STRUCTURAL MECHANISM OF THE T GONDII UPRT, A TARGET FOR STRUCTURAL-BASED DRUG D

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。布伦南实验室的主要研究重点是了解蛋白质的三维结构与其生化和生物学功能之间的关系。这些方法被用于研究原生动物的转录调控、蛋白质-核酸相互作用、多药物识别和结合以及嘌呤和嘧啶回收的机制。我们的兴趣也指向基于结构的药物设计。一种感兴趣的蛋白质是来自弓形虫的尿嘧啶磷酸核糖基转移酶（UPRT），它催化尿嘧啶转移到d -磷酸核糖基焦磷酸（PRPP），释放焦磷酸并产生核苷酸单磷酸UMP。回收的UMP可用于多种代谢途径。这种酶提供了一个很好的目标，可以用来开发针对这种机会性寄生虫的新药。必须充分了解UPRT的底物识别和催化机制。我们实验室之前的研究已经为完整的UPRT结构和机理表征奠定了基础，我们正在进行下一组关键的研究。具体来说，我们正在致力于改进载脂蛋白UPRT的超高分辨率（1.05分辨率）结构和一些关键的二元和三元配合物的高分辨率结构。同时，我们正在对她的野生型蛋白和一些位点定向突变体进行彻底的动力学评估。最后，这些信息开始发展结构参数，将指导我们未来的药物设计尝试。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The major research focus of the Brennan Laboratory is to understand the relationship between the 3-D structures of proteins and their biochemical and biological functions. These approaches are being used to study transcription regulation, protein-nucleic acid interaction, multidrug recognition and binding and the mechanisms of purine and pyrimidine salvage in protozoa. Our interest is also directed in structure-based drug design. One protein of interest is the uracil phosphoribosyltransferase (UPRT) from the opportunistic parasitic protozoan, Toxoplasma gondii, which catalyzes the transfer of uracil to D-phosphoribosyl pyrophosphate (PRPP) releasing pyrophosphate and creating the nucleotide monophosphate UMP. The salvaged UMP can then be used in a number of metabolic pathways. This enzyme presents a very good target to exploit in our attempts to discover new drugs against this opportunistic parasite. The substrate recognition and catalytic mechanisms of UPRT must be understood fully. Previous studies from our lab have set the groundwork for the complete structural and mechanistic characterization of UPRT and we are carrying out the next key set of studies. Specifically, we are working on the refinement of the ultra high resolution (1.05 ¿¿resolution) structure of apo UPRT and the high resolution structures of a number of key binary and ternary complexes. In parallel, we are carrying out a thorough kinetic evaluation of her wild type protein and a number of site directed mutants. Finally, this information starts to develop the structural parameters that will guide our future drug design attempts.