DataSheet_1_Targeting imidazole-glycerol phosphate dehydratase in plants: novel approach for structural and functional studies, and inhibitor blueprinting.pdf

The histidine biosynthetic pathway (HBP) is targeted for herbicide design with preliminary success only regarding imidazole-glycerol phosphate dehydratase (IGPD, EC 4.2.1.19), or HISN5, as referred to in plants. HISN5 catalyzes the sixth step of the HBP, in which imidazole-glycerol phosphate (IGP) is dehydrated to imidazole-acetol phosphate. In this work, we present high-resolution cryoEM and crystal structures of Medicago truncatula HISN5 (MtHISN5) in complexes with an inactive IGP diastereoisomer and with various other ligands. MtHISN5 can serve as a new model for plant HISN5 structural studies, as it enables resolving protein-ligand interactions at high (2.2 Å) resolution using cryoEM. We identified ligand-binding hotspots and characterized the features of plant HISN5 enzymes in the context of the HISN5-targeted inhibitor design. Virtual screening performed against millions of small molecules not only revealed candidate molecules but also identified linkers for fragments that were experimentally confirmed to bind. Based on experimental and computational approaches, this study provides guidelines for designing symmetric HISN5 inhibitors that can reach two neighboring active sites. Finally, we conducted analyses of sequence similarity networks revealing that plant HISN5 enzymes derive from cyanobacteria. We also adopted a new approach to measure MtHISN5 enzymatic activity using isothermal titration calorimetry and enzymatically synthesized IGP.

组氨酸生物合成途径（HBP）是除草剂设计的靶向通路，不过目前仅针对植物来源的咪唑甘油磷酸脱水酶（IGPD，EC 4.2.1.19，即HISN5）取得了初步进展。HISN5催化HBP的第六步反应，将咪唑甘油磷酸（IGP）脱水生成咪唑丙酮醇磷酸。本研究报道了蒺藜苜蓿HISN5（MtHISN5）与失活型IGP非对映异构体及多种其他配体结合的高分辨率冷冻电镜（cryoEM）和晶体结构。MtHISN5可作为植物HISN5结构研究的新型模型，借助该蛋白，我们可通过冷冻电镜以2.2 Å的高分辨率解析蛋白质-配体相互作用。本研究鉴定了配体结合热点位点，并针对靶向HISN5的抑制剂设计场景，表征了植物HISN5酶的特性。针对数百万个小分子开展的虚拟筛选不仅发现了候选活性分子，还鉴定出可结合片段的连接子，且该类连接子的结合活性已通过实验验证。基于实验与计算相结合的研究手段，本研究为设计可结合两个相邻活性位点的对称型HISN5抑制剂提供了指导原则。最后，我们通过序列相似性网络分析证实，植物HISN5酶起源于蓝细菌。本研究还采用了一种新方法：利用等温滴定量热法及酶法合成的IGP来检测MtHISN5的酶活性。