Wu Jun, Chen Ruohong, Zhang Caihui, Li Kangbai, Xu Weiying, Wang Lijuan, Chen Qingmei, Mu Peiqiang, Jiang Jun, Wen Jikai, Deng Yiqun
Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou 510642, China.
Toxins (Basel). 2016 Sep 12;8(9):267. doi: 10.3390/toxins8090267.
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.
由于饲料中不可避免的污染，猪很容易接触到黄曲霉毒素B₁（AFB₁）并中毒，因此受污染的猪肉产品可能会影响人类健康。迄今为止，AFB₁在猪体内的代谢过程仍有待阐明，尤其是负责其激活的主要细胞色素P450氧化酶。在本研究中，我们从猪肝中克隆了CYP3A29并在大肠杆菌中表达，其活性已通过典型的P450一氧化碳还原光谱特征和硝苯地平氧化活性得到证实。重组膜孵育证明重组CYP3A29能够在体外将AFB₁氧化形成AFB₁-exo-8,9-环氧化物。进一步探讨了CYP3A29对AFB₁区域选择性环氧化的结构基础。T309A突变显著降低了AFBO的产生，而F304A对AFB₁的激活作用增强。与诱变研究一致，分子对接模拟表明Thr309通过氢键在稳定活性中心中AFB₁的结合方面发挥了重要作用。此外，Phe304的大苯基可能对AFB₁的结合产生空间位阻。我们的研究证明了猪CYP3A29在体外对AFB₁的生物激活作用，并为理解CYP3A29对AFB₁的区域选择性提供了见解。
