大豆疫霉苏氨酰tRNA合成酶结构与Borrelidin结合位点解析

项目名称： 大豆疫霉苏氨酰tRNA合成酶结构与Borrelidin结合位点解析

项目编号： No.31471801

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 农业科学

项目作者： 张继

作者单位： 东北农业大学

项目金额： 85万元

中文摘要： 氨酰tRNA合成酶是理想的药物靶标。本实验室前期研究发现微生物天然产物Borrelidin是以苏氨酰tRNA合成酶（ThrRS）为靶标的大豆疫霉特效药，抑制作用是防治大豆疫霉商品化药物甲霜灵的62.5倍，是现有杀菌活性最高的化合物。为了深入研究Borrelidin抑制大豆疫霉的分子机理，本项目拟通过气相悬滴扩散法制备ThrRS、ThrRS-ATP和ThrRS-ATP-Borrelidin晶体，收集晶体X射线衍射图谱，采用分子置换法构建晶体结构模型，明确ThrRS上Borrelidin的结合位点和关键氨基酸残基组成，结合分子对接、分子动力学模拟和定点突变分析阐明Borrelidin与ThrRS的相互作用。该研究不仅为基于ThrRS为靶标的药物设计和Borrelidin结构改造奠定基础，而且为从蛋白三维结构差异的角度研究Borrelidin对大豆疫霉抑制作用强于原核生物的分子机制提供新的线索。

中文关键词： 大豆疫霉；苏氨酰tRNA合成酶；结构；Borrelidin；结合位点

英文摘要： The aminoacyl-tRNA synthetase enzymes represent ideal targets for drug discovery. Our previous research demonstrated that Borrelidin is the specific antifungal drug against Phytophthora sojae by targeting threonyl-tRNA synthetase(ThrRS). To the best of our knowledge,Borrelidin is the highest antifungal agent and the inhibition effect of Borrelidin is 62.5-fold higher than that of the widely used and commercial fungicide metalaxyl. In order to further investigate the molecular mechanism of Borrelidin against P. sojae, the crystals of ThrRS, ThrRS-ATP and ThrRS-ATP-Borrelidin were prepared by vapor diffusion approach. After the collection of the various X-ray spectra, the structural models were then constructed by using molecular replacement method. On the basis of the crystal structures, the binding site of Borrelidin and the key amino acids that play important roles in the binding of Borrelidin were identified. Furthermore, the interaction between Borrelidin and ThrRS was elucidated by molecular docking, molecular dynamics simulation, and site-directed mutation. These researches not only pave the way for the drug design using ThrRS as target and the structural modification of Borrelisin, but also provide the clue for investigating the differential inhibition of Borrelidin against eukaryotes P. sojae and prokaryotes from the structural point of view.

英文关键词： Phytophthora sojae;Threonyl-tRNA synthetase;Structure;Borrelidin;Binding site