Mechanisms of Radical-Dependent Biological Methylation

描述（由申请人提供）：RlmN和Cfr分别在C2和C8位点催化细菌核糖体23S rRNA的s -腺苷2503 （A2503）依赖于SAM的甲基化。C2甲基化在细菌中普遍存在，并被认为有助于多肽转移的效率。相比之下，C8甲基化是某些致病菌获得的一种活性，使它们能够抵抗针对细菌核糖体大亚基的七种以上抗生素。C2和C8是亲电的sp2杂化碳，这使得它们对几乎所有其他已知的sam依赖的甲基化酶所使用的催化策略没有反应性。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)通过标准的亲核位移机制将甲基从SAM初始转移到保守的半胱氨酸残基；ii)从生成的甲基半胱氨酸残基中，通过从第二个SAM分子的自由基断裂中得到的5'-脱氧腺苷基5'-自由基（5'-dA）从甲基半胱氨酸残基中提取氢原子；iii)将甲基半胱氨酸自由基中间体加成到核苷酸底物的C2或C8上；（四）决议

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.