Department of Chemistry, University of Utah, Salt Lake City, Utah, USA.
Soliome Inc, San Francisco, California, USA.
J Biol Chem. 2022 Nov;298(11):102548. doi: 10.1016/j.jbc.2022.102548. Epub 2022 Sep 28.
The evolutionarily conserved bacterial proteins MnmE and MnmG collectively install a carboxymethylaminomethyl (cmnm) group at the fifth position of wobble uridines of several tRNA species. While the reaction catalyzed by MnmEG is one of the central steps in the biosynthesis of the methylaminomethyl (mnm) posttranscriptional tRNA modification, details of the reaction remain elusive. Glycine is known to be the source of the carboxy methylamino moiety of cmnm, and a tetrahydrofolate (THF) analog is thought to supply the one carbon that is appended to the fifth position of U. However, the nature of the folate analog remains unknown. This article reports the in vitro biochemical reconstitution of the MnmEG reaction. Using isotopically labeled methyl and methylene THF analogs, we demonstrate that methylene THF is the true substrate. We also show that reduced FAD is required for the reaction and that DTT can replace the NADH in its role as a reductant. We discuss the implications of these methylene-THF and reductant requirements on the mechanism of this key tRNA modification catalyzed by MnmEG.
细菌蛋白 MnmE 和 MnmG 在进化上保守，共同在几种 tRNA 物种的 wobble 尿嘧啶的第五位上安装一个羧甲基氨甲基（cmnm）基团。虽然 MnmEG 催化的反应是甲基氨甲基（mnm）转录后 tRNA 修饰生物合成的中心步骤之一，但该反应的细节仍不清楚。甘氨酸是 cmnm 的羧甲基氨部分的来源，四氢叶酸（THF）类似物被认为提供附加到 U 的第五位的一个碳原子。然而，叶酰谷氨酸类似物的性质仍然未知。本文报道了 MnmEG 反应的体外生化重建。使用同位素标记的甲基和亚甲基 THF 类似物，我们证明亚甲基 THF 是真正的底物。我们还表明，反应需要还原型 FAD，并且 DTT 可以替代 NADH 作为还原剂。我们讨论了这些亚甲基-THF 和还原剂要求对由 MnmEG 催化的这种关键 tRNA 修饰的机制的影响。
