Radical SAM Methytransferases

自由基 SAM 甲基转移酶

• 批准号: 8159594
• 负责人: Danica Galonic Fujimori
• 金额: $ 33.54万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 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/8159594
• 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

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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to NIH's mission because it aims to elucidate the mechanism of modification of ribosomal RNA by methyltransferase Cfr. This modification renders bacteria resistant to several important classes of clinically used antibiotics. Understanding the mechanism of modification is ultimately expected to lead to the development of new treatments for multi-drug resistant pathogens.

描述（由申请人提供）：抗药性决定因素的细菌获取代表了对人类健康的主要威胁。最近发现CFR（氯霉素 - 氯二甲证基因）在多药抗性金黄色葡萄球菌的分离株中是细菌耐药性的一个重要例子。此外，该基因在移动遗传元素上的存在增加了抗药性在人类病原体中传播的可能性，这一含义可能会对人类健康产生毁灭性的影响。 CFR介导的细菌抗性是前所未有的目标修饰策略的结果。 CFR编码一种甲基转移酶，该酶在核糖体RNA中催化甲基添加到腺苷2503（A2503）中。该核苷酸位于大核糖体亚基的肽基转移酶中心，一种常见的抗生素靶标，其通过CFR修饰的修饰排除了抗生素的结合。 CFR及其进化的相对RLMN是自由基SAM（S-腺苷蛋氨酸）超家族的成员。这两种酶都在底物腺苷中修饰胺碳：而RLMN将甲基转移到C2位置CFR甲基酸酯C8碳。芳香族氨基碳和甲基之间的碳碳键是酶学中前所未有的键形成事件。该应用的目的是定义这种新型甲基化模式的机制。中心假设是，该酶能够使用每个引入每个甲基甲基的SAM分子的能力来实现甲基化：一种是辅助因子，是反应性5'-脱氧腺苷自由基的来源，另一个是辅助性的，另一种是cosubstrate和新添加碳的源，并根据我们的原则数据进行了假设。将研究以下具体目的：1。机械信息丰富的底物类似物将用于定义反应的化学机制； 2。催化至关重要的保守残基在甲基转移酶中的作用将受到定向诱变的质疑。和3。这两种酶对A2503的特异性将通过底物调节研究。该方法具有创新性，因为它解决了一种新型且独特的酶促催化模式，如初步数据所预测的那样。拟议的研究很重要，因为它为激进的SAM超家族添加了前所未有的功能。此外，拟议的研究有望提高和扩展对生物学酶甲基化模式的理解。最终，对这种机制的详细理解有可能告知下一代抗生素的发展，这将有助于减轻日益增长的抗生素耐药性问题。 公共卫生相关性：拟议的研究与NIH的使命有关，因为它旨在通过甲基转移酶CFR阐明核糖体RNA修饰的机制。这种修饰使细菌对几种重要类别的临床使用抗生素具有抗性。最终有望理解修饰的机制，导致开发用于多药耐药病原体的新治疗方法。