Radical SAM Methytransferases

自由基 SAM 甲基转移酶

基本信息

批准号：
8665870
负责人：
Danica Galonic Fujimori
金额：
$ 33.35万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-06-15 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8665870
关键词：
Address Adenosine Amidines Antibiotic Resistance Antibiotics Base Sequence Binding Biology Carbon Catalysis Cations Chemicals Chloramphenicol Cysteine Cytosine Data Development Electronics Ensure Enzymatic Biochemistry Enzymes Event Family Genes Goals Health Hospitals Human Hydrogen In Vitro Knowledge Lead Mediating Methionine Methylation Methyltransferase Mission Mobile Genetic Elements Modification Multi-Drug Resistance Mutagenesis Nucleotides Oxazolidinones Peptidyltransferase Phenotype Positioning Attribute RNA, Ribosomal, 23S Reaction Relative (related person)Research Resistance Ribosomal RNA Ribosomes Role Site Site-Directed Mutagenesis Source Specificity Staging Staphylococcus aureus Streptogramins Structure Transferase Translations Uridine analog bacterial resistance base carbene chemical synthesis cofactor combat enolate enzyme substrate florfenicol in vivo innovation lincosamide member methyl group next generation novel novel strategies pathogen pleuromutilin prevent reconstitution research study

项目摘要

DESCRIPTION (provided by applicant): Bacterial acquisition of resistance determinants represents a major threat to human health. The recent discovery of cfr (chloramphenicol-florfenicol resistance gene) in a multidrug-resistant hospital isolate of Staphylococcus aureus is an important recent example of bacterial resistance. Furthermore, the presence of this gene on mobile genetic elements raises the possibility of the spread of the resistance among human pathogens, an implication that could have devastating effects on human health. The bacterial resistance mediated by cfr is a consequence of an unprecedented target modification strategy. Cfr encodes a methyltransferase enzyme which catalyses addition of a methyl group to adenosine 2503 (A2503) in ribosomal RNA. This nucleotide is positioned in the peptidyl transferase center of the large ribosomal subunit, a common antibiotic target, and its modification by Cfr precludes binding of antibiotics. Cfr and its evolutionary relative RlmN are members of the Radical SAM (S-adenosyl methionine) superfamily. Both enzymes modify amidine carbons in the substrate adenosine: while RlmN transfers the methyl group to the C2 position, Cfr methylates C8 carbon. The formation of a carbon-carbon bond between the aromatic amidine carbon and the methyl group is an unprecedented bond-forming event in enzymology. The goal of this application is to define the mechanism of this novel mode of methylation. The central hypothesis is that methylation is enabled by the enzyme's ability to use two molecules of SAM per each methyl group introduced: one as a cofactor and a source of the reactive 5'-deoxyadenosyl radical, and the other as a cosubstrate and a source of newly added carbon, a hypothesis formulated on the basis of our preliminary data. The following specific aims will be investigated: 1. mechanistically informative substrate analogues will be used to define the chemical mechanism of the reaction; 2. Roles of catalytically crucial conserved residues in methyltransferases will be interrogated by site-directed mutagenesis; and 3. the specificity of both enzymes towards A2503 will be investigated through substrate modulation. The approach is innovative because it addresses a novel and unique mode of enzymatic catalysis, as predicted by the preliminary data. The proposed research is significant because it adds an unprecedented function to the Radical SAM superfamily. Moreover, the proposed research is expected to advance and expand understanding of modes of enzymatic methylation in biology. Ultimately, detailed understanding of this mechanism has the potential to inform the development of next generation antibiotics that will help alleviate the growing problem of antibiotic resistance.

描述（由申请人提供）：细菌获得耐药性决定因素对人类健康构成重大威胁。最近在医院分离的多重耐药金黄色葡萄球菌中发现了 CFR（氯霉素-氟苯尼考耐药基因），这是细菌耐药性的一个重要例子。此外，该基因在移动遗传元件上的存在增加了人类病原体中耐药性传播的可能性，这可能对人类健康产生毁灭性影响。 CFR 介导的细菌耐药性是前所未有的靶点修饰策略的结果。 Cfr 编码甲基转移酶，该酶催化甲基基团添加到核糖体 RNA 中的腺苷 2503 (A2503) 上。该核苷酸位于核糖体大亚基（常见的抗生素靶点）的肽基转移酶中心，其 Cfr 修饰可阻止抗生素的结合。 Cfr 及其进化相关 RlmN 是 Radical SAM（S-腺苷甲硫氨酸）超家族的成员。两种酶都会修饰底物腺苷中的脒碳：RlmN 将甲基转移到 C2 位，而 Cfr 则甲基化 C8 碳。芳香脒碳和甲基之间碳-碳键的形成是酶学中前所未有的成键事件。本申请的目标是定义这种新型甲基化模式的机制。核心假设是，甲基化是通过酶能够在每个引入的甲基基团上使用两个 SAM 分子来实现的：一个作为辅因子和反应性 5'-脱氧腺苷自由基的来源，另一个作为共底物和 5'-脱氧腺苷自由基的来源。新添加的碳，这是根据我们的初步数据提出的假设。将研究以下具体目标： 1. 提供机械信息的底物类似物将用于定义反应的化学机制； 2. 通过定点诱变来探究甲基转移酶中催化关键保守残基的作用； 3. 将通过底物调节研究两种酶对 A2503 的特异性。该方法具有创新性，因为它解决了一种新颖且独特的酶催化模式，正如初步数据所预测的那样。这项研究意义重大，因为它为 Radical SAM 超家族添加了前所未有的功能。此外，拟议的研究有望促进和扩大对生物学中酶促甲基化模式的理解。最终，对这一机制的详细了解有可能为下一代抗生素的开发提供信息，从而有助于缓解日益严重的抗生素耐药性问题。