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 characteriz ed in microsomal preparations from cell cultures of Coleus blumei, Cin namic acid 4-hydroxylase introduces the 4-hydroxyl group into cinnamic acid and forms 4-coumaric acid. This enzyme from Coleus blumei displa yed saturation concentrations of 0.5 mM for both cinnamic acid and NAD PH. The apparent K-m-values were determined to be at 35 and 40 mu M, r espectively. Hydroxycinnamoylhydroxyphenyllactate 3- and 3'-hydroxylas es 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'-hydroxyphenyl lactate. 4-Coumaric acid and its CoA-ester as well as 4-hydroxyphenylp yruvate and 4-hydroxyphenyllactate were not accepted as substrates. 3- Hydroxylase was saturated with 250 mu M 4-coumaroyl-3',4'-dihydroxyphe nyllactate and had an apparent K-m-value of 12.5 mu M for this substra te. The respective values for 3'-hydroxylase and the substrate caffeoy l-4'-hydroxyphenyllactate were 100 and 7 mu M. The order of introducti on of the 3- and 3'-hydroxyl groups could not be determined. The 3- an d 3'-hydroxylations are dependent on O-2 and NADPH; the saturation con centration 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 dep endent on cytochrome P450 by inhibition experiments with cytochrome c, ancymidol, metyrapone, miconazole and tetcyclacis as well as by inhib ition of the reactions in a gas phase containing CO besides O-2 and th e partial reversion of this inhibition after illumination with light a t 450 nm wavelength. (C) 1997 Elsevier Science Ltd. All rights reserve d.