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 Seedlings¹

René Lessire^*, Sylvette Chevalier, Karine Lucet-Levannier, Jean-Paul Lellouche, Charles Mioskowski, and Claude Cassagne

Laboratoire de Biogenèse Membranaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5544, Université V. Ségalen Bordeaux 2, 146 Rue Léo Saignat, 33076 Bordeaux cedex, France (R.L., S.C., C.C.); Département de Biologie Cellulaire et Moléculaire, Service des Molécules Marquées, Centre d'études nucléaires-Saclay, Bat 547, 91191 Gif-sur-Yvette cedex, France (K.L.-L., C.M.); and Ben-Gurion University of the Negev, The Institutes for Applied Research, E.D. Bergman Campus, P.O. Box 653, 84105 Beer-Sheva, Israel (J.-P.L.)

(R,S)-[1-¹⁴C]3-Hydroxy eicosanoyl-coenzyme A (CoA) has been chemically synthesized to study the 3-hydroxy acyl-CoA dehydratase involved in the acyl-CoA elongase of etiolated leek (Allium porrum L.) seedling microsomes. 3-Hydroxy 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 have determined the optimal conditions of the dehydration and also resolved the stereospecificity requirement of the dehydratase for (R)-3-OH C20:0-CoA. Isotopic dilution experiments showed that 3-hydroxy acyl-CoA dehydratase had 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 [2-¹⁴C]malonyl-CoA was higher than that using the (S) isomer, whatever the malonyl-CoA and the 3-OH C20:0-CoA concentrations. We have also used [1-¹⁴C]3-OH C20:0-CoA to investigate the reductant requirement of the enoyl-CoA reductase of the acyl-CoA elongase complex. In the presence of NADPH, [1-¹⁴C]3-OH C20:0-CoA conversion was stimulated. Aside from the product of dehydration, i.e. (E)-2,3 eicosanoyl-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 NADPH or NADH, [1-¹⁴C]3-OH C20:0-CoA led to the synthesis of very-long-chain fatty acids. This synthesis was measured using [1-¹⁴C]3-OH C20:0-CoA and malonyl-CoA or (E)-2,3 eicosanoyl-CoA and [2-¹⁴C]malonyl-CoA. In both conditions and in the presence of NADPH, the acyl-CoA elongation activity was about 60 nmol mg¹ h¹, which is the highest ever reported for a plant system.

Plant Physiol. (1999) 119: 1009-1016
Copyright Clearance Center:   0032-0889/99/119//08
© 1999 American Society of Plant Physiologists

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