Mechanism-Driven Enzyme Reshaping: A Catalytic Switch Dictates Product Specificity in Sesquiterpene Synthases

Abstract Sesquiterpene synthases generate diverse terpenoid skeletons through carbocation-driven cyclization cascades. Here, we performed a comparative mechanistic study of Artemisia argyisesquiterpene synthase (AaCPS) and Zingiber zerumbet synthase 1 (ZSS1), which exhibit reversed major-product profiles. GC–MS analysis showed that AaCPS predominantly produces β-caryophyllene, whereas ZSS1 mainly generates α-humulene. QM/MM simulations revealed that this product divergence originates from distinct terminal deprotonation pathways. In ZSS1, Cys441 facilitates the dominant proton-transfer process by shaping the local catalytic environment, while PPi primarily contributes to the minor-product pathway. Conversely, AaCPS relies mainly on PPi-mediated deprotonation, which is associated with minor-product formation. Mutagenesis experiments further demonstrated that substitutions at this key position significantly altered product distributions and reduced catalytic efficiency, with C441A and A440C mutations nearly reversing the product profiles. Structural analysis suggests that this conserved active-site locus represents a critical determinant of carbocation quenching and product selectivity across sesquiterpene synthases. These findings reveal how residue–PPi interactions regulate catalytic fidelity and promiscuity, providing mechanistic insights into terpene synthase evolution and engineering. Structural and sequence analyses identify this conserved locus as a key regulator of carbocation quenching and product selectivity. These findings reveal how residue–PPi interactions shape terpene synthase fidelity and promiscuity.