Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong 510642, China.
Biochem Pharmacol. 2013 Nov 1;86(9):1357-65. doi: 10.1016/j.bcp.2013.08.014. Epub 2013 Sep 2.
Aflatoxin B₁ (AFB₁) is a severe threat to human and animal health. The aflatoxin B₁ aldehyde reductase (AFAR) family specifically catalyzes AFB₁-dialdehyde, a toxic metabolic intermediate of AFB₁, producing a nontoxic dialcohol. Although several AFARs have been found and characterized, the binding specificity of the family for AFB₁-dialdehyde remains unclear. Herein, according to the published sequence, we cloned a porcine AFAR gene. Recombinant porcine AFAR was expressed and purified from Escherichia coli as hexa-histidine tagged fusion protein. Using the cloned porcine AFAR as a model, site-directed mutagenesis combined with high performance liquid chromatography studies revealed that the substitution of Trp266 with Ala resulted in almost complete loss of catalytic activity for AFB₁-dialdehyde. Interestingly, the substitution of Met86 with Ala exhibited an obviously increased activity to the dialdehyde. Based on these results and by using molecular docking simulations, this work provides a structural explanation for why the AFAR family exhibits high specificity for AFB₁-dialdehyde. The Trp266 residue in porcine AFAR plays a critical role in stabilizing the binding of AFB₁-dialdehyde in the active pocket through the hydrophobic interaction of the side-chain indole ring of Trp266 with the fused coumarin rings of the dialdehyde molecule. The enhanced activity of M86A may be attributed to the formed π-π stacking interaction between Trp266 and the dialdehyde. In addition, other hydrophobic residues (e.g. Phe and Trp) around the dialdehyde molecule also stabilize the substrate binding. The findings may contribute to understanding the substrate specificity of the AFAR family for AFB₁-dialdehyde.
黄曲霉毒素 B₁（AFB₁）是严重威胁人类和动物健康的物质。黄曲霉毒素 B₁醛还原酶（AFAR）家族特异性催化 AFB₁-二醛，AFB₁的有毒代谢中间产物，生成无毒的二醇。尽管已经发现并表征了几种 AFAR，但该家族对 AFB₁-二醛的结合特异性仍不清楚。在此，根据已发表的序列，我们克隆了猪的 AFAR 基因。从大肠杆菌中表达和纯化了重组猪 AFAR，作为六组氨酸标记融合蛋白。使用克隆的猪 AFAR 作为模型，定点突变结合高效液相色谱研究表明，将色氨酸 266 替换为丙氨酸几乎完全丧失了对 AFB₁-二醛的催化活性。有趣的是，将蛋氨酸 86 替换为丙氨酸表现出明显增加的对二醛的活性。基于这些结果并通过分子对接模拟，这项工作为 AFAR 家族对 AFB₁-二醛表现出高特异性提供了结构解释。猪 AFAR 中的色氨酸 266 残基通过色氨酸 266 侧链吲哚环与二醛分子融合的香豆素环之间的疏水相互作用，在稳定活性口袋中 AFB₁-二醛的结合中起关键作用。M86A 的增强活性可能归因于色氨酸 266 和二醛之间形成的π-π堆积相互作用。此外，二醛分子周围的其他疏水性残基（如苯丙氨酸和色氨酸）也稳定了底物结合。这些发现可能有助于理解 AFAR 家族对 AFB₁-二醛的底物特异性。
