Although the enzymatic mechanisms of terpene synthases have been extensively characterized through experimental and computational studies, the atomistic details underlying the product release process have remained elusive. In this study, we present the first atomistic simulations of the initial stages of product release in a terpene synthase, using the bacterial diterpene cyclase CotB2 as a model system. CotB2 catalyzes the complex cyclization of geranylgeranyl diphosphate (GGDP) to the tricyclic diterpene cyclooctat-9-en-7-ol in a single active site through an 11-step reaction cascade. Our MD simulations focus on three model systems representing CotB2 with bound GGDP and cyclooctat-9-en-7-ol, the latter in two states—with the diphosphate fully deprotonated (P2O74−) and protonated diphosphate (HP2O73−). Analysis of the MD trajectories clearly shows that product release is initiated by the dislocation of the diphosphate group, which in turn triggers active site opening via coordinated C-terminal motions. Notably, protonation of the diphosphate moiety appears to be the key event that weakens its interactions with the active site and enables product release. These findings provide crucial mechanistic insight into the final phase of terpene biosynthesis and open new avenues for rational enzyme engineering targeting product release.
