Cytochrome P450-dependent hydroxylation in the biosynthesis of rosmarinic acid in Coleus

Three membrane-bound, cytochrome P450-dependent hydroxylases which are involved in the biosynthesis of rosmarinic acid have been characterized in microsomal preparations from cell cultures of Coleus blumei, Cinnamic acid 4-hydroxylase introduces the 4-hydroxyl group into cinnamic acid and forms 4-coumaric acid. This enzyme from Coleus blumei displayed saturation concentrations of 0.5 mM for both cinnamic acid and NADPH. The apparent K-m-values were determined to be at 35 and 40 mu M, respectively. Hydroxycinnamoylhydroxyphenyllactate 3- and 3'-hydroxylases introduce the 3- and 3'-hydroxyl groups into the aromatic rings of rosmarinic acid-like esters like 4-coumaroyl-4'-hydroxyphenyllactate, 4-coumaroyl-3',4'-dihydroxyphenyllactate and caffeoyl-4'-hydroxyphenyllactate. 4-Coumaric acid and its CoA-ester as well as 4-hydroxyphenylpyruvate and 4-hydroxyphenyllactate were not accepted as substrates. 3-Hydroxylase was saturated with 250 mu M 4-coumaroyl-3',4'-dihydroxyphenyllactate and had an apparent K-m-value of 12.5 mu M for this substrate. The respective values for 3'-hydroxylase and the substrate caffeoyl-4'-hydroxyphenyllactate were 100 and 7 mu M. The order of introduction of the 3- and 3'-hydroxyl groups could not be determined. The 3- and 3'-hydroxylations are dependent on O-2 and NADPH; the saturation concentration for both enzymes for NADPH was at 0.5 mM and the apparent K-m values at 30 mu M. All three hydroxylases were determined to be dependent on cytochrome P450 by inhibition experiments with cytochrome c, ancymidol, metyrapone, miconazole and tetcyclacis as well as by inhibition of the reactions in a gas phase containing CO besides O-2 and the partial reversion of this inhibition after illumination with light at 450 nm wavelength. (C) 1997 Elsevier Science Ltd. All rights reserved.