Study of the 3-hydroxy eicosanoyl-coenzyme A dehydratase and (E)-2,3 enoyl-coenzyme A reductase involved in acyl-coenzyme A elongation in etiolated leek seedlings

(R,S)-[1 -C-14]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically sy nthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl- CoA elongase of etiolated reek (Allium porrum L.) seedling microsomes. 3-Hy droxy eicosanoyl-CoA (3-OH C20:0-CoA) dehydration led to the formation of ( E)-2,3 eicosanoyl-CoA, which has been characterized. Our kinetic studies ha ve determined the optimal conditions of the dehydration and also resolved t he stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase h ad a marked preference for (R)3-OH C20:0-CoA. Moreover, the very-long-chain synthesis using (R)-3-OH C20:0-CoA isomer and [2-C-14]malonyl-CoA was high er than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C2 0:0-CoA concentrations. We have also used [1-C-14]3-OH C20:0-CoA to investi gate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA e longase complex. In the presence of NADPH, [1-C-14]3-OH C20:0-CoA conversio n was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicos anoyl-CoA, we detected eicosanoyl-CoA resulting from the reduction of (E)-2 ,3 eicosanoyl-CoA. When we replaced NADPH with NADH, the eicosanoyl-CoA was 8- to 10-fold less abundant. Finally, in the presence of malonyl-CoA and N AD PH or NAD H, [1-C-14] 3-OH C20: 0-CoA led to the synthesis of very-long- chain fatty acids. This synthesis was measured using [1-C-14]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-C-14]malonyl-CoA, In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg(-1) h(-1), which is the highest ever reported for a pl ant system.