STRUCT STUD OF ENZYMES INVOLVED IN COFACTOR BIOSYNTHESIS AND NUCLEOSIDE SALVAGE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们的研究集中在酶的三维结构上，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。在嘌呤和嘧啶核苷方面，我们利用Semet MAD相变技术确定了在嘌呤挽救途径中起关键作用的两种不同晶型的AMP核苷酶的结构。此外，抑制物结合的AMP核苷酶的结构有助于更好地了解该蛋白家族的生物学功能和酶机制。尿苷磷酸化酶(UDP)是一种对嘧啶和嘌呤具有特异性的大肠杆菌PNP同源物。UDP与5-氟尿苷、尿嘧啶和核糖-1-磷酸的共晶体清楚地显示了重要的活性中心残基，也显示了第一个结构表征的氧卡宾中间体。利用SEMET SAD阶段法确定了鼠伤寒沙门氏菌必需的多功能酶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。这种结构将使人们能够深入了解蛋白质/蛋白质的相互作用和进化。枯草芽孢杆菌的结构被MR解析，并且在嘌呤代谢中起着关键作用。收集了来自Thermatoga maritima的小PUL的SAD数据，结构溶液正在进行中。利用磁共振仪对醋酸菌纯品进行了溶解，为进一步了解纯品和一般蛋白酸稳定性的机理提供了依据。用Semet SAD比色法求解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。该结构将提供对这两条途径和核糖激酶超家族进化的洞察。在多胺生物合成方面，我们用核磁共振技术确定了两个丙酮酰依赖的精氨酸脱羧酶(PvlArgDC)突变体的结构。这些结构可能有助于设计干扰人类S腺苷甲硫氨酸脱羧酶(ADOMetDC)自动加工的抑制剂。在辅因子生物合成方面，我们用核磁共振确定了人磷酸泛硫半胱氨酸脱羧酶(PPC脱羧酶)的结构，该酶催化磷酸泛酸合成辅酶A的第二步。这种酶是一种潜在的药物设计靶点。ThiSG的结构已经通过SAD分期确定，正在寻找各种突变体来提供对机制的见解。

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.