Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B1
Jun Wu,
Ruohong Chen,
Caihui Zhang,
Kangbai Li,
Weiying Xu,
Lijuan Wang,
Qingmei Chen,
Peiqiang Mu,
Jun Jiang,
Jikai Wen,
Yiqun Deng
Affiliations
Jun Wu
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Ruohong Chen
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Caihui Zhang
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Kangbai Li
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Weiying Xu
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Lijuan Wang
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Qingmei Chen
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Peiqiang Mu
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Jun Jiang
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Jikai Wen
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Yiqun Deng
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1.
