Mechanisms of Radical-Dependent Biological Methylation

自由基依赖性生物甲基化机制

基本信息

批准号：
8841377
负责人：
SQUIRE J. BOOKER
金额：
$ 23.15万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2017-04-30
项目状态：
已结题

项目摘要

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分别催化了C2和C8处细菌核糖体的23S rRNA的腺苷2503（A2503）23S rRNA的S-腺苷（SAM）依赖性甲基化。 C2甲基化始终在整个细菌中都发现，并被认为有助于肽基转移的效率。相比之下，C8甲基化是某些致病细菌获得的活性，它赋予它们对七类抗生素的抗性，这些抗生素针对细菌核糖体的较大亚基。 C2和C8是亲电的SP2杂交碳，它使它们对几乎所有其他已知的SAM依赖性甲基酶使用的催化策略没有反应。实际上，我们已经证明了这些反应是通过自由基机制进行的，涉及i）通过标准的亲核位移机制将甲基从SAM从SAM从SAM转移到保守的囊这基残基； ii）通过从第二个SAM分子的自由基碎片衍生出的5'-脱氧腺苷（5'-da）从产生的甲基半胱天需要的氢原子中抽象； iii）添加甲基椎体自由基中间体中核苷酸底物的C2或C8；和iv）解决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。