Bommisetti Praneeth, Bandarian Vahe
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States.
J Am Chem Soc. 2023 Dec 13;145(49):26947-26961. doi: 10.1021/jacs.3c10182. Epub 2023 Dec 5.
The evolutionarily conserved bacterial proteins MnmE and MnmG (and their homologues in Eukarya) install a 5-carboxymethylaminomethyl (cmnm) or a 5-taurinomethyl (τm) group onto wobble uridines of several tRNA species. The MnmE binds guanosine-5'-triphosphate (GTP) and methylenetetrahydrofolate (CHTHF), while MnmG binds flavin adenine dinucleotide (FAD) and a reduced nicotinamide adenine dinucleotide (NADH). Together with glycine, MnmEG catalyzes the installation of cmnm 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[N═CH] as a central intermediate in the MnmEG reaction. Using a synthetic FADH[N═CD] analogue, the intermediacy of the FAD in the transfer of the methylene group from CHTHF to the C5 position of U 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 U-modified tRNAs, showing FAD[N═CH] 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.
进化上保守的细菌蛋白MnmE和MnmG（以及它们在真核生物中的同源物）会在几种tRNA种类的摆动尿苷上安装一个5-羧甲基氨基甲基（cmnm）或一个5-牛磺酸甲基（τm）基团。MnmE结合鸟苷-5'-三磷酸（GTP）和亚甲基四氢叶酸（CHTHF），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，在一个还需要GTP水解的反应中催化cmnm的安装。在这封信中，我们使用多种生化技术研究了MnmEG反应的关键步骤。我们展示了多条证据支持黄素亚胺鎓FADH[N═CH]作为MnmEG反应中的核心中间体。使用一种合成的FADH[N═CD]类似物，明确证明了FAD在亚甲基从CHTHF转移到U的C5位置的过程中起到中间体作用。此外，含有氘代黄素亚胺鎓中间体和诸如牛磺酸和氨等替代亲核试剂的MnmEG反应也导致了预期的U修饰tRNA的形成，表明FAD[N═CH]是所有MnmEG同源物的通用中间体。此外，还鉴定出一种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突变变体被证明能还原FAD，但它们无法促进修饰tRNA的形成。总体而言，这项研究为MnmEG对tRNA进行修饰的生化机制提供了关键见解。
