Discovery of a novel eunicellane synthase unveils the relationship between stereochemistry and flexibility among eunicellanes

Eunicellane diterpenoids, containing a typical 6,10-bicycle, are bioactive compounds widely distributed in marine corals as well as rarely discovered in bacteria and plants, attracting the attention of scientists in the pharmaceutical field. The intrinsic macrocycle exhibits innate structural flexibility resulting in dynamic conformational changes. However, the intriguing mechanisms controlling flexibility remain unknown. To shed light on this, a genome mining-based discovery of a terpene synthase, MicA, that is responsible for the biosynthesis of a non-flexible eunicellane skeleton, was presented, enabling us to propose a feasible theory that configurations of bridging carbons and their adjacent double bond govern flexibility in eunicellane structures. Notably, isotopic labeling experiments, together with density functional theory calculations, revealed that bacteria-derived MicA follows the catalytic route mainly as coral-derived eunicellane synthase through consecutive 1,14-ring closure, two 1,2-hydride shifts, 1,10-cyclization, and final deprotonation. Furthermore, structural analysis of the artificial intelligence-based MicA model and mutational studies provided an insightful basis for the enzymatic mechanism, featuring a new 2E-configured eunicellane scaffold formation by mutant MicAV220A. Finally, parallel studies of all eunicellane synthases in nature discovered to date, including 2Z-GGPP incubations and DFT-based Boltzmann population computations, revealed that a trans-fused bicycle with a 2Z-configured alkene restricts conformational flexibility resulting in a non-flexible eunicellane skeleton. Our findings presented a new eunicellane synthase, providing new insights into the eunicellane formation and the theory governing flexibility among eunicellane skeletons.