Substrate Conformational Switch Enables the Stereoselective Dimerization in P450 NascB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations.

P450 NascB catalyzes the coupling of cyclo-(l-tryptophan-l-proline) (1) to generate (-)-naseseazine C (2) through intramolecular C-N bond formation and intermolecular C-C coupling. A thorough understanding of its catalytic mechanism is crucial for the engineering or design of P450-catalyzed C-N dimerization reactions. By employing MD simulations, QM/MM calculations, and enhanced sampling, we assessed various mechanisms from recent works. Our study demonstrates that the most favorable pathway entails the transfer of a hydrogen atom from N7-H to Cpd I. Subsequently, there is a conformational change in the substrate radical, shifting it from the Re-face to the Si-face of N7 in Substrate 1. The Si-face conformation of Substrate 1 is stabilized by the protein environment and the π-π stacking interaction between the indole ring and heme porphyrin. The subsequent intermolecular C3-C6' bond formation between Substrate 1 radical and Substrate 2 occurs via a radical attack mechanism. The conformational switch of the Substrate 1 radical not only lowers the barrier of the intermolecular C3-C6' bond formation but also yields the correct stereoselectivity observed in experiments. In addition, we evaluated the reactivity of the ferric-superoxide species, showing it is not reactive enough to initiate the hydrogen atom abstraction from the indole NH group of the substrate. Our simulation provides a comprehensive mechanistic insight into how the P450 enzyme precisely controls both the intramolecular C-N cyclization and intermolecular C-C coupling. The current findings align with the available experimental data, emphasizing the pivotal role of substrate dynamics in governing P450 catalysis.