STRUCT STUD OF ENZYMES INVOLVED IN COFACTOR BIOSYNTHESIS AND NUCLEOSIDE SALVAGE

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。我们的研究重点是酶的三维结构，重点是药物设计，蛋白质工程，酶机制的确定和蛋白质的进化。在嘌呤和嘧啶核苷领域，我们使用SeMet MAD相位确定了两种不同晶体形式的AMP核苷酶的结构，AMP核苷酶在嘌呤回收途径中起关键作用。此外，抑制剂结合的AMP核苷酶的结构允许更好地了解蛋白质家族的生物学功能和酶促机制。尿苷磷酸化酶（UDP）是大肠杆菌PNP同源物，对嘧啶和嘌呤具有特异性。UDP与5-氟吡啶、尿嘧啶和核糖-1-磷酸共晶清楚地显示了重要的活性位点残基，也显示了第一个结构表征的氧羰基中间体。利用SeMet SAD相位法测定鼠伤寒沙门菌必需多功能酶PurL的结构，该酶由三个结构域组成，与其他生物中的三个独立蛋白同源。这种结构将允许深入了解蛋白质/蛋白质相互作用和进化。枯草芽孢杆菌PurS在嘌呤代谢中起关键作用。收集了来自Thermatoga maritima的小型PurL的SAD数据，结构解决方案正在进行中。利用磁共振成像技术对乙酰醋酸杆菌（Acetobacter aceti PurE）进行了解析，对其与一般蛋白质的稳定性机制有了更深入的了解。采用SeMet SAD相法分析了AirS激酶的结构。这种酶在嘌呤和硫胺素生物合成途径的界面上起作用。该结构将为两种途径和核糖激酶超家族的进化提供见解。在多胺生物合成领域，我们利用磁共振成像技术确定了两个pyruvol -dependent arginine decarboxylase （PvlArgDC）突变体的结构。这些结构可能有助于设计干扰人s -腺苷蛋氨酸脱羧酶（AdoMetDC）自动加工的抑制剂。在辅助因子生物合成方面，我们利用MR确定了人磷蚁囊酸甲酰半胱氨酸脱羧酶（PPC脱羧酶）的结构，该酶催化磷酸蚁囊酸酯合成辅酶A的第二步。这种酶是一种潜在的药物设计靶点。ThiSG的结构是由SAD分期决定的，人们正在寻找各种突变体来深入了解其机制。

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. Our research focuses on the three-dimensional structures of enzymes, with emphasis on drug design, protein engineering, determination of enzyme mechanism and protein evolution. In the area of purine and pyrimidine nucleosides, we used SeMet MAD phasing to determine the structure of two different crystal forms of AMP nucleosidase, which plays a key role in the purine salvage pathway. In addition, structures of inhibitor bound AMP nucleosidase allow for greater understanding of the biological functions and enzymatic mechanism of the protein family. Uridine phosphorylase (UDP) is an E. coli PNP homolog with specificity for pyrimidines versus purines. Co-crystals of UDP with 5-fluorouridine and with uracil and ribose-1-phosphate clearly show the important active site residues and also show the first structurally characterized oxocarbenium intermediate. SeMet SAD phasing was used to determine the structure of the essential multifunctional enzyme PurL from Salmonella typhimurium, which consists of three domains homologous to three separate proteins in other organisms. This structure will allow for insights into protein/protein interactions and evolution. The structure of Bacillus subtilis PurS was solved by MR and plays a key role in purine metabolism. SAD data was collected for small PurL from Thermatoga maritima and the structure solution is progressing. Acetobacter aceti PurE was solved using MR and provides a deeper understanding into the mechanism of PurE and general protein acid stability. SeMet SAD phasing was used to solve the structure of AirS kinase. This enzyme functions at the interface of the purine and thiamin biosynthetic pathways. The structure will provide insights into both pathways and into the evolution of the ribokinase superfamily. In the area of polyamine biosynthesis, we used MR to determine the structures of two mutants of pyruvoyl-dependent arginine decarboxylase (PvlArgDC). The structures may aid in designing inhibitors that interfere with the autoprocessing of human S-adenosylmethionine decarboxylase (AdoMetDC). In the area of cofactor biosynthesis, we used MR to determine the structure of human phosphopantothenoylcysteine decarboxylase (PPC decarboxylase), which catalyzes the second step in the synthesis of Coenzyme A from phosphopantothenate. This enzyme is a potential drug design target. The structure of ThiSG has been determined by SAD phasing and various mutants are being sought to provide insights into mechanisms.