Cytochrome P450 GcoA is an enzyme that catalyses the guaiacol unit of lignin during the lignin breakdown via aryl-O-demethylation reaction. This reaction is intriguing and is of commercial importance for its potential application in the production of biofuel and plastic from biomass feedstock. Recently, the F169A mutation in the P450 GcoA elicits a promiscuous activity for syringol while maintaining the native activity for guaiacol. Using comprehensive MD simulations and hybrid QM/MM calculations we address, herein, the origin of promiscuity in P450 GcoA and its relevance to the specific activity toward lignin-derived substrates. Our study shows a crucial role of an aromatic dyad, F169, and F395 through regulating the water access to the catalytic center. The F169A mutation opens a water aqueduct and hence increases the native activity for the G-lignin. We show that syringol binds very tightly in the WT enzyme which blocks the conformational rearrangement needed for the second step of O-demethylation. The F169A creates an extra room favoring the conformational rearrangement in the demethylated syringol (3MC) and second dose of the dioxygen insertion. Therefore, using MD simulations and complemented by thorough QM/MM calculations, our study shows how does a single site mutation re-architect active site engineering for promiscuous syringol activity.
Supporting Information contains QM optimized geometries, RMSD, RMSF, diffusion etc.