Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activitiesand identification of cytochrome P450CYP72A1 as secologanin synthase

The molecular characterization of CYP72A1 from Catharanthus roseus (Madagas car periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemi stry that the expression in immature leaves is epidermis-specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their bios ynthesis. The early reactions in that pathway, i.e. from geraniol to strict osidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7-deoxyloganin to loganin (deox yloganin 7-hydroxylase, DL7H) and the oxidative ring cleavage converting lo ganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been i dentified as P450 activity in one other plant. We show for the first time t hat both enzyme activities are present in microsomes from C. roseus cell cu ltures. We then tested whether CYP72A1 expressed in E. coli as a translatio nal fusion with the C. roseus P450 reductase (P450Red) has one or both of t hese activities. The results show that CYP72A1 converts loganin into secolo ganin.