Radical SAM Methytransferases

描述（由申请人提供）：细菌获得耐药性决定因素是对人类健康的主要威胁。最近在一种多药耐药金黄色葡萄球菌医院分离物中发现的氯霉素-氟苯尼考耐药基因是细菌耐药的一个重要例子。此外，这种基因出现在可移动的遗传元件上，增加了耐药性在人类病原体中传播的可能性，这可能对人类健康产生破坏性影响。由cfr介导的细菌耐药性是一种前所未有的靶向修饰策略的结果。Cfr编码一种甲基转移酶，该酶催化核糖体RNA中腺苷2503 （A2503）上甲基的添加。该核苷酸位于大核糖体亚基的肽基转移酶中心，这是一种常见的抗生素靶点，Cfr对其进行修饰可阻止抗生素的结合。Cfr及其进化亲戚RlmN是Radical SAM （s -腺苷蛋氨酸）超家族的成员。这两种酶都修饰底物腺苷中的氨基碳：RlmN将甲基转移到C2位置，Cfr将C8碳甲基化。芳烃酰胺碳与甲基之间形成碳-碳键是酶学中前所未有的成键事件。本应用程序的目标是定义这种新型甲基化模式的机制。核心假设是，甲基化是通过酶的能力来实现的，每个甲基都使用两个SAM分子：一个作为辅助因子和活性5'-脱氧腺苷自由基的来源，另一个作为共底物和新添加的碳的来源，这是基于我们的初步数据制定的假设。将调查以下具体目标：机械信息底物类似物将用于确定反应的化学机理；2. 甲基转移酶中催化关键的保守残基的作用将通过位点定向诱变被询问；和3。这两种酶对A2503的特异性将通过底物调节来研究。正如初步数据所预测的那样，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。这项研究意义重大，因为它为Radical SAM超家族增加了一个前所未有的功能。此外，提出的研究有望推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。

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.