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首页> 外文学位 >Structural and Functional Studies of Bacillus anthracis Enzymes in de novo Purine Synthesis.
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Structural and Functional Studies of Bacillus anthracis Enzymes in de novo Purine Synthesis.

机译：从头合成嘌呤中炭疽杆菌酶的结构和功能研究。

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摘要

The de novo purine biosynthesis is an essential life sustaining process in many organisms, including bacterial pathogens. My work investigates the structural and functional properties of enzymes from this pathway in Bacillus anthracis (Ba) towards the identification of antimicrobials. The first enzyme I studied was PurK, which carboxylates aminoimidazole ribonucleotide (AIR), with bicarbonate in the presence of ATP, to N5-carboxyaminoimidazole ribonucleotide (N5-CAIR). PurK is unique to prokaryotes and lower eukaryotes. Divergence at this step in the pathway makes it an appealing target for antimicrobial development. I used x-ray crystallography to solve the structure of BaPurK. My work also produced several ligand-bound BaPurK structures; of which included bicarbonate in the active site, a first for any PurK structure. Based on these structures, a reaction mechanism was proposed. The second enzyme I focused on was PurC. BaPurC catalyzes the conversion of carboxyaminoimidazole ribonucleotide (CAIR) and aspartate (L-Asp) to succinoaminoimidazolecarboxamide ribonucleotide (SAICAR), with the use of ATP. Studies focused on the large difference in the purification yield between Bacillus anthracis and Streptococcus pneumoniae PurC. Although their amino acid sequences are very similar the recombinant protein yields differed by more than 10-fold. Results from biophysical studies with CD and fluorescence spectroscopies, and molecular modeling suggest that the variances in exposed hydrophobic surfaces are the cause of the difference in purification yield. BaPurC was targeted in high throughput screening, using malachite green, a common phosphate detection assay, in effort to find antimicrobials that inhibit this enzyme from Bacillus anthracis. A set of hit molecules was obtained from the screening of activity inhibition. While analyzing the data an unusual trend appeared in the controls of the assay, further investigation of this event reveals that PurC exhibits substrate-independent ATPase activity, which provides insights into the reaction mechanism.
机译：从头开始的嘌呤生物合成是许多生物（包括细菌病原体）中必不可少的生命维持过程。我的工作调查了炭疽芽孢杆菌（Ba）中此途径中通往抗微生物药鉴定的酶的结构和功能特性。我研究的第一个酶是PurK，它在ATP存在的情况下用碳酸氢盐将氨基咪唑核糖核苷酸（AIR）羧化为N5-羧基氨基咪唑核糖核苷酸（N5-CAIR）。 PurK是原核生物和低等真核生物所独有的。该途径这一步骤的差异性使其成为抗微生物发展的诱人目标。我使用X射线晶体学来解决BaPurK的结构。我的工作还产生了几个配体结合的BaPurK结构。其中在活性位点中包含碳酸氢盐，这是任何PurK结构的首创。基于这些结构，提出了一种反应机理。我关注的第二种酶是PurC。 BaPurC使用ATP催化羧氨基咪唑核糖核苷酸（CAIR）和天冬氨酸（L-Asp）转化为琥珀酰胺基咪唑羧酰胺核糖核苷酸（SAICAR）。研究集中在炭疽芽孢杆菌和肺炎链球菌PurC的纯化产率上有很大差异。尽管它们的氨基酸序列非常相似，但重组蛋白的产量相差十倍以上。 CD和荧光光谱的生物物理研究结果以及分子模型表明，疏水表面暴露的差异是纯化产率差异的原因。 BaPurC的目标是使用孔雀石绿（一种常见的磷酸盐检测测定法）进行高通量筛选，以寻找可抑制炭疽杆菌中抑制该酶的抗菌剂。从活性抑制的筛选中获得了一组命中分子。在分析数据时，分析控件中出现了异常的趋势，对此事件的进一步研究表明，PurC表现出与底物无关的ATPase活性，从而提供了对反应机理的认识。

著录项

  • 作者

    Tuntland, Micheal L.;

  • 作者单位

    University of Illinois at Chicago.;

  • 授予单位 University of Illinois at Chicago.;
  • 学科 Chemistry.
  • 学位 Ph.D.
  • 年度 2015
  • 页码 151 p.
  • 总页数 151
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 遥感技术;
  • 关键词

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