Radical SAM Methytransferases

自由基 SAM 甲基转移酶

• 批准号: 8281447
• 负责人: Danica Galonic Fujimori
• 金额: $ 33.48万
• 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/8281447
• 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.

描述(申请人提供)：细菌获得耐药性决定因素是对人类健康的主要威胁。最近在一株多重耐药的金黄色葡萄球菌中发现了cfr(氯霉素-氟苯尼考耐药基因)，这是近年来细菌耐药的一个重要例子。此外，该基因在可移动的遗传元件上的存在增加了抗药性在人类病原体之间传播的可能性，这意味着可能对人类健康产生毁灭性的影响。CFR介导的细菌耐药性是前所未有的靶向修饰策略的结果。CFR编码一种甲基转移酶，催化核糖体RNA中的腺苷2503(A2503)加成甲基。这种核苷酸位于大的核糖体亚基的肽基转移酶中心，这是一个常见的抗生素靶标，它被CFR修饰后排除了抗生素的结合。CFR及其进化相关基因R1mN是自由基SAM(S-腺苷蛋氨酸)超家族的成员。这两种酶都能修饰底物腺苷中的嘧啶碳：当RlmN将甲基转移到C2位时，CFR甲基化C8碳。芳香酰胺碳和甲基之间的碳-碳键的形成是酶学中前所未有的成键事件。本申请的目的是确定这种新的甲基化模式的机制。中心假说是，甲基化是由于酶能够在每个引入的甲基中使用两个SAM分子：一个作为辅因子和反应性5‘-脱氧腺苷自由基的来源，另一个作为辅基和新添加碳的来源，这是一个基于我们初步数据的假设。将研究以下具体目标：1.机械信息底物类似物将被用来确定反应的化学机制；2.催化关键保守残基在甲基转移酶中的作用将通过定点突变来询问；以及3.两种酶对A2503的特异性将通过底物调节来研究。正如初步数据预测的那样，该方法具有创新性，因为它解决了一种新颖而独特的酶催化模式。这项拟议的研究具有重要意义，因为它为激进的SAM超家族增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，这将有助于缓解日益严重的抗生素耐药性问题。