A CYTOCHROME-P-450 MONOOXYGENASE CATALYZES THE FIRST STEP IN THE CONVERSION OF TABERSONINE TO VINDOLINE IN CATHARANTHUS-ROSEUS

Hydroxylation at the C-16 position of the indole alkaloid tabersonine has been suggested as the first step toward vindoline biosynthesis in Catharanthus roseus. Tabersonine 16-hydroxylase (16-OH) activity was d etected in total protein extracts from young leaves of C. roseus using a novel coupled assay system. Enzyme activity was dependent on NADPH and molecular oxygen and was inhibited by CO, clotrimazole, miconazole , and cytochrome c. 16-OH was localized to the endoplasmic reticulum b y linear sucrose density gradient centrifugation. These data suggest t hat 16-OH is a cytochrome P-450-dependent monooxygenase. The activity of 16-OH reached a maximum in seedlings 9 d postimbibition and was ind uced by light. The leaf-specific distribution of 16-OH in the mature p lant is consistent with the localization of other enzymes in the taber sonine to vindoline pathway. However, in contrast to enzymes that cata lyze the last four steps of vindoline biosynthesis, enzymes responsibl e for the first two steps from tabersonine (16-OH and 16-O-methyltrans fersase) were detected in C. roseus cell-suspension cultures. These da ta complement the complex model of vindoline biosynthesis that has evo lved with respect to enzyme compartmentalization, metabolic transport, and control mechanisms.