A Simple Method for Enzymatic Synthesis of Unlabeled and Radiolabeled Hydroxycinnamate-CoA

摘要

Hydroxycinnamate coenzyme A (CoA) thioesters are substrates for biosynthesis of lignin and hydroxycinnamate esters of polysaccharides and other polymers. Hence, a supply of these substrates is essential for investigation of cell wall biosynthesis. In this study, three recombinant enzymes, caffeic acid 3-O-methyltransferase, 4-coumarate-CoA ligase 1, and 4-coumarate-CoA ligase 5, were cloned from wheat, tobacco, and Arabidopsis, respectively, and were used to synthesize ~(14)C-feruloyl-CoA, caffeoyl-CoA, p-coumaroyl-CoA, feruloyl-CoA, and sinapoyl-CoA. The corresponding hydroxycinnamoyl-CoA thioesters were high-performance liquid chromatography purified, the only extraction/purification step necessary, with total yields between 88-95%. Radiolabeled ~(14)C-feruloyl-CoA was generated from caffeic acid and S-adenosyl-~(14)C-methionine under the combined action of caffeic acid 3-O-methyltransferase and 4-coumarate-CoA ligase 1. About 70% of ~(14)C-methyl groupsrnfrom S-adenosyl methionine were incorporated into the final product. The methods presented are simple, fast, and efficient for the preparation of the hydroxycinnamate thioesters.