Voacangine/coronaridine hydroxylases catalyze C19-hydroxylation in iboga alkaloid synthesis

The combination of bioinformatics and functional approaches was used to identify three cytochrome P450 from the CYP71 clan that perform the 19-hydroxylation of two monoterpene indole alkaloids (coronaridine and voacangine) from Voacanga thouarsii.  Voacanga thouarsii is a medicinal plant from the Apocynaceae family producing monoterpene indole alkaloids (MIAs), notably iboga-type ones, to cope with their environment. Iboga MIAs, including ibogaine or voacristine, display a wide range of pharmacological properties explaining the ever-growing interest in these compounds. Although much of the iboga biosynthetic pathway has been resolved, the enzymes responsible for the C19-hydroxylation of coronaridine and voacangine, which leads to heyneanine and voacristine, remain unknown. Using newly available genomic and transcriptomic resources, we performed an orthology-guided strategy to identify candidate cytochrome P450 enzymes from the CYP71 family. Functional characterization through transient expression in Nicotiana benthamiana revealed three closely related enzymes capable of stereospecific C19-hydroxylation of both substrates. These enzymes, named voacangine/coronaridine hydroxylases (VCOH1-3), display moderate substrate specificity and a preferential activity toward coronaridine. Subcellular localization confirmed their association with the endoplasmic reticulum, consistent with other MIA-related P450s. Homology modeling and molecular docking analyses identified key residues that constrain substrate orientation and explain the observed stereochemical outcome of hydroxylation. Together, these findings complete the biosynthetic pathways of voacristine and heyneanine and highlight convergent evolutionary solutions for C19-hydroxylation within MIAs. By identifying and mechanistically characterizing the enzymes responsible for this missing biosynthetic step, this work significantly advances the state of the art in iboga MIA biosynthesis and provides new molecular tools for their metabolic engineering and the sustainable production of these valuable compounds.