Enzymatic Biosynthesis of Vomilenine, a Key Intermediate of the Ajmaline Pathway, Catalyzed by a Novel Cytochrome P 450-Dependent Enzyme from Plant Cell Cultures of Rauwolfia serpentina

Abstract

Microsomal preparations from Rauwolfia serpentina Benth. cell suspension cultures catalyze a key step in the biosynthesis of ajmaline - the enzymatic hydroxylation of the indole alkaloid vinorine at the allylic C-21 resulting in vomilenine. Vomilenine is an important branch-point intermediate, leading not only to ajmaline but also to several side reactions of the biosynthetic pathway to ajmaline. The investigation of the taxonomical distribution of the enzyme indicated that vinorine hydroxylase is exclusively present in ajmaline-producing plant cells. The novel enzyme is strictly dependent on NADPH2 and 02 and can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole and carbon monoxide (the effect of CO is reversible with light of 450 nm). This suggests that vinorine hydroxylase is a cytochrome P450-dependent monooxygenase. A pH optimum of 8.3 and a temperature optimum of 40 °C were found. The Kmvalue was 3 μM for NADPH2 and 26 μM for vinorine. The optimum enzyme activity could be determined at day 4 after inoculation of the cell cultures in AP I medium. Vinorine hydroxylase could be stored with 20% sucrose at -28 °C without significant loss of activity. © 1995, Verlag der Zeitschrift für Naturforschung. All rights reserved.