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Metabolism and Enzymology

Isolation and Characterization of a 2-Oxoglutarate Dependent Dioxygenase Involved in the Second-to-Last Step in Vindoline Biosynthesis ^1,2

Département de Sciences Biologique, Université de Montréal, Saskatoon, Saskatchewan Canada S7N 0W9, Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan Canada S7N 0W9

Young leaves from Catharanthus roseus plants contain the enzymes which convert the monoterpenoid indole alkaloid, tabersonine by three hydroxylations, two methylations, and one acetylation step to vindoline. A novel direct enzyme assay has been developed for a hydroxylase involved in vindoline biosynthesis, which catalyzes the C4-hydroxylation of 2,3-dihydro-3-hydroxy-N(1)-methyltabersonine to the 3,4-dihydroxy derivative. The enzyme showed an absolute requirement for 2-oxoglutarate and enzymatic activity was enhanced by ascorbate, establishing it as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). The hydroxylase exhibited specificity for position 4 of various alkaloid substrates. The enzyme exhibited a pH optima between 7 and 8 and an apparent molecular weight of 45,000. The appearance of 4-hydroxylase activity was developmentally regulated and was shown to be inducible by light treatment of seedlings. Substrate specificity studies of this enzyme for indole alkaloid substrate suggested that hydroxylation at position 3 and N-methylation occur prior to hydroxylation at position 4. This is in agreement with previous studies which suggest that C4-hydroxylation is the second to last step in vindoline biosynthesis in Catharanthus roseus.

¹ This work was supported by the National Research Council of Canada (NRCC) (F.C., J.B., and V.D.L.), Natural Sciences and Engineering Research Council of Canada (V.D.L.), and the Department of Higher Education, Government of Quebec (FCAR: V.D.L. and E.D.C.). E.D.C. was the recipient of a Faculte des Etudes Superieures scholarship.

² Present address: University of British Columbia Medical School, 156 Main Mall Vancouver, British Columbia Canada V6T 1W5. This is study NRCC No. 32452.

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