Regio- and Substrate-Specific Oxidative Metabolism of Terpinolene by Cytochrome P450 Monooxygenases in <i>Cupressus lusitanica</i> Cultured Cells

Many of monoterpenes produced in plants contribute to defenses against herbivores, insects and microorganisms. Among those compounds, β-thujaplicin formed in Cupressaceae plants has a unique conjugated seven-membered ring and some useful biological activities, e.g. fungicide, repellent, insecticide and so on. The biosynthesis pathway of β-thujaplicin has not yet been revealed; we have been trying to uncover it using Cupressus lusitanica cultured cells as a model. In our previous study, terpinolene was identified as a potential β-thujaplicin intermediate at the branching point to terpenoids. In this article, terpinolene metabolism in C. lusitanica cultured cells was investigated, and it was shown that the microsomal fraction from cells oxidized terpinolene into the hydroxylated compound, 5-isopropylidene-2-met-hylcyclohex-2-enol (IME). Then, IME was further oxidized by microsomal fraction to the epoxidized compound, 1,6-epoxy-4(8)-p-menthen-2-ol (EMO). These were the only two products detected from the microsomal reactions, respecttively. Moreover, microsomal reactions with monoterpenes other than terpinolene produced nothing detectable. These results show that the enzymes of these reactions had strict substrate specificity and regio-selectivity. Experiments on kinetics and with specific inhibitors confirmed that these reactions were caused by cytochrome P450 monooxygenases, respectively. These results support our hypothesis that terpinolene is a putative intermediate of β-thujaplicin biosynthesis and show that IME and EMO are also putative intermediates.