The overexpression and characterisation of Enoyl ACP Reductase from Brassica. napus: a component enzyme of fatty acid synthetase in plants

Abstract

The main objective of this research is to advance our understanding at the structural and expressional level of plant enoyl ACP reductase (ER), the enzyme which catalyses the last reductive step in the de-novo biosynthesis of fatty acids. Two different substrates (CoA and ACP) can be used for this enzyme. Large scale preparations of crotonyl CoA were prepared using crotonic anhydride and CoA and the reaction product was quantified and authenticated. ACP (E.coli) was prepared, using standard literature methods, and the homogeneity of the product was verified by SDS/PAGE and N-terminal amino acid sequence determination. Recombinant plant ER, under the control of the T7 promoter and the T7 polymerase, was produced in large quantities in E.coli, and purified to homogeneity using a procedure developed in this study. The recombinant protein was authenticated by both amino acid analysis and N-terminal amino acid analysis. Spectral determinations were performed which allowed for the first time an extinction coefficient (2.95 x 10(^5)) of the enzyme to be determined. Antibodies were raised in mice against denatured recombinant ER and two dimensional western blotting was performed to establish the complexity of isoform expression in rape seed and leaf material. CD spectroscopy was carried out on the protein, to make structural predictions of a-helix and β-sheet which were correlated with those determined directly from X-ray crystallographic studies. In collaboration with the University of Sheffield, plant ER was successfully crystallised, several heavy metal derivatives prepared and the complete 3-dimensional structure of the NADH enzyme complex at 1.9 Angstrom resolution was determined. Inhibitor studies using phenylglyoxal were performed and it was found that ACP (500µM) and CoA (l0mM) would give total protection against loss of biological activity. Contrary to expectations no evidence could be obtained for the specific modification of a single arginine residue using phenylglyoxal, suggesting that this reagent may not be as specific as earlier reported.