Inhibitor and substrate activities of sesquiterpene olefins toward (+)-d-cadinene-8-hydroxylase, a cytochrome P450 monooxygenase (CYP706B1) Yan-Hong Wang 1 , Margaret Essenberg ⇑ Department of Biochemistry and Molecular Biology, Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, OK 74078-3035, USA article info Article history: Received 6 May 2010 Received in revised form 23 July 2010 Keywords: Gossypium arboreum Malvaceae Cotton Enzyme inhibition Substrate specificity Cytochrome P450 monooxygenase Sesquiterpene Clotrimazole Miconazole d-Cadinene a-Muurolene a-Cubebene a-Copaene a-Humulene abstract Several lines of evidence indicate that (+)-d-cadinene-8-hydroxylase (CYP706B1) plays an important role in biosynthesis of gossypol in Gossypium arboreum L. (Luo et al., 2001; Wang et al., 2003). The catalytically active enzyme has been expressed in yeast microsomes. Some microsomal preparations conjugated the hydroxylated (+)-d-cadinene to a moiety that has not yet been identified. However, when microsomes were treated with n-octyl-b-D-glucoside (OG), a non-ionic detergent, (+)-d-cadinene was reproducibly converted to the free alcohol, 8-hydroxy-(+)-d-cadinene. OG had little effect on K m and slightly stimulated apparent V max . Enzymic activity was more than 10-fold more sensitive to inhibition by the N-substituted imidazole clotrimazole than to miconazole. Sesquiterpene olefins (À)-d-cadinene, (À)-a-cubebene, (À)- a-muurolene, a-humulene, and a mixture of (À)- and (+)-a-copaene were inhibitory to hydroxylation of (+)-d-cadinene. In addition, (À)-a-cubebene, (À)-a-muurolene, a-humulene, and, to a smaller extent, (À)-d-cadinene served as alternative substrates for (+)-d-cadinene-8-hydroxylase and were converted to mono-hydroxylated products. Of the five olefins tested, a-humulene and a-copaene are found in lysigen- ous glands of cotton (Elzen et al., 1985), which are also the site of gossypol accumulation (Bell et al., 1978; Mace et al., 1976) and the probable site of its biosynthesis. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction In plants, cytochrome P450 monooxygenases play essential roles in the biosynthesis of phenylpropanoids, terpenoids, alka- loids, and other natural products and in the catabolism of herbi- cides and other xenobiotics (Bolwell et al., 1994; Nelson, 2006; Schuler, 1996). P450-dependent monooxygenases have been shown to introduce molecular oxygen, via hydroxylation, into ter- penoids of various sizes, including the monoterpenes geraniol (Madyastha et al., 1976), (+)-sabinene (Karp et al., 1987), and (À)-limonene (Karp et al., 1990), the sesquiterpenes (+)-d-cadinene (1a)(Fig. 1)(Luo et al., 2001), 5-epi-aristolochene and 1-deoxycap- sidiol (Ralston et al., 2001), and the diterpenoids taxa- 4(20),11(12)-dien-5a-ol and taxa-4(20),11(12)-dien-5a-yl acetate (Wheeler et al., 2001). In contrast with some well-characterized P450 monooxygenases of mammals and microorganisms, most of the plant P450 monooxygenases that have been characterized ex- hibit fairly high substrate and product specificity (Jennewein et al., 2001; Schuler, 1996). (+)-d-Cadinene, (1a) a bicyclic sesquiterpene that is derived from farnesyl diphosphate by (+)-d-cadinene synthase, is an inter- mediate in biosynthesis of gossypol and related cadinane-type compounds that contribute to defense of the cotton plant against herbivory and infectious disease (Alchanati et al., 1998; Chen et al., 1995; Chen et al., 1996; Davis et al., 1996; Davis and Essen- berg, 1995; Liang et al., 2000; Liu et al., 1999; Meng et al., 1999; Tan et al., 2000). Each of these defense compounds is characterized by having an oxygen function at either C-7 or C-8, but not at both positions. Thus it appears likely that regio-specific 7- and 8- hydroxylases determine partitioning of the plant’s (+)-d-cadinene (1a) pool into two biosynthetic pathway branches. A cDNA for a cytochrome P450 monooxygenase, CYP706B1, has been cloned from the diploid cotton Gossypium arboreum L. and ex- pressed in Saccharomyces cerevisiae (Luo et al., 2001). Its expressed protein catalyzes the conversion of (+)-d-cadinene (1a) to the cor- responding 8-hydroxy product (2)(Fig. 1). Expression patterns of the CYP706B1 gene in various tissues of glanded and glandless cot- ton plants during seed development and in cotton cell suspension cultures following elicitation correlated well with production of 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.07.014 ⇑ Corresponding author. Tel.: +1 541 343 1733; fax: +1 662 915 7989. E-mail address: margaret.essenberg@okstate.edu (M. Essenberg). 1 Present address: National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, Oxford, MS 38677, USA. Phytochemistry 71 (2010) 1825–1831 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem