Enzyme Tunnel Dynamics and Catalytic Mechanism of Norcoclaurine Synthase: Insights from a Combined LiGaMD and DFT Study.

This study conducts a systematic investigation into the catalytic mechanism of norcoclaurine synthase (NCS), a key enzyme in the biosynthesis of tetrahydroisoquinolines (THIQs) with therapeutic applications. By integration of LiGaMD and DFT calculations, the reaction pathway of NCS is mapped, providing detailed insights into its catalytic activity and selectivity. Our findings underscore the critical role of E103 in substrate capture and reveal the hitherto unappreciated influence of nonpolar residues M183 and L76 on tunnel dynamics. A prominent discovery is the identification of a high-energy barrier (44.2 kcal/mol) associated with the aromatic electrophilic attack, which pinpoints the rate-limiting step. Moreover, we disclose the existence of dual transition states leading to different products with the energetically favored six-membered ring formation consistent with experimental evidence. These mechanistic revelations not only refine our understanding of NCS but also advocate for a renewed emphasis on enzyme tunnel engineering for optimizing THIQs biosynthesis. The research sets the stage for translating these findings into practical enzyme modifications. Our results highlight the potential of NCS as a biocatalyst to overcome the limitations of current synthetic methodologies, such as low yields and environmental impacts, and provide a theoretical contribution to the efficient, eco-friendly production of THIQs-based pharmaceuticals.