substrates or co-factor requirements have been partially purified from developing oil seeds (Domergue et al 1999(Domergue et al , 2000. Furthermore, three KCSs have been shown to be involved in the formation of the cuticle in Arabidopsis: CER6 (Millar et al 1999;Fiebig et al 2000;Hooker et al 2002), KCS1 (Todd et al 1999) and FDH (Yephremov et al 1999;Pruitt et al 2000).…”

Mutations in the fatty acid elongation 1 gene are associated with a loss of β‐ketoacyl‐CoA synthase activity in low erucic acid rapeseed

“…A 15 000× g pellet isolated from immature seeds according to [13] was used as the enzyme source. The acyl‐CoA elongation was measured by incubating 60 μg protein in 30 μM C18:1‐CoA, 0.5 mM NADPH, 0.5 mM NADH, 1 mM MgCl 2 , 2 mM dithiothreitol (DTT) and 34 μM [2‐ 14 C]malonyl‐CoA, 0.08 M HEPES buffer pH 7.2 for 1 h. The ATP‐dependent elongase was performed using the same conditions except that C18:1‐CoA was replaced by 1 mM ATP.…”

“…The 3‐ketoacyl‐CoA synthase activity was measured under the same conditions reported in [13]. The 3‐hydroxyacyl‐CoA dehydratase activity was determined by incubating 60 μg of microsomal proteins in the presence of 2 mM DTT, 1 mM MgCl 2 , 150 μM Triton X‐100 and 11 μM [1‐ 14 C]3‐hydroxy‐C20‐CoA, 80 mM HEPES buffer (pH 7.2) in a volume of 0.1 ml for 15 min at 30°C.…”

High‐value oils from plants

“…longer than C18), and one such fatty acid, erucic acid (22:1D 13 ), is dominant in the seed oil of many Brassicaceae species. These elongation steps are performed while the acyl group is attached to CoA in a reaction sequence analogous to those of de novo fatty acid synthesis in plastids, except that all of the extra-plastidial enzyme activities are membrane-bound, whereas in the plastid they are soluble and utilize acyl-ACP as substrates (Domergue et al, 2000). For industrial use, erucic acid must be obtained in high concentration in order to reduce purification costs.…”