Global DynamicDesign (GDD) Empowers Cytochrome P450sfor Improved Catalytic Performance

Protein engineering is a powerful tool to enhance the catalytic performance of enzymes. State-of-the-art methodologies usually focus on identifying positions in localized regions, such as the binding pockets and substrate access tunnels. Here, we presented a global dynamic design strategy to evolve cytochrome P450s for improved catalytic performance by identifying distant residues. Three potential positions (D164, A195, and E405) were identified and subjected to site saturation mutagenesis (SSM), resulting in the beneficial variant 8G8/D164N/A195P, which exhibited 7- and 148-fold improved <i>k</i>_cat values compared to variants 8G8 and WT, respectively. Dynamic tunnel and conformational dynamic analyses revealed that substitutions distant from substrate access tunnels increased substrate translocation through the substrate access tunnels of CYP116B3, improving the catalytic performance. General applicability was demonstrated by transferring the global dynamic design strategy to an additional P450 enzyme, namely P450 BM3. The proposed global design strategy advances state-of-the-art P450 engineering by improving the catalytic performance of P450 by identifying distant correlated positions that enhance substrate translocation through access tunnels.