Structure-based modeling reveals molecular basis for CYP153A6’s novel activity toward toluene derivatives

CYP153A6 activity towards a set of toluene derivatives, along with the underlying molecular recognition. Initial in vivo evaluation of CYP153A6 activity, conducted using both whole cells and cell-free extracts, showed efficient conversion of toluene derivatives with apolar or slightly polar substituents, while no detectable activity was observed for derivatives bearing more polar groups. A homology model of CYP153A6 3D structure was built and validated, revealing key structural features and molecular tunnels. An ensemble docking strategy was used to identify the most effective docking setup. Molecular dynamics simulations and binding free energy calculations further confirmed the hydrophobic nature of the active site. QM/MM calculations supported the different reactivity observed between p-chlorotoluene and p-nitrotoluene. Toluene derivatives bearing a hydroxyl or nitro group on the aromatic ring exhibit reduced binding affinity, adopting unfavorable orientations and non-productive distances between the methyl group and the enzyme’s heme iron center. These computational findings agree with experimental data. Overall, this study provides valuable insights into CYP153A6 molecular recognition mechanism and lay a strong foundation for future protein engineering to extend CYP153A6 enzyme substrate scope and/or enhance the product yield.