Enzymatic synthesis and purification of caffeoyl-CoA, p-coumaroyl-CoA, andferuloyl-CoA

An enzyme preparation from wheat seedlings containing p-coumaroyl:CoA ligas e activity was used to synthesize caffeoyl-CoA, p-coumaroyl-CoA, and ferulo yl-CoA. The same enzyme preparation also contains caffeic acid-3-O-methyl t ransferase and caffeoyl-CoA-3-O-methyl transferase activities. The maximum activity was found in enzyme preparation from 2-day-old seedlings, where 15 -20% of the hydroxy cinnamic acid could be converted into the corresponding thioester. This yield is a result of an equilibrium between the ligase and a thioesterase also present in the crude enzyme preparation. The activity of caffeic acid 3-O-methyl transferase and caffeoyl-CoA 3-O-methyl transfer ase enables the production of C-14-labeled feruloyl-CoA when using S-adenos yl-L-[methyl-C-14]-methionine as methyl donor. The produced thioesters can be purified by reverse phase HPLC using a phosphoric acid-acetonitrile grad ient. (C) 2000 Academic Press.