Structure-guided switching of carbocation quenching in sesquiterpene synthases for accessing aromadendrane and guaiane frameworks

Abstract The extraordinary structural diversity of terpenoid natural products arises from enzyme-controlled carbocation rearrangements during cyclization. Understanding how sesquiterpene synthases (STSs) control these reactive intermediates is a key challenge in enzymatic catalysis. In this study, we demonstrate the rational control of carbocation-quenching pathways in aromadendrane-type STSs through structure-guided mutagenesis. Mutational analysis of 2 key residues (S75 and T95) in CpSTS9 enabled us to switch the product profile from viridiflorol (via water quenching) to 9-alloaromadendrene (via deprotonation), and further to γ-gurjunene with an altered carbon-skeleton framework. Structural modeling indicates that subtle reshaping of the active site redirects the cyclization and quenching pathways. These results establish a mechanism for modulating the cyclization pathway and provide a basis for designing artificial enzymes capable of controlling complex carbon-skeleton formation.