Chicken CYP1A5 is able to hydroxylate aflatoxin B₁ to aflatoxin M₁

Aflatoxin B₁ (AFB₁), a naturally-occurring mycotoxin, can cause severe toxicological and carcinogenic effects in livestock and humans. Given that the chicken is one of the most important food-producing animals, knowledge regarding AFB₁ metabolism and enzymes responsible for AFB₁ transformation in the chicken has important implications for chicken production and food safety. Previously, we have successfully expressed chicken CYP1A5 and CYP3A37 monooxygenases in E. coli, and reconstituted them into a functional CYP system consisting of CYP1A5 or CYP3A37, CPR and cytochrome b5. In this study, we aimed to investigate the roles of CYP1A5 and CYP3A37 in the bioconversion of AFB₁ to AFM₁. Our results showed that chicken CYP1A5 was able to hydroxylate AFB₁ to AFM₁. The formation of AFM₁ followed the typical Michaelis-Menten kinetics. The kinetics parameters of V_max and K_m were determined as 0.83 ± 0.039 nmol/min/nmol P450 and 26.9 ± 4.52 μM respectively. Docking simulations further revealed that AFB₁ adopts a "side-on" conformation in chicken CYP1A5, facilitating the hydroxylation of the C9a atom and the production of AFM₁. On the other hand, AFB₁ assumes a "face-on" conformation in chicken CYP3A37, leading to the displacement of the C9a atom from the heme iron and explaining the lack of AFM₁ hydroxylation activity. The results demonstrate that chicken CYP1A5 possesses efficient hydroxylase activity towards AFB₁ to form AFM₁.