Dehydration of 3-hydroxy icosanoyl-CoA and reduction of (E) 2,3 icosenoyl-CoA are required for elongation by leek microsomal elongase(s)

Abstract

Very-long-chain fatty acids (VLCFA) are synthesized by the acyl-CoA elongase which catalyses the addition of two carbon units from malonyl-CoA to an acyl-CoA. This elongation mechanism is thought to involve four successive reactions: condensation, reduction, dehydration and reduction. We provide here biochemical evidence supporting this pathway. Two intermediates of the elongation process — (R,S) 3-hydroxyicosanoyl-CoA and (E) 2,3 eicosenoyl-CoA — were chemically synthesized and used as primers for elongation. They were elongated by leek microsomes in the presence of [2- 14C] malonyl-CoA, NADPH and Triton X-100 with the same efficiency as using C 20:0-CoA as primer. The elongation of both intermediates increased as a function of time (up to 20 min), proteins (up to 30 μg) and substrate concentration (up to 16 μM). The resulting products were saturated straight-chain acyl-CoAs. No activity of 3-oxoacyl-CoA synthase was detected when using the 3-hydroxyicosanoyl-CoA or the (E) 2,3 eicosenoyl-CoA demonstrating that these intermediates were not substrates of the first enzyme (condensing enzyme) of the acyl-CoA elongase complex. The results establish that the 3-hydroxyacyl-CoA is a substrate of the 3-hydroxyacyl-CoA dehydrase and the (E) 2,3 icosenoyl-CoA is a substrate of the enoyl-CoA reductase.