Structure determination of reaction intermediates in macromolecular complexes

大分子复合物中反应中间体的结构测定

• 批准号: 8225797
• 负责人: Amie K Boal
• 金额: $ 8.26万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8225797
• 关键词: Active Sites; Adenine; Aerobic; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Binding; Carbon; Catalysis; Charge; Cocrystallography; Complex; DNA biosynthesis; Data; Dependence; Dependency; Development; Distant; Elements; Engineering; Enzymes; Escherichia coli; Eukaryota; Evolution; Flavins; Foundations; Freezing; Funding; Goals; Growth; Housekeeping; Human; Imagery; Isotopes; Knowledge; Macromolecular Complexes; Measurement; Mediating; Metals; Methionine; Methods; Methylation; Methyltransferase; Modification; Molecular Conformation; Monitor; Mutagenesis; Nature; Nucleotides; Operating System; Organism; Oxidants; Oxygen; Pathway interactions; Peptidyltransferase; Phase; Play; Positioning Attribute; Predisposition; Production; Prokaryotic Cells; Property; Proteins; RNA; RNA Folding; Reaction; Reaction Time; Regulation; Research; Resistance; Resolution; Ribonucleotide Reductase; Ribosomal RNA; Ribosomes; Roentgen Rays; Role; Site; Site-Directed Mutagenesis; Small RNA; Solutions; Solvents; Source; Specificity; Staphylococcus aureus; Structure; Substrate Specificity; Surface; System; Techniques; Temperature; Translations; Variant; Work; analog; base; cofactor; design and construction; enzyme mechanism; enzyme structure; in vivo; insight; macromolecule; metalloenzyme; novel; nucleotide metabolism; pathogen; preference; protein complex; research study; tRNA Methyltransferases; tool

DESCRIPTION (provided by applicant): The objective of the proposed research is to determine structures of reaction intermediates in two separate metalloenzyme systems that operate within macromolecular complexes. The first specific aim will determine structures of intermediates in oxygen-mediated activation of class Ib ribonucleotide reductase, found only in prokaryotes and recently discovered to employ a novel dimanganese(III)-tyrosyl radical cofactor for catalysis. This project will be completed during the K99 funding period and will crystallographically characterize early reaction intermediates via freeze trapping and mutagenesis techniques. Later intermediates will be stabilized by exploiting the pH and temperature dependence of the reaction and its susceptibility to isotope effects. Spectroscopic characterization of reaction intermediates in the crystal will provide independent verification of structures. The essential nature of the enzyme and its function as the primary mode of deoxynucleotide production in a number of human pathogens makes its activation reaction a possible new avenue for novel antibiotic development. The second aim will explore substrate-bound structures of an RNA methylase that uses a [4Fe-4S] cluster, S-adenosyl-L-methionine (SAM) cofactor to catalyze a mechanistically novel methyl transfer reaction at an unactivated carbon center. The enzyme to be studied (Escherichia coli RlmN) specifically methylates a position that imparts the capacity to modulate translation within the peptidyl transferase center of the large subunit (23S) of the ribosome. RlmN is related to a methylase (Staphylococcus aureus Cfr) with a slightly different site selectivity. Cfr-mediated methylation of the 23S ribosom is implicated in resistance to antibiotics that target the PTC. RlmN and Cfr target a specific adenine site within the 23S subunit and are most active in the context of large fragments of the ribosome. The goal of the proposed work is to gain structural information about RlmN bound to minimal and increasingly large fragments of its substrate and to investigate the structures of trapped reaction intermediates. This work will begin during the K99 funding period and will continue during the independent phase. Understanding the structure of the enzyme bound to its substrate and at various states in the reaction pathway will provide critical information about the structural basis for mechanism and specificity and will lay the foundation to elucidate evolution of antibiotic resistance in Cfr. PUBLIC HEALTH RELEVANCE: The proposed work, determining X-ray crystal structures of reaction intermediates in two distinct prokaryotic enzymes that operate within macromolecular complexes, is motivated by the information it will provide about the mechanistic details of the reactions catalyzed, both of which are completely novel. The important roles these enzymes play in nucleotide metabolism and regulation in prokaryotes, with connections to analogous enzymes in human pathogens, may allow the information gained in this study to be exploited in the development of new antibiotic therapies.

描述（由申请人提供）：拟议研究的目的是确定在大分子复合物内操作的两个独立金属酶系统中反应中间体的结构。第一个具体的目标将确定中间体的结构，在氧介导的激活Ib类核糖核苷酸还原酶，发现只有在原核生物和最近发现采用一种新的二锰（III）-酪氨酰基自由基辅因子的催化。该项目将在K99资助期间完成，并将通过冷冻捕获和诱变技术对早期反应中间体进行晶体学表征。随后的中间体将通过利用反应的pH和温度依赖性及其对同位素效应的敏感性来稳定。晶体中反应中间体的光谱表征将提供结构的独立验证。酶的基本性质及其作为许多人类病原体中脱氧核苷酸生产的主要模式的功能使其活化反应成为新型抗生素开发的可能新途径。第二个目标将探索RNA甲基化酶的底物结合结构，该酶使用[4Fe-4S]簇，S-腺苷-L-甲硫氨酸（SAM）辅因子在未活性碳中心催化机械新颖的甲基转移反应。待研究的酶（大肠杆菌RlmN）特异性甲基化赋予调节核糖体大亚基（23 S）的肽基转移酶中心内的翻译的能力的位置。RlmN与甲基化酶（金黄色葡萄球菌Cfr）相关，具有略微不同的位点选择性。Cfr介导的23 S核糖体甲基化与对靶向PTC的抗生素的耐药性有关。RlmN和Cfr靶向23 S亚基内的特定腺嘌呤位点，并且在核糖体的大片段的情况下最活跃。建议的工作的目标是获得有关RlmN绑定到最小和越来越大的片段，其基板的结构信息，并调查被困的反应中间体的结构。这项工作将在K99供资期间开始，并将在独立阶段继续进行。了解与其底物结合的酶的结构以及反应途径中的不同状态将提供有关酶的关键信息。 为阐明Cfr耐药机制和特异性的结构基础奠定了基础。 公共卫生关系：拟议的工作，确定X射线晶体结构的反应中间体在两个不同的原核生物酶内的大分子复合物的操作，是出于它将提供有关催化的反应，这两者都是完全新颖的机械细节的信息。这些酶在原核生物的核苷酸代谢和调节中发挥着重要作用，与人类病原体中的类似酶有关，这可能使本研究中获得的信息能够用于开发新的抗生素疗法。