Unraveling stereochemical control mechanisms of neuroactive securinine via isotope-guided spatial transcriptomic strategy

Securinine ( 1 ) and its three stereoisomers, allosecurinine ( 2 ), virosecurinine ( 3 ) and viroallosecurinine ( 4 ), can be produced in the medicinal plant Flueggea suffruticosa . Their stereochemistry critically impacts biological activity: only securinine acts as a potent GABA A receptor antagonist and was once clinically used as a central nervous system stimulant. However, the mechanism underlying their stereochemical control has remained elusive. Here, through isotope labeling, we elucidated that (±)-menisdaurilide and (±)-aquilegiolide serve as key intermediates. They can non-enzymatically couple with Δ 1 -piperideine to form neosecurinane alkaloids, subsequently undergoing intramolecular rearrangement to generate 1 – 4 . Notably, the C6–OH configuration of (±)-menisdaurilide and (±)-aquilegiolide serves as a gatekeeper for controlling selective production of levorotatory products 1 / 2 and dextrorotatory products 3 / 4 . To identify enzymes governing this stereoselective formation of C6–OH, we employed an isotope-guided spatial transcriptomic strategy, which revealed a critical spatial discrepancy: biosynthesis of securinine and its stereoisomers occurs in stems, whereas they accumulate in roots. Leveraging this spatial localization insight, we ultimately identified three stem-preferentially expressed ketoreductases that mediate stereoselective biosynthesis of these alkaloids. This study comprehensively delineates the mechanistic basis for stereochemical control of securinine and highlights the utility of the isotope-guided spatial transcriptomic strategy in efficiently identifying biosynthetic genes of plant natural products.