Discovery of Stereoselective UrCYP71AU Oxidases Catalyzing the Final Biosynthetic Step of Rhynchophylline and Mechanistic Insights into 7R and 7S Spiroindole Skeleton Formation

Abstract Rhynchophylline (RHY) is a spiroindole alkaloid that acts on the cardiovascular and central nervous systems. Based on the distribution patterns of 7R and 7S spiroindole alkaloids in U. rhynchophylla, we hypothesize the existence of hirsutine oxidase responsible for 7R and 7S RHY biosynthesis, respectively. However, the stereoselective oxidase has not been identified, and the mechanism underlying spiroindole formation remains unclear. In this study, we identify three hirsutine oxidases that catalyze the final step of RHY biosynthesis in U. rhynchophylla. CYP71AU491 and CYP71AU492 stereoselectively convert hirsutine (a secoyohimbine scaffold) into RHY with a 7R spiroindole configuration, whereas CYP71AU557 specifically catalyzes the formation of the 7S spiroindole scaffold. In planta validation confirms that CYP71AU491 and CYP71AU492 contribute to 7R RHY biosynthesis in U. rhynchophylla, while CYP71AU557 is involved in 7S RHY production. These enzymes are predominantly enriched in roots and hooks and are localized to the endoplasmic reticulum. Enzymatic activity is substantially enhanced by 87.3, 91.3, and 81.6%, respectively, through a combined mutation strategy (CYP71AU491-F131G + V322S, CYP71AU492-T327G + L388S, and CYP71AU557-F131G + L391S). QM/MM simulations indicate that product formation proceeds via a key Compound I−mediated epoxidation intermediate, followed by a concerted dual ring-opening process involving simultaneous C−O and C−C bond cleavage, revealing a previously unrecognized reaction mechanism. Furthermore, the chiral selectivity of the products arises from differences in substrate recognition conformations dictated by distinct binding-pocket architectures in CYP71AU492 and CYP71AU557. Overall, this study reports the identification of stereoselective hirsutine oxidases involved in RHY biosynthesis and elucidates the mechanism underlying 7R and 7S spiroindole scaffold formation. These findings resolve a key bottleneck in RHY biosynthesis and provide a foundation for advancing its biomanufacturing.