The critical role of porcine cytochrome P450 3A46 in the bioactivation of aflatoxin B1

Aflatoxin B-1 (AFB(1)) is bioactivated by cytochrome P450 (CYP) 3A isoforms in humans to generate the highly reactive epoxide intermediate AFB(1)-8,9-epoxide (AFBO), causing hepatotoxicity and hepatocarcinoma. Due to the unavoidable contamination in their feed, pigs are more likely to be exposed to AFB(1) and indirectly harm human health. Therefore, identifying the porcine CYP3A isoforms involved in AFB(1)-8,9-epoxidation is critical. In this study, we used codon optimization and N-terminal coding sequence modification to modify a CYP3A46 recombinant protein that exhibits good structure and catalytic activities and revealed its strong AFB(1)-8,9-epoxidase activity for the first time. Site-directed mutagenesis, kinetics and docking analyses were performed and demonstrated that residues Phe-108, Ser-119, Phe-215, Phe-304 and Thr-309 play important roles in AFB(1)-8,9-epoxidation and its responsiveness to alpha-naphthoflavone. Interestingly, we uncovered the dual and reverse roles of Phe-304 in CYP3A46, CYP3A5 and CYP3A4 in AFBI oxidation. Unlike the pi-pi interaction between the Phe-304 phenyl of CYP3A4 and the AFBI aromatic ring, Phe-304 of CYP3A46 may function to provide steric hindrance to bind AFB(1). Phe-108 and Phe-215 could stabilize AFB(1) with a potentially productive orientation through van der Waals interactions with AFBI. Ser-119 and Thr-309 are likely to function to form H-bonds with AFB(1). This study broadens our knowledge of AFB(1) bioactivation in pigs and may contribute to reduce the deleterious effects of AFB(1) in pigs and humans.