Mechanistic Characterization of Mollisia scopiformis Mycolaurene Synthase, an Enzyme Showing Little but Decisive Intermediate Stabilization

Abstract A fungal multiproduct sesquiterpene synthase from Mollisia scopiformis (MsMS), a laurane-type sesquiterpene synthase that generates a 6/5 bicyclic skeleton, is reported. Using a combination of experimental and theoretical techniques, including isotopic labelings, DFT calculations, molecular docking, site-directed mutagenesis, and quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations, we elucidated the detailed enzyme mechanisms diverging from the (S)-bisabolyl cation as a central intermediate toward all the observed products. For the main product, mycolaurene, we identified an energetically feasible mechanism that is in line with the results of isotopic-labeling experiments and solves the problem of laurane sesquiterpene biosynthesis raised previously in the literature. The MsMS product molisidiene expands the known chemical diversity arising from the bisabolyl cation, a flexible species known to enter diverse reaction paths depending on its conformation. QM/MM MD simulations in comparison to DFT computations reveal that most intermediates along the cyclization cascade are not stabilized through direct engagement of the enzyme but only through intramolecular interactions. Only the bisabolyl cation as a key intermediate receives stabilization through cation–π interaction with F188, and the transition state of a 1,2-hydride shift is guided by the effector G183. Taken together, this work reveals a distinct but minimized enzyme-substrate interplay for sesquiterpene diversification, enriches the cyclization mechanism network and the diversity of sesquiterpene products, identifies key residues involved in mycolaurene formation, and provides a blueprint for structure-guided enzyme engineering.