Plant Physiol. Biochem., 1999,37 (2), 139-144 The effects of phenobarbital and ketoconazole on the alkaloid biosynthesis in Catharanthus roseus cell suspension cultures Adriana Contin, Graziella ColIu, Robert van der Heijden*, Robert Verpoorte Division of Pharmacognosy, LeidenIAmsterdam Center for Drug Research, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, the Netherlands. * Author to whom correspondence should be addressed (fax +31 71 527 45 11; e-mail Heijden@lacdr.leidenuniv.nl) (Received August 17, 1998; accepted November 29, 1998) Abstract - The cytochrome P450 enzyme geraniol IO-hydroxylase plays an important role in the biosynthesis of terpenoid indole alkaloids in suspension cultures of Cutharnnthus roseus. The activity of this enzyme was induced by the treatment of cells with phenobarbital, and inhibited by treatment with ketoconazole. The alkaloid accumulation increased after phenobarbital treatment whereas it decreased after ketoconazole treatment. Phenobarbital and ketoconazole did not affect the in vivo conver- sion rate of loganin to secologanin, a reaction proposed to be catalyzed by a cytochrome P450 enzyme. 0 Elsevier, Paris Alkaloids / Catharanthus roseus I cytochrome P450 I geraniol lo-bydroxylase I iridoids I ketoconazole I phenobarbital DMSO, dimetbylsulphoxide / DW, dry weight / EDTA, ethylenedinitrilo tetraacetic acid / FW, fresh weight / GlOH, geraniol lo-hydroxylase I NAA, naphthalene acetic acid 1. INTRODUCTION Cutharunthus ~OS~US (L.) G. Don cell suspension cultures have been extensively investigated for the production of pharmaceutically important terpenoid indole alkaloids. Strictosidine is the common precur- sor for the terpenoid alkaloids; it arises from the con- densation of the indole tryptamine with the iridoid glucoside secologanin. Although several approaches have been followed in order to increase the accumula- tion of alkaloids in cell cultures of C. roseus, the yields obtained so far, are too low to allow commer- cial production. The low levels of alkaloids in suspen- sion cultures of C. rOSeus have frequently been associated with the low activities of some enzymes involved in the biosynthesis of secologanin [22]. Recently, it was demonstrated that secologanin is bio- synthesized via the novel triose phosphate/pyruvate pathway in C. ~WZUS [6]. The cytochrome P4.50 monooxygenase geraniol IO-hydroxylase (GlOH), involved in an early step of the biosynthesis of secolo- ganin, has been suggested as a potential site for regu- latory control [22]. Later steps in the terpenoid indole alkaloid biosyn- thesis have not all been elucidated yet, and there is evidence for the participation of other cytochrome P450 enzymes in the secondary metabolism of C. ~OS~US cell cultures [ 111. The last step in the bio- synthesis of secologanin, the conversion of the iridoid loganin to secologanin is of particular interest. The enzyme catalyzing this unusual reaction is still unknown and only in vivo conversion has been achieved while no intermediates were detected with a radioimmunoassay [18]. The reaction consists of an oxidative cleavage of a carbon-carbon single bond in the cyclopentane ring of loganin, and up to now, only very few enzymes which perform such type of reac- tions have been isolated and characterized. Common to all of them is the presence of an iron atom and the requirement of molecular oxygen. This type of reac- tion may indicate a cytochrome P450 enzyme [2, 191. Moreover, the conversion of loganin aglucone- l-O- methylether into seco-type compounds through lead tetraacetate oxydation may be considered as a possible model reaction mimicking the biosynthetic step, where the cleavage via an oxidative radical process has been proposed [ 141. The manipulation of the alkaloid biosynthesis in C. roseus cells by cytochrome P450 inducers and inhibitors was first studied by Simpson and Kelly Plant Physiol. Biochem., 0981-9428/99/2/O Elsevier, Paris