Cloning, Expression and Functional Characterization of Cytochrome P450 3A37 from Turkey Liver with High Aflatoxin B1 Epoxidation Activity

Cytochrome P450s (P450) play an important role in the formation of carcinogenic and mutagenic electrophilic intermediates from a wide range of xenobiotics, including naturally occurring dietary compounds. The pathogenesis of hepatotoxic and hepatocarcinogenic action of the mycotoxin aflatoxin B1 (AFB1) involves initial bioactivation by P450s to a reactive and electrophilic intermediate exo-aflatoxin B1-8,9-epoxide (exo-AFBO). Poultry, especially turkeys are extremely sensitive to AFB1, a condition due, in part, to efficient epoxidation by P450 1A and 3A enzymes. We previously reported the discovery of P450 1A5 from turkey liver, which like its human homologue, 1A2, bioactivated AFB1 to exo-AFBO and aflatoxin M1 (AFM1). Here, we describe P450 3A37, the 3A4 homologue from turkey liver. This gene has an open reading frame (ORF) of 1512 bp, and the protein is predicted to be 504 amino acids with 97% identity to chicken P450 3A37. A truncated construct of the turkey P450 3A37 gene with 11 amino acids deleted from the hydrophobic N-terminal region was heterologously expressed in Escherichia coli, the protein from which exhibited a CO difference spectrum typical of P450s. Like human P450 3A4, 3A37 biotransformed AFB1 to exo-AFBO and aflatoxin Q1 (AFQ1) and possessed nifedipine oxidation activity, both of which were inhibited by the P450 3A4 inhibitor 17α-ethynylestradiol. Oxidation of AFB1 to exo-AFBO and AFQ1 by P450 3A37 followed sigmoidal Hill kinetics, suggestive of an allosteric interaction between the enzyme and AFB1. The Hill coefficient (n) value was 1.9 for exo-AFBO and 1.6 for AFQ1, indicative of positive cooperativity. The calculated K m and V max values for the formation of exo-AFBO were 287 ± 21 μM and 1.45 ± 0.07 nmol/min/nmol P450, respectively, whereas those of AFQ1 formation were 302 ± 51 μM and 7.86 ± 0.75 nmol/min/nmol P450, respectively. These data strongly suggest that P450 3A37, along with P450 1A5, plays an important role in AFB1 epoxidation in turkey liver.