Evolution of CYP71D as a driving force of the diversification of monoterpene indole alkaloid biosynthesis

Besseau, S.; Durand, M.; Zamar, D.-L.; Lezin, E.; Meteignier, L.-V.; Birer Williams, C.; Dutilleul, C.; Perrot, T.; Lanoue, A.; Oudin, A.; Papon, N.; Sun, C.; St-Pierre, B.; Courdavault, V.

doi:10.1101/2025.01.09.632084

bioRxiv · 10.1101/2025.01.09.632084

Evolution of CYP71D as a driving force of the diversification of monoterpene indole alkaloid biosynthesis

Besseau, S.·Durand, M.·Zamar, D.-L.·Lezin, E.·Meteignier, L.-V.·Birer Williams, C.·Dutilleul, C.·Perrot, T.·Lanoue, A.·Oudin, A.·Papon, N.·Sun, C.·St-Pierre, B.·Courdavault, V.

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Abstract

Monoterpene indole alkaloids (MIAs) constitute a vast group of plant natural products synthesized within the Gentianales order. MIAs possess outstanding pharmacological properties that explain their wide use in the treatment of human diseases. These biological activities result from the complex structure of MIAs that originate from intricated biosynthetic pathways involving several enzyme families and notably cytochrome P450s. The early steps of MIA synthesis involve several P450s from the 71 clade, which catalyse the conversion of various reduced strictosidine aglycones into MIAs from the mavacurane, strychnane, akuammilane, sarpagane, and heteroyohimane groups. An extensive study of these P450 distribution in genomic resources reveals that they all belong to a single evolutionary lineage named GAS clade, restricted to the MIA producing Gentianales (Gelsemiaceae and Rauvolfioideae). A related but distinct P450 lineage (named sister GAS clade) was found in non-MIA producing Gentianales and species producing structurally distinct MIAs (Rubiaceae). Functional characterization of 36 members of the GAS clade revealed that these enzymes felt into four main groups of activity depending on substrate acceptance (ex: tetrahydroalstonine, geissoschizine, ajmalicine...) and the nature of the catalyzed reaction (aromatization, cyclisation). These characterizations also lead to the identification of a yohimbane aromatization activity for several of these P450s, increasing the number of MIA scaffolds associated with GAS activity to six. Lastly, the chronology of emergence of these GAS activities was assessed by using ancestral sequence reconstitution, establishing that the initial ancestor of the whole GAS clade exhibits a main alstonine synthase activity. Subsequent gene duplication events, combined with neofunctionalization, facilitated the progressive emergence of eight additional activities, variably distributed among the four GAS subgroups. This comprehensive enzyme characterization establishes the evolutionary trajectory of the GAS clade, demonstrating how its diversification has progressively shaped the chemical diversity of MIAs in Gentianales.

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Besseau, S., Durand, M., Zamar, D.-L., Lezin, E., Meteignier, L.-V., Birer Williams, C., Dutilleul, C., Perrot, T., Lanoue, A., Oudin, A., Papon, N., Sun, C., St-Pierre, B., Courdavault, V.. 2025-01-11. Evolution of CYP71D as a driving force of the diversification of monoterpene indole alkaloid biosynthesis. https://doi.org/10.1101/2025.01.09.632084

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