Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B₁.
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.
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. wujun@scau.edu.cn.
Jun
J
Wu
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. Ruohongchen@hotmail.com.
Ruohong
R
Chen
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. zhangcaihui11@gmail.com.
Caihui
C
Zhang
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. kangboli@gmail.com.
Kangbai
K
Li
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. grace87330@163.com.
Weiying
W
Xu
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. wlj322@scau.edu.cn.
Lijuan
L
Wang
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. chenqingmei@scau.edu.cn.
Qingmei
Q
Chen
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. mpeiqiang@scau.edu.cn.
Peiqiang
P
Mu
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. jiangjun@scau.edu.cn.
Jun
J
Jiang
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. jkwen@scau.edu.cn.
Jikai
J
Wen
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China. yqdeng@scau.edu.cn.
Yiqun
Y
Deng
D065767:Activation, Metabolic; D016604:Aflatoxin B1 / Q000031:analogs & derivatives* / Q000378:metabolism; D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes; D008099:Liver / Q000201:enzymology*; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D034421:Sus scrofa
D007527:Isoenzymes; D011994:Recombinant Proteins; C006426:aflatoxin B1-2,3-oxide; D016604:Aflatoxin B1; D051544:Cytochrome P-450 CYP3A
AFB1; CYP3A29; bioactivation; cytochrome P450; regioselectivity