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 B(1) (AFB(1)) involves initial bioactivation by P450s to a reactive and electrophilic intermediate exo-aflatoxin B(1)-8,9-epoxide (exo-AFBO). Poultry, especially turkeys are extremely sensitive to AFB(1), 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 AFB(1) to exo-AFBO and aflatoxin M(1) (AFM(1)). 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 AFB(1) to exo-AFBO and aflatoxin Q(1) (AFQ(1)) and possessed nifedipine oxidation activity, both of which were inhibited by the P450 3A4 inhibitor 17alpha-ethynylestradiol. Oxidation of AFB(1) to exo-AFBO and AFQ(1) by P450 3A37 followed sigmoidal Hill kinetics, suggestive of an allosteric interaction between the enzyme and AFB(1). The Hill coefficient (n) value was 1.9 for exo-AFBO and 1.6 for AFQ(1), indicative of positive cooperativity. The calculated K(m) and V(max) values for the formation of exo-AFBO were 287 +/- 21 muM and 1.45 +/- 0.07 nmol/min/nmol P450, respectively, whereas those of AFQ(1) formation were 302 +/- 51 muM 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 AFB(1) epoxidation in turkey liver.
Rawal
Sumit
S
Department of Veterinary Sciences, Graduate Program in Toxicology, Utah State University, Logan, UT 84322-4620, USA.
Yip
Shirley S M
SS
Coulombe
Roger A
RA
D016604:Aflatoxin B1 / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism; D065692:Cytochrome P-450 CYP3A Inhibitors; D004997:Ethinyl Estradiol / Q000494:pharmacology; D020869:Gene Expression Profiling; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D014422:Turkeys
D065692:Cytochrome P-450 CYP3A Inhibitors; D011994:Recombinant Proteins; D004997:Ethinyl Estradiol; D016604:Aflatoxin B1; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human