Phe-125 and Phe-226 of pig cytochrome P450 1A2 stabilize the binding of aflatoxin B1 and 7-ethoxyresorufin through the key CH/π interactions

Abstract Cytochrome P450 1A2 (CYP1A2) plays important roles in the metabolism of many planar and aromatic drugs and also contributes to the bioactivation of aflatoxin B 1 (AFB 1 ) in vivo . To date, the structural basis for CYP1A2′s preference to the planar substrates remains unclear. Herein, we investigated the structure-activity relationships for pig CYP1A2 catalyzing AFB 1 and 7-ethoxyresorufin (7-ER). A molecular docking study was performed based on a constructed model of pig CYP1A2, which predicted the contributions of Thr-118, Thr-124, Phe-125, Phe-226, Leu-260, and Asp-313 to the substrate catalysis. Site-directed mutagenesis and kinetic analyses exhibited the common grounds: Phe-125, Phe-226 and Asp-313 were vital to AFB 1 oxidation (including exo-epoxidation and 9A-hydroxylation) and ethoxyresorufin O -deethylation. Meanwhile, Phe-125 and Phe-226 formed CH/π interactions with AFB 1 /7-ER, and Asp-313 formed hydrogen bonds with them. Based on other published reports, this study further emphasizes the critical roles of Phe-125 and Phe-226 in recognizing the planar substrates. Our findings highlight the structural basis of pig CYP1A2 specifically catalyzing AFB 1 and 7-ER, and may help to elucidate the underlying mechanism of CYP1A2′s metabolic preference to the planar and aromatic substrates.