How the Conformational Movement of the Substrate Drives the Regioselective C–N Bond Formation in P450 TleB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations

P450 TleB catalyzes the oxidative cyclization of the dipeptide N-methylvalyl-tryptophanol into indolactam V through selective intramolecular C–H bond amination at the indole C4 position. Understanding its catalytic mechanism is instrumental for the engineering or design of P450-catalyzed C–H amination reactions. Using multiscale computational methods, we show that the reaction proceeds through a diradical pathway, involving a hydrogen atom transfer (HAT) from N1–H to Cpd I, a conformational transformation of the substrate radical species, and a second HAT from N13–H to Cpd II. Intriguingly, the conformational transformation is found to be the key to enabling efficient and selective C–N coupling between N13 and C4 in the subsequent diradical coupling reaction. The underlined conformational transformation is triggered by the first HAT, which proceeds with an energy-demanding indole ring flip and is followed by the facile approach of the N13–H group to Cpd II. Detailed analysis shows that the internal electric field (IEF) from the protein environment plays key roles in the transformation process, which not only provides the driving force but also stabilizes the flipped conformation of the indole radical. Our simulations provide a clear picture of how the P450 enzyme can smartly modulate the selective C–N coupling reaction. The present findings are in line with all available experimental data, highlighting the crucial role of substrate dynamics in controlling this highly valuable reaction.