STRUCT STUD OF ENZYMES INVOLVED IN COFACTOR BIOSYNTHESIS AND NUCLEOSIDE SALVAGE

参与辅因子生物合成和核苷回收的酶的结构研究

• 批准号: 7598531
• 负责人: STEVEN E EALICK
• 金额: $ 3.5万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598531
• 关键词: 5 fluorouridine; AMP nucleosidase; Acetobacter; Acids; Active Sites; Adenosylmethionine Decarboxylase; Air; Anabolism; Area; Arginine decarboxylase; Bacillus subtilis; Binding; Biological Process; Carboxy-Lyases; Coenzyme A; Computer Retrieval of Information on Scientific Projects Database; Data; Depth; Drug Design; Enzymes; Escherichia coli; Evolution; Funding; Grant; Homologous Gene; Human; Institution; L-Selenomethionine; Nucleosides; Organism; Pathway interactions; Phase; Phosphopantothenoylcysteine decarboxylase; Phosphotransferases; Play; Polyamines; Protein Engineering; Protein Family; Proteins; Purines; Pyrimidine; Pyrimidine Nucleosides; Pyrimidines; Research; Research Personnel; Resources; Ribokinase; Role; Salmonella typhimurium; Solutions; Source; Specificity; Structure; Thiamine; United States National Institutes of Health; Uracil; Uridine Phosphorylase; cofactor; design; enzyme mechanism; inhibitor/antagonist; insight; mutant; protein protein interaction; purine; purine metabolism; ribose 1-phosphate; three dimensional structure

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们的研究主要集中在酶的三维结构，重点是药物设计，蛋白质工程，酶机制的确定和蛋白质进化。 在嘌呤和嘧啶核苷领域，我们使用SeMet MAD定相来确定在嘌呤补救途径中起关键作用的两种不同晶体形式的AMP核苷酶的结构。 此外，抑制剂结合AMP核苷酶的结构允许更好地理解蛋白质家族的生物学功能和酶促机制。 尿苷磷酸化酶（UDP）是一种E. coli PNP同源物，对嘧啶和嘌呤具有特异性。 UDP与5-氟尿苷以及与尿嘧啶和核糖-1-磷酸的共晶体清楚地显示了重要的活性位点残基，并且还显示了第一个结构特征为oxocarbenium中间体。 SeMet SAD定相用于确定来自鼠伤寒沙门氏菌的必需多功能酶PurL的结构，所述多功能酶PurL由与其他生物体中的三个单独蛋白质同源的三个结构域组成。 这种结构将允许深入了解蛋白质/蛋白质相互作用和进化。 利用核磁共振技术解析了枯草芽孢杆菌PurS的结构，该蛋白在嘌呤代谢中起关键作用。 收集了来自海栖热袍菌（Thermatoga maritima）的小PurL的SAD数据，结构解决方案正在进行中。 利用核磁共振技术对醋酸杆菌PurE进行了解析，为深入了解PurE的作用机制和一般蛋白质的酸稳定性提供了理论依据。 SeMet SAD定相用于解析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处起作用。 该结构将提供深入了解这两种途径和核糖激酶超家族的进化。 在多胺生物合成方面，我们用核磁共振技术测定了两个精氨酸脱羧酶（PvlArgDC）突变体的结构。 这些结构可能有助于设计干扰人S-腺苷甲硫氨酸脱羧酶（SNAMetDC）自动加工的抑制剂。 在辅因子生物合成领域，我们使用MR来确定人磷酸泛酸半胱氨酸脱羧酶（PPC脱羧酶）的结构，该酶催化从磷酸泛酸合成辅酶A的第二步。 该酶是一个潜在的药物设计靶点。ThiSG的结构已经通过SAD定相确定，并且正在寻找各种突变体以提供对机制的见解。