Cytochrome P450s CYP6D1 and CYP6G4 are versatile players in insecticide metabolism: Metabolic capacity and structural insights.

The use of various insecticides has resulted in insecticide resistance in many populations of pest insects. CYP6D1 and CYP6G4 are two cytochrome P450s that have been recruited to confer insecticide resistance in the house fly populations, yet the biochemical basis for their roles in insecticide metabolism is not fully understood. Here, we used recombinant enzymes to explore the capacity of the two field-evolved resistance-conferring P450s from house flies in the metabolism of 16 commonly used insecticides. The chemical structure of the detectable metabolite was identified by liquid chromatography coupled with mass spectrometry. Moreover, we analyzed the structural features associated with substrate selectivity between the two P450s through AlphaFold-based modeling and substrate docking. We found that both CYP6D1 and CYP6G4 were able to metabolize multiple classes of structurally diverse insecticides via substrate-dependent catalytic reactions including dealkylation, desulfurization and hydroxylation. Molecular modeling predicted that CYP6D1 possesses a larger solvent-accessible V-shaped pocket that accommodates bulky substrates such as esfenvalerate, whereas CYP6G4 has a more compact architecture that favors smaller substrates like acetamiprid and methomyl. We propose that the intrinsically broad substrate spectra of CYP6D1 and CYP6G4, together with their regulatory evolvability and negligible fitness costs, collectively predispose these two P450s to recurrent selection among the 146 P450 genes in the housefly genome in field populations. These findings advance our understanding of the evolutionary principles governing P450-mediated insecticide resistance, and provide a biochemical framework for designing evidence-based pest management strategies.