A Simple Method for Enzymatic Synthesis of Unlabeled and Radiolabeled Hydroxycinnamate-CoA
- Author(s): Rautengarten, Carsten
- Baidoo, Edward
- Keasling, Jay D.
- Scheller, Henrik Vibe
- et al.
Published Web Locationhttps://doi.org/10.1007/s12155-010-9085-3
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 14C-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 14C-feruloyl-CoA was generated from caffeic acid and S-adenosyl-14C-methionine under the combined action of caffeic acid 3-O-methyltransferase and 4-coumarate-CoA ligase 1. About 70% of 14C-methyl groups from S-adenosyl methionine were incorporated into the final product. The methods presented are simple, fast, and efficient for the preparation of the hydroxycinnamate thioesters.