Determinants of Stereoselectivity in Monoterpene Synthases

High Resolution Image Download MS PowerPoint Slide Monoterpene synthases (MTSs) catalyze the conversion of an achiral prenyl diphosphate precursor, most commonly geranyl diphosphate (GPP), into structurally diverse, often chiral, products. However, the mechanistic basis of stereoselectivity remains poorly understood. Here, we combine enzymatic assays with six MTSs and selected variants, along with extensive molecular dynamics simulations, to investigate the mechanistic basis of stereoselectivity. We demonstrate that the preferred helical binding conformation of GPP, determined from free-energy calculations and selected crystal structures of MTSs, correlates well with the experimentally determined stereoselectivity of MTSs, whereas only a poor correlation is observed between the binding of enantiomers of linalyl diphosphate (LPP), a chiral intermediate of MTS catalysis, and the stereochemical reaction outcomes. Free energy maps indicate that enzyme-bound GPP conformers preferentially occupy regions that enable a direct and stereochemically faithful transition from GPP to subsequent carbocations. In contrast, LPP frequently populates regions of the energy surface where conformational scrambling can occur, thus leading to a lack of correlation between LPP enantiomer binding and product stereochemistry. Overall, these findings provide the strongest evidence yet that the binding conformation of GPP is a primary contributor to stereochemical outcomes in MTSs, providing a broader comparative framework for designing stereoselective terpene synthases.