Dynamic Analysis of the Substrate Tolerance Mechanism and Domain Synergistic Engineering of CYP450 Fusion Protein (CYP _LY )

CYP450 enzymes are important in natural product biosynthesis and industrial catalysis. A significant challenge in their engineering is alleviating substrate inhibition and improving electron transfer properties. Here, multitemplate homology modeling and molecular dynamics (MD) simulations are integrated to elucidate the gating mechanisms of substrate tunnels in the chimeric enzyme CYP153A/M228L-CPR_BM3 (CYP_LY), revealing its direct linkage to substrate inhibition. Concurrently, we established that conformational dynamics critically regulate electron transfer efficiency. Leveraging deep learning-based mutational predictions, we identified the variant CYP_LY-N243D, which alleviated substrate inhibition by 34-fold. Additionally, the CYP_LY-T592N mutation in the electron transfer domain achieved a 49.1% conversion at a substrate concentration of 0.8 g/L, which was 6 h earlier than that observed for CYP_LY. Most remarkably, the mutant CYP_LY-N243D/T592N delivered 65.8% conversion with a 4 h reduction in reaction time and alleviated substrate inhibition by 61-fold. This study provides insights into synergistic gating-dynamics-electron transfer regulatory model for industrial CYP450 engineering.