Hydroxyphenylpyruvate Reductase from Cell Suspension Cultures of Coleus blumei Benth

Cell suspension cultures o f Coleus blumei Benth. producing high amounts o f rosmarinic acid were used to study the biosynthetic pathway o f this caffeic acid ester. One o f the involved en­ zymes, the hydroxyphenylpyruvate reductase (HPPR), is characterized in this paper. HPPR catalyzes the NAD(P)H dependent reduction o f /^-hydroxyphenylpyruvate to /»-hydroxyphenyllactate. The enzyme developed maximal activity at an incubation temperature o f 37 °C and at a pH o f 6.5 to 7.0. The reaction proceeded linearly for an incubation time o f 60 min and up to a protein concentration o f 0.2 mg per assay. As electron donor HPPR accepted N A D H and N A D PH with Âm-values o f 190 jim and 95 |im respectively. The enzyme reduced differently substituted hydroxyphenylpyruvates but not ß-phenylpyruvate. The apparent Âm-values for the various substrates were at 10 for /^-hydroxyphenylpyruvate, at 130^xm for 3,4-dihydroxyphenylpyruvate and at 250 |iM for 3-methoxy-4-hydroxyphenylpyruvate. HPPR was competitively inhibited by rosmarinic acid and pyruvate with /^-values o f 210 |iM and 200 |IM respectively. Caffeic acid,/>-coumaric acid and cinnamic acid did not affect the enzyme activity but/>-coumaroyl-CoA inhibited HPPR.