ARTICLE OBC www.rsc.org/obc Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450 cam and P450 BM-3 Rebecca J. Sowden, Samina Yasmin, Nicholas H. Rees, Stephen G. Bell and Luet-Lok Wong* Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, UK OX1 3QR. E-mail: luet.wong@chem.ox.ac.uk Received 25th August 2004, Accepted 11th October 2004 First published as an Advance Article on the web 18th November 2004 The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450 cam from Pseudomonas putida, and of P450 BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450 cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450) −1 min −1 , with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450 BM-3 and mutants had higher activities (up to 43 min −1 ) than P450 cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450 cam but multiple orientations in P450 BM-3 . Introduction Terpenoid hydrocarbons (C 5 H 8 )n are biosynthesised by the coupling of isoprene units in the form of isopentenyl py- rophosphate (IPP) to give polyenyl pyrophosphates which are then transformed to the terpenes by terpene synthases. These synthase enzymes catalyze the dissociation of pyrophosphate to form polyenyl carbocations which can undergo complex rearrangements that are controlled by the enzyme active site, resulting in the diverse structures of the terpenes (linear, cyclic, polycyclic). Coupling of two IPP molecules gives geranyl pyrophosphate (GPP, Fig. 1), which is the precursor to all monoterpenes (C 10 H 16 ). Addition of another isoprene unit to GPP gives farnesyl pyrophosphate (FPP), from which all Fig. 1 The biosynthesis pathway of geranyl (GPP) and farnesyl pyrophosphate (FPP) via the coupling of isoprene units. The structures of (+)-valencene 1, (+)-nootkatone 2, and related examples of sesquiterpenoid compounds are shown. sesquiterpenes (C 15 H 24 ) are derived. Diterpenoid compounds include the gibberellins, which are plant hormones, while the triterpene squalene is the precursor to all sterols. The sesquiterpenes are the largest class of terpenoid com- pounds and common constituents of plant essential oils. Many sesquiterpenes and their alcohol, aldehyde and ketone derivatives, are biologically active or precursors to metabo- lites with biological functions, while others have desirable fragrance, flavouring and medicinal properties. 1 For example the phytoalexin capsidiol is derived from the sesquiterpene 5- epi-aristolochene, 2 and amorpha-4,11-diene is the precursor to the potent antimalarial compound artemisinin (Fig. 1). 3,4 (+)-Valencene (1) is found in orange oil (Fig. 1) while its ketone derivative (+)-nootkatone (2), is a sought after fragrance DOI: 10.1039/b413068e This journal is © The Royal Society of Chemistry 2005 Org. Biomol. Chem. , 2005, 3 , 57–64 57