Molecular cloning and expression of a novel cytochrome p450 from turkey liver with aflatoxin b1 oxidizing activity.
Cytochromes P450 are members of a superfamily of oxidative hemoprotein enzymes that metabolize a variety of endogenous and exogenous compounds. Previous studies in our laboratory have shown that efficient P450-mediated activation underlies the extreme sensitivity of poultry, specifically turkeys, to the toxic effects of the mycotoxin aflatoxin B1 (AFB1). Using 3'- and 5'-rapid amplification of cDNA ends (RACE), we amplified from turkey liver RNA a full-length 1.73 kb cDNA predicted to be 528 amino acids with 94.7% sequence identity to a CYP1A5 from chicken liver. A truncated construct of the turkey CYP1A5 gene with 29 amino acids deleted from the hydrophobic NH2-terminal region was cloned and heterologously expressed in Escherichia coli. The expressed protein from E. coli membranes had a CO-binding spectrum typical of P450s, and it catalyzed the O-dealkylation of the CYP1A prototype substrates ethoxyresorufin and methoxyresorufin. CYP1A5-mediated O-dealkylation of methoxyresorufin was completely inhibited by alpha-naphthoflavone, a specific CYP1A inhibitor. Inhibitors to other mammalian P450s (3A4, 2D, 2E, and 3A1) either slightly inhibited this activity or not at all. CYP1A5 oxidized AFB1 to form two metabolites: the reactive intermediate, AFB1 -8,9-epoxide (AFBO), and aflatoxin M1 (AFM1). Because of the importance of AFBO and AFM1 in the toxicity of AFB1, we conclude that this P450 probably plays some role in the well-known hypersensitivity of turkeys to AFB1. To our knowledge, this is the first P450 cloned and sequenced from turkeys, the species in which the toxicity of AFB1 was first discovered.
Yip
Shirley S M
SS
Graduate Program in Toxicology, Department of Veterinary Sciences, Utah State University, Logan, Utah 84322-4620, USA.
Coulombe
Roger A
RA
D016604:Aflatoxin B1 / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014422:Turkeys
D011994:Recombinant Proteins; D016604:Aflatoxin B1; D010088:Oxidoreductases; D001189:Aryl Hydrocarbon Hydroxylases; C108015:cytochrome P-450 CYP1A5