Stereochemical Insights into Sarpagan and Akuammiline Alkaloid Biosynthesis

Abstract The Apocynaceae family produces a diverse array of monoterpenoid indole alkaloids (MIAs) with significant pharmaceutical value. Among these, sarpagan and akuammiline alkaloids stand out for their complex stereochemistry, derived from the enzymatic cyclization and rearrangement of geissoschizine. This study investigates the stereochemical outcomes of sarpagan bridge enzymes (SBEs) and rhazimal synthases (RHS), key players in geissoschizine cyclization and MIA diversification. Using two known and five newly identified enzymes from six plant species, we show that RHS enzymes from Alstonia scholaris, Vinca minor, and Amsonia tabernaemontana exclusively produce the 16R rhazimal stereoisomer. Meanwhile, SBEs from Catharanthus roseus, Tabernaemontana elegans, Vinca minor, and Rauvolfia serpentina likely generate 16R polyneuridine aldehyde; however, downstream aldehyde reductase, deformylase, and esterase activities further epimerize and alter the C16 stereochemistry, yielding naturally occurring alkaloids with distinct C16 stereochemistry across species. These findings, supported by in vitro assays and in planta silencing of C. roseus CrSBE after we reroute biosynthetic flux toward mutated sarpagan MIAs, further reveal enzymatic control over C16 stereochemistry in sarpagan MIA biosynthesis. By elucidating the transformation of diastereomeric intermediates, this work provides key insights into the stereochemical and enzymatic diversification of MIAs in nature. Competing Interest Statement The authors have declared no competing interest. Footnotes New experiments on chemical reduction and reformulation of aldehyde intermediates are included. The relevant figures, results, and discussion have been updated accordingly.