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

Graphical abstract Abstract 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 α-naphthoflavone. Interestingly, we uncovered the dual and reverse roles of Phe-304 in CYP3A46, CYP3A5 and CYP3A4 in AFB 1 oxidation. Unlike the π-π interaction between the Phe-304 phenyl of CYP3A4 and the AFB 1 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 AFB 1. 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. [ABSTRACT FROM AUTHOR]