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

Several lines of evidence indicate that (+)-δ-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 (+)-δ-cadinene to a moiety that has not yet been identified. However, when microsomes were treated with n-octyl-β-d-glucoside (OG), a non-ionic detergent, (+)-δ-cadinene was reproducibly converted to the free alcohol, 8-hydroxy-(+)-δ-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 (-)-δ-cadinene, (-)-α-cubebene, (-)-α-muurolene, α-humulene, and a mixture of (-)- and (+)-α-copaene were inhibitory to hydroxylation of (+)-δ-cadinene. In addition, (-)-α-cubebene, (-)-α-muurolene, α-humulene, and, to a smaller extent, (-)-δ-cadinene served as alternative substrates for (+)-δ-cadinene-8-hydroxylase and were converted to mono-hydroxylated products. Of the five olefins tested, α-humulene and α-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.