Abstract
The evolutionarily conserved bacterial proteins MnmE and MnmG (and their homologues in Eukarya) install a 5-carboxymethylaminomethyl (cmnm(5)) or a 5-taurinomethyl (τm(5)) group onto wobble uridines of several tRNA species. The Escherichia coli MnmE binds guanosine-5'-triphosphate (GTP) and methylenetetrahydrofolate (CH(2)THF), while MnmG binds flavin adenine dinucleotide (FAD) and a reduced nicotinamide adenine dinucleotide (NADH). Together with glycine, MnmEG catalyzes the installation of cmnm(5) 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(5)═CH(2)](+) as a central intermediate in the MnmEG reaction. Using a synthetic FADH[N(5)═CD(2)](+) analogue, the intermediacy of the FAD in the transfer of the methylene group from CH(2)THF to the C5 position of U(34) 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(34)-modified tRNAs, showing FAD[N(5)═CH(2)](+) 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.