Discovery of a novel cytochrome P450, (+)-vincadifformine 19-hydroxylase (V19H), distinguishes separate branch pathways forming aspidosperma-type monoterpenoid indole alkaloids in Catharanthus roseus roots

Abstract

Investigation of Catharanthus roseus monoterpenoid indole alkaloid (MIA) biosynthesis and accumulation has been important in elucidating the formation of the antineoplastic drugs, vinblastine and vincristine. These pharmaceuticals are formed by the condensation of the MIAs catharanthine and vindoline, which accumulate in C. roseus leaves. While we had completed and expressed the seven-step pathway from the aspidosperma-type MIA (-)-tabersonine to vindoline in yeast, little was known about the reactions involved in the metabolism of aspidosperma-type MIAs in roots. C. roseus roots convert (-)-tabersonine to lochnericine, the precursor for a major root alkaloid hörhammericine, and the reasons for the production of different aspidosperma MIAs in above and below ground plant organs is unknown. The molecular and biochemical characterization of minovincinine-19-O-acetyltransferase (MAT), tabersonine-19-hydroxylase (T19H), tabersonine-6,7-epoxidase (TEX1/2), and tabersonine 19-O-acetyltransferase (TAT) suggests that biosynthesis of hörhammericine and its derivative, 19-O-acetyl-hörhammericine, involves an ordered series of reactions. Bioinformatic analysis led to the identification of a root specific homolog of tabersonine-3-oxygenase (T3O), a cytochrome P450 (P450) involved in the formation of tabersonine 2,3-epoxides, as part of the vindoline pathway in leaves. Characterization of the T3O-homolog revealed that it converts (+)-vincadifformine to its 19-hydroxyderivative, (+)-minovincinine, and it was named (+)-vincadifformine 19-hydroxylase (V19H). V19H did not accept (-)-tabersonine or tabersonine-derived (-)-vincadifformine. T19H, another root-specific P450, hydroxylates (-)-tabersonine and its derivatives, including (-)-vincadifformine, to their respective 19-hydroxyderivatives, but does not accept (+)-vincadifformine. TAT will only acetylate the (-)-tabersonine derivatives, whereas MAT only turns over the (+)-vincadifformine derivative to form (+)-echitovenine. This shows that two distinct aspidosperma pathways exist in C. roseus since endogenous vincadifformine must be the (+)-enantiomer instead of the tabersonine derived (-)-vincadifformine. Modelling studies revealed that V19H activity is competitively inhibited by (-)-vincadifformine, suggesting that the (-)-aspidosperma backbone could still be incorporated into the binding site, albeit without hydroxylation. Models of T3O and T19H were generated to compare their binding pockets with that of V19H, and there were four conserved residues in T3O and T19H that were missing in V19H. Using site-directed mutagenesis (SDM) of V19H at those four residues, the binding pocket became more T3O-like, and V19H gained T3O-like activity without the loss of V19H activity.