Mechanisms of Radical-Dependent Biological Methylation

描述（由申请人提供）：RlmN和Cfr分别催化细菌核糖体的23 S rRNA的腺苷2503（A2503）在C2和C8处的S-腺苷甲硫氨酸（SAM）依赖性甲基化。C2甲基化在整个细菌中发现，并且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，赋予它们对靶向细菌核糖体大亚基的七类以上抗生素的抗性。C2和C8是亲电子的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起反应。事实上，我们已经表明这些反应通过自由基机制发生，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提取氢原子; iii）将甲基半胱氨酰基自由基中间体添加至核苷酸底物的C2或C8;以及 所得到的蛋白质-核酸通过二硫键形成交联。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，并为反应中的每个假定中间体提供生化和/或结构证据。

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.