Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania, USA.
Nat Chem Biol. 2013 Jul;9(7):422-7. doi: 10.1038/nchembio.1251. Epub 2013 May 5.
Cfr-dependent methylation of C8 of A2503 in 23S ribosomal RNA confers bacterial resistance to an array of clinically important antibiotics that target the large subunit of the ribosome, including the synthetic oxazolidinone antibiotic linezolid. The key element of the proposed mechanism for Cfr, a radical S-adenosylmethionine enzyme, is the addition of a methylene radical, generated by hydrogen-atom abstraction from the methyl group of an S-methylated cysteine, onto C8 of A2503 to form a protein-nucleic acid crosslinked species containing an unpaired electron. Herein we use continuous-wave and pulsed EPR techniques to provide direct spectroscopic evidence for this intermediate, showing a spin-delocalized radical with maximum spin density at N7 of the adenine ring. In addition, we use rapid freeze-quench EPR to show that the radical forms and decays with rate constants that are consistent with the rate of formation of the methylated product.
Cfr 依赖性甲基化 23S 核糖体 RNA 的 A2503 C8 赋予细菌对一系列临床重要抗生素的耐药性，这些抗生素靶向核糖体大亚基，包括合成的噁唑烷酮类抗生素利奈唑胺。Cfr（一种自由基 S-腺苷甲硫氨酸酶）提出的机制的关键要素是从 S-甲基半胱氨酸的甲基中抽取氢原子，在 A2503 的 C8 上生成亚甲基自由基，形成一种含有未配对电子的蛋白-核酸交联物种。在此，我们使用连续波和脉冲 EPR 技术为该中间体提供直接的光谱证据，显示出具有最大自旋密度在腺嘌呤环 N7 的自旋离域自由基。此外，我们使用快速冷冻淬灭 EPR 表明，自由基的形成和衰减速率常数与甲基化产物的形成速率一致。
