Stereochemical insights into sarpagan and akuammiline alkaloid biosynthesis.

Sarpagan and akuammiline monoterpenoid indole alkaloids (MIAs) are a class of bioactive plant-derived compounds with significant pharmaceutical potential. These alkaloids are formed through oxidative cyclization of geissoschizine, catalyzed by homologous cytochrome P450 monooxygenases (CYP) known as sarpagan bridge enzymes (SBEs) and rhazimal synthases (RHSs). However, a long-standing discrepancy exists between the expected C16 stereochemistry of enzymatic products and the stereochemistry of naturally occurring derivatives. This study investigates C16 stereochemistry outcomes after geissoschizine cyclization and subsequent enzymatic transformations in six MIA-producing species. We combine comparative gene discovery, in planta gene silencing of Catharanthus roseus SBE, in vitro enzyme characterization, and intermediate nuclear magnetic resonance analysis. We identify and characterize five new CYPs and a new akuammidine aldehyde reductase alongside two known enzymes from Alstonia scholaris, Amsonia tabernaemontana, Catharanthus roseus, Rauvolfia serpentina, Tabernaemontana elegans, and Vinca minor. RHS enzymes consistently produce the 16R rhazimal stereoisomer. SBEs likely yield 16R polyneuridine aldehyde and, to a lesser extent, its 16S epimer akuammidine aldehyde. Downstream aldehyde reductases, deformylases, and decarbomethoxylases further epimerize C16, generating species-specific stereochemical diversity. This work demonstrates the enzymatic tailoring steps that govern C16 stereochemistry in sarpagan MIA biosynthesis, offering new insight into stereochemical diversification and pathway evolution in plant specialized metabolism.