4-COUMAROYL COENZYME-A 3-HYDROXYLASE ACTIVITY FROM CELL-CULTURES OF LITHOSPERMUM-ERYTHRORHIZON AND ITS RELATIONSHIP TO POLYPHENOL OXIDASE

A 4-coumaroyl-CoA 3-hydroxylase activity was purified 4600-fold from c ell cultures of Lithospermum erythrorhizon. The enzyme showed a molecu lar mass of 42,400 +/- 1700 Da in gel chromatography and required asco rbate, NADH, or NADPH as cofactors, 4-Coumaroyl-CoA, 4-coumarate, p-cr esol, and several other phenolic substances, but not tyrosine, were ac cepted as substrates for the hydroxylation. Besides hydroxylase activi ty, the enzyme showed diphenol oxidase activity, Both activities were inhibited by diethyldithiocarbamate or beta-mercaptoethanol, although at different concentrations. The enzyme showed striking similarity to a 4-coumaroyl-glucose 3-hydroxylase from sweet potato (Ipomoe batatas) roots, which has reportedly been purified to homogeneity and identifi ed as a specific enzyme of chlorogenic acid biosynthesis. Close examin ation and comparison to a commercially available polyphenol oxidase, h owever, suggest that the enzyme activities purified from both Lithospe rmum and sweet potato are polyphenol oxidases rather than specific enz ymes of secondary metabolism. (C) 1997 Academic Press.