Stereochemical Insights into Sarpagan and Akuammiline Alkaloid Biosynthesis

The Apocynaceae family produces a remarkable diversity of monoterpenoid indole alkaloids (MIAs), many of which possess significant pharmaceutical value. Among these, sarpagan and akuammiline alkaloids represent distinct subclasses characterized by their intricate stereochemical frameworks, derived through enzymatically controlled cyclization and rearrangement of the key precursor geissoschizine. In this study, we systematically investigated the products and stereochemical outcomes of sarpagan bridge enzymes (SBEs) and rhazimal synthases (RHS), key enzymes involved in geissoschizine cyclization and MIA diversification. Using two previously characterized enzymes alongside five newly identified enzymes from six plant species, we demonstrate that RHS enzymes from Alstonia scholaris , Vinca minor , and Amsonia tabernaemontana exclusively produce the 16 R rhazimal stereoisomer. In contrast, SBEs from Catharanthus roseus , Vinca minor , Tabernaemontana elegans , and Rauvolfia serpentina generate both 16 S akuammidine aldehyde and 16 R polyneuridine aldehyde diastereomers in varying ratios. Remarkably, downstream esterases and deformylases epimerize and alter the stereochemical configuration, yielding naturally occurring alkaloids bearing distinct C16 stereochemistry in different species. These findings are supported by both in vitro assays and in planta silencing of the newly identified C. roseus CrSBE, after we redirected the MIA flux to the otherwise muted sarpagan pathway. Our results provide a comprehensive understanding of the enzymatic control over C16 stereochemistry during SBE-mediated and RHS-mediated transformations. By elucidating how diastereomeric intermediates contribute to sarpagan MIA biosynthesis, this work addresses long-standing questions surrounding the stereochemical dynamics and enzymatic diversification of MIAs in nature.