Inhibitor and substrate activities of sesquiterpene olefins toward (+)-δ-cadinene-8-hydroxylase, a cytochrome P450 monooxygenase (CYP706B1)

Several lines of evidence indicate that (+)-delta-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 (+)-delta-cadinene to a moiety that has not yet been identified. However, when microsomes were treated with n-octyl-beta-D-glucoside (OG), a non-ionic detergent, (+)-delta-cadinene was reproducibly converted to the free alcohol, 8-hydroxy-(+)-delta-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 (-)-delta-cadinene, (-)-alpha-cubebene, (-)-alpha-muurolene, alpha-humulene, and a mixture of (-)- and (+)-alpha-copaene were inhibitory to hydroxylation of (+)-delta-cadinene. In addition, (-)-alpha-cubebene, (-)-alpha-muurolene, alpha-humulene, and, to a smaller extent, (-)-delta-cadinene served as alternative substrates for (+)-delta-cadinene-8-hydroxylase and were converted to mono-hydroxylated products. Of the five olefins tested, alpha-humulene and alpha-copaene are found in lysigenous 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.(C) 2010 Elsevier Ltd. All rights reserved.