Detection and characterization of p-coumaric acid hydroxylase in mung bean, Vigna mungo, seedlings

A new p-coumaric acid (4-hydroxycinnamic acid) hydroxylase was detected in mung bean seedlings treated with tentoxin, a fungal toxin, in which polyphenol oxidase that hydroxylates a wide variety of monophenols in vitro was completely eliminated. The enzyme required molecular oxygen and showed a pH optimum of 5.0. The enzyme acted only on p-coumaric acid (Km, 3.0 X 10(-5) M), while its specificity for the electron donor was rather broad. The Km value for NADPH (1.5 X 10(-4) M) was much lower than that for L-ascorbic acid (1.0 X 10(-2) M), although the Vmax value was almost the same with both electron donors. The enzyme was potently inhibited by beta-mercaptoethanol (Ki, 3.5 X 10(-6) M) and diethyldithiocarbamate (Ki, 2.3 X 10(-4) M), but was insensitive to p-chloromercuribenzoate. The enzyme was localized in the cell organelles which sedimented between mitochondria and endplasmic reticulum on sucrose density gradient centrifugation. The enzyme activity in the seedling was changed in response to induction by light in a manner suggesting its involvement in biosynthesis of phenolic compounds in mung bean seedlings.