Mechanisms of Radical-Dependent Biological Methylation

自由基依赖性生物甲基化机制

• 批准号: 8649058
• 负责人: SQUIRE J. BOOKER
• 金额: $ 27.06万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8649058
• 关键词: Active Sites; Adenosine; Affinity; Anti-Bacterial Agents; Antibiotics; Attention; Bacteria; Binding; Binding Sites; Biochemical; Biological; Carbon; Catalysis; Chemistry; Conserved Sequence; Cysteine; Enzymes; Epigenetic Process; Eubacterium; Freezing; Future; Glutamine; Goals; Hydrogen; Imagery; Isotope Labeling; Kinetics; Label; Linezolid; Mediating; Metabolism; Methionine; Methods; Methylation; Methyltransferase; Mutation; Nucleotides; Oxazolidinones; Play; Positioning Attribute; Predisposition; Process; Proteins; RNA; RNA Binding; Reaction; Resistance; Resolution; Ribosomal RNA; Ribosomes; Roentgen Rays; Role; S-Adenosylmethionine; Scheme; Signal Transduction; Site; Solvents; Structure; Techniques; Technology; Variant; Work; X-Ray Crystallography; abstracting; analog; base; crosslink; disulfide bond; environmental adaptation; enzyme structure; methyl group; pathogenic bacteria; programs

DESCRIPTION (provided by applicant): RlmN and Cfr catalyze S-adenosylmethionine (SAM)-dependent methylation of adenosine 2503 (A2503) of 23S rRNA of the bacterial ribosome at C2 and C8, respectively. C2 methylation is found throughout bacteria, and is believed to aid in the efficiency of peptidyltransfer. By contrast, C8 methylation is an activity acquired by certain pathogenic bacteria that confers upon them resistance to over seven classes of antibiotics that target the large subunit of the bacterial ribosome. C2 and C8 are electrophilic sp2-hybridized carbons, which renders them unreactive toward the catalytic strategy used by almost all other known SAM-dependent methylases. In fact, we have shown that these reactions take place via radical mechanisms, involving i) initial transfer of a methyl group from SAM to a conserved cysteinyl residue via a standard nucleophilic displacement mechanism; ii) abstraction of a hydrogen atom from the resulting methylcysteinyl residue by a 5'-deoxyadenosyl 5'-radical (5'-dA) derived from radical fragmentation of a second SAM molecule; iii) addition of the methylcysteinyl radical intermediate to C2 or C8 of the nucleotide substrate; and iv) resolution of the resulting protein-nucleic cross-link by disulfide-bond formation. We will characterize this reaction further using a variety of kinetic, spectroscopic, and biochemical techniques, and provide biochemical and/or structural evidence for each of the postulated intermediates in the reaction.

描述（由申请人提供）：RlmN和Cfr分别催化细菌核糖体23S rRNA的C2和C8处的腺苷2503（A2503）的S-腺苷甲硫氨酸（SAM）依赖性甲基化。 C2 甲基化存在于整个细菌中，并且被认为有助于提高肽基转移的效率。相比之下，C8 甲基化是某些病原菌获得的一种活性，使它们对针对细菌核糖体大亚基的七类以上抗生素具有抗性。 C2 和 C8 是亲电子 sp2 杂化碳，这使得它们对几乎所有其他已知的 SAM 依赖性甲基化酶所使用的催化策略不发生反应。事实上，我们已经证明这些反应是通过自由基机制发生的，包括 i) 通过标准亲核置换机制将甲基从 SAM 初始转移到保守的半胱氨酰残基； ii)通过衍生自第二个SAM分子的自由基断裂的5'-脱氧腺苷5'-自由基(5'-dA)从所得甲基半胱氨酰残基中夺取氢原子； iii)将甲基半胱氨酰基中间体添加至核苷酸底物的C2或C8； iv) 决议 通过二硫键形成产生的蛋白质-核交联。我们将使用各种动力学、光谱和生化技术进一步表征该反应，并为反应中每个假定的中间体提供生化和/或结构证据。