Red clover coumarate 3'-hydroxylase (CYP98A44) is capable of hydroxylating p-coumaroyl-shikimate but not p-coumaroyl-malate: implications for the biosynthesis of phaselic acid.

Red clover (Trifolium pratense) leaves accumulate several mumol of phaselic acid [2-O-caffeoyl-L-malate] per gram fresh weight. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases (PPO) prevents breakdown of forage protein during storage. Forages like alfalfa (Medicago sativa) lack both foliar PPO activity and o-diphenols. Consequently, breakdown of their protein upon harvest and storage results in economic losses and release of excess nitrogen into the environment. Understanding how red clover synthesizes o-diphenols such as phaselic acid will help in the development of forages utilizing this natural system of protein protection. We have proposed biosynthetic pathways in red clover for phaselic acid that involve a specific hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase. It is unclear whether the transfer reaction to malate to form phaselic acid involves caffeic acid or p-coumaric acid and subsequent hydroxylation of the resulting p-coumaroyl-malate. The latter would require a coumarate 3'-hydroxylase (C3'H) capable of hydroxylating p-coumaroyl-malate, an activity not previously described. Here, a cytochrome P450 C3'H (CYP98A44) was identified and its gene cloned from red clover. CYP98A44 shares 96 and 79% amino acid identity with Medicago truncatula and Arabidopsis thaliana C3'H proteins that are capable of hydroxylating p-coumaroyl-shikimate and have been implicated in monolignol biosynthesis. CYP98A44 mRNA is expressed in stems and flowers and to a lesser extent in leaves. Immune serum raised against CYP98A44 recognizes a membrane-associated protein in red clover stems and leaves and cross-reacts with C3'H proteins from other species. CYP98A44 expressed in Saccharomyces cerevisiae is capable of hydroxylating p-coumaroyl-shikimate, but not p-coumaroyl-malate. This finding indicates that in red clover, phaselic acid is likely formed by transfer of a caffeoyl moiety to malic acid, although the existence of a second C3'H capable of hydroxylating p-coumaroyl-malate cannot be definitively ruled out.