Biosynthesis of Stizolobinic Acid and Stizolobic Acid in Higher Plants

Two new enzymes, which catalyze the synthesis of stizolobinic acid and stizolobic acid through oxidative ring cleavage of 3,4‐dihydroxyphenylalanine, subsequent recyclization and dehydrogenation of the resultant possible products were isolated from the soluble protein extract of Stizolobium hassjoo seedlings and were purified approximately 100‐fold by ammonium sulfate fractionation followed by column chromatography on Sephadex G‐100, DEAE‐cellulose and hydroxyapatite. The apparent molecular weights of both enzymes as determined by Sephadex G‐100 column chromatography were 45 000. The pH optima were 7.6 and 7.4, apparent K m values for 3,4‐dihydroxyphenylalanine as substrate were 1.67 mM and 1.39 mM for the enzymes to synthesize stizolobinic acid and stizolobic acid, respectively. Both enzymes required NADP + for the activities. They were activated by Zn 2+ and inhibited by sulfhydryl binding and zinc chelating agents. Both exhibited a strict specificity for 3,4‐dihydroxyphenylalanine as substrate and showed no affinities for other o ‐dihydric phenols tested. The enzymes were very labile with a half‐life of 3–5 h at 30°C but they could be stored at −20°C for about 3 months under nitrogen gas with 0.1 mM dithiothreitol without appreciable loss of the catalytic enzyme activities. These enzymes have never been reported in any biological material. The trivial names, stizolobinic acid synthase and stizolobic acid synthase, are proposed.