Malonyl-CoA:Acyl Carrier Protein Transacylase from Developing Seeds of Cuphea lanceolata

Abstract

Malonyl-CoA:acyl carrier protein (ACP) transacylase (MAT; E.C. 2.3.1.39.) was partially purified from developing seeds of Cuphea lanceolata , a crop accumulating up to 90% decanoic acid in storage triacylglycerols. Upon treatment of a cell-free seed extract with ammonium sulfate, most of the enzyme activity was precipitated between 40–75% ammonium sulfate saturation. Further purification was accomplished in 4 steps that included gel filtration on Sephacryl S 200 HR, anion exchange chromatography on Mono Q (0.0 to 0.2M LiBr gradient), rechromatography on Mono Q (0.00 to 0.12M LiBr gradient) and gel filtration on Sephacryl S 100 HR. The MAT-preparation obtained after anion exchange chromatography was free from any β-ketoacyl-ACP synthase (KAS; E.C. 2.3.1.41.) activity and could be used in the synthesis of malonyl-ACP to study medium-chain fatty acid biosynthesis. The molecular mass of the MAT was 27.5 kDa based on gel filtration and 23.5 kDa based on Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-aminoterminal sequence of this enzyme (VAVAELQVE-FI; -,not determined), for the first time determined for a MAT from a higher plant, showed a 25% homology to the enzyme from Escherichia coli .