DataSheet_1_Crystal Structure of the Chloroplastic Glutamine Phosphoribosylpyrophosphate Amidotransferase GPRAT2 From Arabidopsis thaliana.pdf

Chloroplastic glutamine phosphoribosylpyrophosphate amidotransferase (GPRATase) catalyzes the first committed step of de novo purine biosynthesis in Arabidopsis thaliana, and DAS734 is a direct and specific inhibitor of AtGPRAT, with phytotoxic effects similar to the leaf beaching phenotypes of known AtGPRAT genetic mutants, especially cia1 and atd2. However, the structure of AtGPRAT and the inhibition mode of DAS734 still remain poorly understood. In this study, we solved the structure of AtGPRAT2, which revealed structural differences between AtGPRAT2 and bacterial enzymes. Kinetics assay demonstrated that DAS734 behaves as a competitive inhibitor for the substrate phosphoribosyl pyrophosphate (PRPP) of AtGPRAT2. Docking studies showed that DAS734 forms electrostatic interactions with R264 and hydrophobic interactions with several residues, which was verified by binding assays. Collectively, our study provides important insights into the inhibition mechanism of DAS734 to AtGPRAT2 and sheds light on future studies into further development of more potent herbicides targeting Arabidopsis GPRATases.

叶绿体谷氨酰胺磷酸核糖焦磷酸酰胺转移酶（GPRATase）可催化拟南芥（Arabidopsis thaliana）体内从头嘌呤生物合成的首个决定性步骤；DAS734是拟南芥GPRAT（AtGPRAT）的直接特异性抑制剂，其植物毒性效应与已报道的AtGPRAT遗传突变体（尤其是cia1和atd2）的叶片漂白表型相似。然而，目前学界对AtGPRAT的结构及其抑制剂DAS734的作用模式仍缺乏深入了解。本研究解析了AtGPRAT2的晶体结构，揭示了AtGPRAT2与细菌同源酶之间的结构差异。动力学测定结果表明，DAS734对AtGPRAT2的底物磷酸核糖焦磷酸（PRPP）表现为竞争性抑制作用。分子对接研究显示，DAS734可与R264形成静电相互作用，并与多个氨基酸残基产生疏水相互作用，这一结论通过结合实验得到了验证。综上，本研究为阐明DAS734对AtGPRAT2的抑制机制提供了重要见解，同时为未来开发靶向拟南芥GPRAT酶的高效除草剂指明了研究方向。