SOLUBLE AND MEMBRANE-BOUND COMPONENTS OF PLANT LIPID-SYNTHESIS

Enoyl ACP reductase (ENR) catalyzes the NADH dependent reduction of tr ans enoyl ACP to form saturated acyl ACPs; it is an essential componen t of the multisubunit type II fatty acid synthetase which is highly ex pressed in a temporal specific manner in seeds. The enzyme has been pu rified from rape, extensively sequenced, its cDNA cloned, and the prot ein overexpressed and crystallized. The complete 3-dimensional structu re of the enzyme has been determined at 1.9 Angstrom. Difference Fouri er analysis has shown that crotonyl ACP is a better substrate than cro tonyl CoA as the latter also binds to the NADH pocket of the enzyme an d thereby acts as an enzyme inhibitor. The potential active site has b een identified from the position of conserved residues and by the loca tion of the position of the nicotinamide ring of NADH. In addition ext ensive structural similarity has been found between ENR and the 3 alph a-20 beta-hydroxysteroid dehydrogenase. This has provided insights int o the catalytic mechanisms which are being tested by site directed mut agenesis. In an attempt to gain insight into membrane bound enzymes of lipid biosynthesis we have employed a complementation cloning techniq ue in E. coli to isolate the membrane bound 2-acyltransferase which ha s defied conventional purification techniques. In the first instance w e cloned a 2-acyltransferase (2-AT) from maize and more recently we ha ve cloned two 2-acyltransferases from Limnanthes douglasii. One of the se shows distinct substrate specificity differences to the E. coli 2-A T. Introduction of the cDNA encoding this 2-AT into a high erucic acid rape line has allowed the synthesis of trierucin in the transgenic se ed. Analysis of the transgenes and other acyltransferases is in progre ss.