Molecular Cloning and Expression of a Novel Cytochrome P450 from Turkey Liver with Aflatoxin B1 Oxidizing Activity

Authors

Shirley S. M. Yip, Utah State University
Roger A. Coulombe Jr., Utah State UniversityFollow

Document Type

Article

Journal/Book Title/Conference

Chemical Research in Toxicology

Volume

19

Issue

1

Publisher

American Chemical Society

Publication Date

2006

First Page

30

Last Page

37

Abstract

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 α-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.

Comments

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Copyright © 2006 American Chemical Society.

Recommended Citation

Yip, S.S.M. and R.A. Coulombe, Jr. (2006) Molecular cloning and expression of a novel cytochrome P450 from turkey liver with aflatoxin B1 metabolizing activity. Chemical Research in Toxicology 19:30-37.