Genome-wide analysis of cytochrome P450 genes discovers two oxidases responsible for the biosynthesis of oxindole alkaloids in Uncaria rhynchophylla

Uncaria rhynchophylla (Gou-teng, UR) produces numerous structurally diverse tetracyclic monoterpene oxindole-type alkaloids (tMOAs) with notable pharmacological activities. Cytochromes P450 (CYPs) play crucial roles in generating the structural diversity of specialized metabolites. However, the CYP superfamily and its biochemical function in tMOAs biosynthesis in UR remains poorly characterized, largely due to the complexity of P450 genes. In this study, a total of 460 UrCYP genes were identified in the UR genome and clustered into nine clans and 43 families by a comprehensive genome-wide analysis. Furthermore, phylogenetic relationships, gene structure, conserved motifs, and duplication events driving the expansion of these UrCYPs were revealed, respectively. Using gene coexpression analysis and yeast functional assays, two tandemly duplicated CYP71 members were discovered to catalyze the regio-specific C-2’ oxindole and rearrangement of tetracyclic corynanthe-type intermediates (hirsutine, hirsuteine) to form tMOAs, including rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine. Meanwhile, overexpression and RNA interference of UrCYP71A22.4 and UrCYP71A22.5 substantially increased and decreased the accumulation of these oxindole alkaloids in hairy roots, respectively. Subcellular localization revealed that UrCYP71A22.4/5-eGFP fusion proteins localize to endoplasmic reticulum membranes. Docking analysis identified four conserved candidate residues (S134/130, D319/318, R447/459, P450/462) surrounding UrCYP71A22.4/5 binding pockets that likely serve as key active sites for 2-oxindole structure formation. Together, this study not only provides critical insights into the previously unknown biosynthesis of tMOAs in UR but also offers a potential strategy for the sustainable production of these pharmaceutically valuable alkaloids.