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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

基本信息

批准号：
8423691
负责人：
Amie K Boal
金额：
$ 2.75万
依托单位：
NORTHWESTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-03-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8423691
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

项目摘要

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.

描述(申请人提供)：拟议研究的目标是确定在大分子复合体内运行的两个独立的金属酶系统中反应中间体的结构。第一个特定目标将确定氧介导的Ib类核糖核苷酸还原酶激活的中间产物的结构，该酶仅在原核生物中发现，最近发现使用一种新的二锰(III)-酪氨酸自由基辅因子用于催化。该项目将在K99资助期内完成，并将通过冷冻捕获和诱变技术对早期反应中间体进行结晶学表征。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。晶体中反应中间产物的光谱表征将提供结构的独立验证。该酶的基本性质及其在许多人类病原体中作为脱氧核苷酸产生的主要方式的功能使其激活反应可能成为开发新型抗生素的新途径。第二个目标是探索核糖核酸甲基酶的底物结合结构，它使用一个[4Fe-4S]簇，S-腺苷-L-蛋氨酸(SAM)辅因子在未活化的碳中心催化一个机械上新颖的甲基转移反应。要研究的酶(EscherichiaColiRlmN)特异性地甲基化一个位置，该位置赋予了在核糖体大亚基(23S)的多肽转移酶中心内调节翻译的能力。RlmN与一种甲基酶(金黄色葡萄球菌CFR)相关，具有略有不同的位点选择性。CFR介导的23S核糖体甲基化与靶向PTC的抗生素的耐药性有关。R1mN和CFR靶向23S亚基内的特定腺嘌呤位置，在核糖体大片段的背景下最活跃。这项拟议工作的目标是获得与其底物的最小片段和越来越大的片段结合的RlmN的结构信息，并研究捕获的反应中间产物的结构。这项工作将在K99资助期开始，并将在独立阶段继续进行。了解与底物结合的酶的结构以及反应途径中的各种状态将提供有关为阐明CFR耐药机制和特异性奠定了结构基础，为阐明CFR耐药的进化奠定了基础。