The evolutionarily conserved bacterial proteins MnmE and MnmG (and their homologues in Eukarya) install a 5-carboxymethylaminomethyl (cmnm⁵) or a 5-taurinomethyl (τm⁵) group onto wobble uridines of several tRNA species. The Escherichia coli MnmE binds guanosine-5′-triphosphate (GTP) and methylenetetrahydrofolate (CH₂THF), while MnmG binds flavin adenine dinucleotide (FAD) and a reduced nicotinamide adenine dinucleotide (NADH). Together with glycine, MnmEG catalyzes the installation of cmnm⁵ in a reaction that also requires hydrolysis of GTP. In this letter, we investigated key steps of the MnmEG reaction using a combination of biochemical techniques. We show multiple lines of evidence supporting flavin-iminium FADH[N⁵═CH₂]⁺ as a central intermediate in the MnmEG reaction. Using a synthetic FADH[N⁵═CD₂]⁺ analogue, the intermediacy of the FAD in the transfer of the methylene group from CH₂THF to the C5 position of U₃₄ was unambiguously demonstrated. Further, MnmEG reactions containing the deuterated flavin-iminium intermediate and alternate nucleophiles such as taurine and ammonia also led to the formation of the anticipated U₃₄-modified tRNAs, showing FAD[N⁵═CH₂]⁺ as the universal intermediate for all MnmEG homologues. Additionally, an RNA-protein complex stable to urea-denaturing polyacrylamide gel electrophoresis was identified. Studies involving a series of nuclease (RNase T1) and protease (trypsin) digestions along with reverse transcription experiments suggest that the complex may be noncovalent. While the conserved MnmG cysteine C47 and C277 mutant variants were shown to reduce FAD, they were unable to promote the modified tRNA formation. Overall, this study provides critical insights into the biochemical mechanism underlying tRNA modification by the MnmEG.

中文翻译：

---

深入了解 tRNA 修饰酶 MnmE 和 MnmG 安装 5-羧甲基氨基甲基尿苷超修饰的机制

进化上保守的细菌蛋白 MnmE 和 MnmG（及其在真核生物中的同源物）将 5-羧甲基氨基甲基 (cmnm ⁵ ) 或 5-牛磺酸甲基 (τm ⁵ ) 基团安装到几种 tRNA 物种的摆动尿苷上。大肠杆菌MnmE 结合鸟苷 5'-三磷酸 (GTP) 和亚甲基四氢叶酸 (CH ₂ THF)，而 MnmG 结合黄素腺嘌呤二核苷酸 (FAD) 和还原型烟酰胺腺嘌呤二核苷酸 (NADH) 。^{MnmEG 与甘氨酸一起催化 cmnm 5}的安装，该反应也需要 GTP 的水解。在这封信中，我们结合生化技术研究了 MnmEG 反应的关键步骤。我们展示了多种证据支持黄素亚胺 FADH[N ⁵ =CH ₂ ] ⁺作为 MnmEG 反应的中心中间体。使用合成的FADH[N ⁵ =CD ₂ ] ⁺类似物，明确证明了FAD在亚甲基从CH _{2 THF转移至U 34}的C5位置中的中间作用。此外，含有氘化黄素-亚胺中间体和替代亲核试剂（例如牛磺酸和氨）的 MnmEG 反应也导致了预期的 U ₃₄修饰 tRNA 的形成，表明 FAD[N ⁵ =CH ₂ ] ⁺作为所有 MnmEG 的通用中间体同系物。此外，还鉴定出了对尿素变性聚丙烯酰胺凝胶电泳稳定的RNA-蛋白质复合物。涉及一系列核酸酶（RNase T1）和蛋白酶（胰蛋白酶）消化以及逆转录实验的研究表明，该复合物可能是非共价的。虽然保守的 MnmG 半胱氨酸 C47 和 C277 突变体被证明可以减少 FAD，但它们无法促进修饰的 tRNA 形成。总的来说，这项研究为 MnmEG 修饰 tRNA 的生化机制提供了重要的见解。