I-helix modifications reveal functional determinants of Apis mellifera CYP9Q3 and its impact on insecticide...

Cytochrome P450 enzymes (P450s) play a central role in insecticide detoxification with Apis mellifera CYP9Q3 recently demonstrated to mediate the metabolism of several insecticidal chemotypes, including N-cyanoamidine neonicotinoids and the butenolide flupyradifurone. While substrate-recognition sites (SRSs) are known to influence substrate access and binding, the contribution of individual amino acid residues to the catalytic competence of CYP9Q3 remains poorly understood. Here, a phylogenetically informed analysis of specificity-determining positions (SDPs) combined with protein modeling identified the I-helix as a key determinant of CYP9Q enzyme function. Guided by these in silico predictions, ten CYP9Q3 variants spanning amino acid residues T302 and G306-V314 were generated by single alanine substitutions and functionally characterized upon recombinant expression. Substitutions at G307, F308 and D309 emerged as dominant determinants of CYP9Q3 metabolic activity, consistently impairing metabolism of coumarin model substrates and insecticides, including thiacloprid and flupyradifurone. In contrast, other substitutions within the I-helix produced substrate-dependent effects. Together, these results revealed a short I-helix/SRS4 segment as a critical structural determinant governing substrate access, oxygen activation and catalytic competence of CYP9Q3. This systematic dissection of residue-level contributions provides new insights into the structure-function relationships of insect P450 detoxification enzymes and the conserved substrate specificity within P450 subfamilies.