DEHYDRATION OF 3-HYDROXY ICOSANOYL-COA AND REDUCTION OF (E)2,3-ICOSENOYL-COA ARE REQUIRED FOR ELONGATION BY LEEK MICROSOMAL ELONGASE(S)

Very-long-chain fatty acids (VLCFA) are synthesized by the acyl-CoA el ongase which catalyses the addition of two carbon units from malonyl-C oA to an acyl-CoA. This elongation mechanism is thought to involve fou r successive reactions: condensation, reduction, dehydration and reduc tion. We provide here biochemical evidence supporting this pathway. Tw o intermediates of the elongation process -(R,S) 3-hydroxyicosanoyl-Co A and (E) 2,3 eicosenoyl-CoA-were chemically synthesized and used as p rimers for elongation. They were elongated by leek microsomes in the p resence of [2-C-14] malonyl-CoA, NADPH and Triton X-100 with the same efficiency as using C20:0-CoA as primer. The elongation of both interm ediates increased as a function of time (up to 20 min), proteins (up t o 30 mu g) and substrate concentration (up to 16 mu M). The resulting products were saturated straight-chain acyl-CoAs. No activity of 3-oxo acyl-CoA synthase was detected when using the 3-hydroxyicosanoyl-CoA o r the (E) 2,3 eicosenoyl-CoA demonstrating that these intermediates we re 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-acyl-CoA dehydrase and the (E) 2, 3 icosenoyl-CoA is a substrate of the enoyl-CoA reductase. (C) Elsevie r, Paris.