PMID- 38050996 OWN - NLM STAT- MEDLINE DCOM- 20231216 LR - 20240214 IS - 1520-5126 (Electronic) IS - 0002-7863 (Print) IS - 0002-7863 (Linking) VI - 145 IP - 49 DP - 2023 Dec 13 TI - Insights into the Mechanism of Installation of 5-Carboxymethylaminomethyl Uridine Hypermodification by tRNA-Modifying Enzymes MnmE and MnmG. PG - 26947-26961 LID - 10.1021/jacs.3c10182 [doi] AB - The evolutionarily conserved bacterial proteins MnmE and MnmG (and their homologues in Eukarya) install a 5-carboxymethylaminomethyl (cmnm(5)) or a 5-taurinomethyl (taum(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) horizontal lineCH(2)](+) as a central intermediate in the MnmEG reaction. Using a synthetic FADH[N(5) horizontal lineCD(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) horizontal lineCH(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. FAU - Bommisetti, Praneeth AU - Bommisetti P AUID- ORCID: 0000-0003-2321-0839 AD - Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States. FAU - Bandarian, Vahe AU - Bandarian V AUID- ORCID: 0000-0003-2302-0277 AD - Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States. LA - eng GR - R35 GM126956/GM/NIGMS NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20231205 PL - United States TA - J Am Chem Soc JT - Journal of the American Chemical Society JID - 7503056 RN - 0 (Escherichia coli Proteins) RN - WHI7HQ7H85 (Uridine) RN - EC 3.6.1.- (GTP Phosphohydrolases) RN - 146-14-5 (Flavin-Adenine Dinucleotide) RN - 86-01-1 (Guanosine Triphosphate) RN - 9014-25-9 (RNA, Transfer) RN - EC 3.6.1.- (MnmE protein, E coli) RN - EC 2.1.- (MnmG protein, E coli) RN - EC 2.1.- (One-Carbon Group Transferases) SB - IM MH - *Escherichia coli Proteins/chemistry MH - Uridine/metabolism MH - GTP Phosphohydrolases/metabolism MH - Flavin-Adenine Dinucleotide/metabolism MH - Escherichia coli/metabolism MH - Guanosine Triphosphate/metabolism MH - RNA, Transfer/chemistry MH - One-Carbon Group Transferases/chemistry/metabolism PMC - PMC10723064 COIS- The authors declare no competing financial interest. EDAT- 2023/12/05 17:44 MHDA- 2023/12/17 17:42 PMCR- 2024/12/05 CRDT- 2023/12/05 08:23 PHST- 2024/12/05 00:00 [pmc-release] PHST- 2023/12/17 17:42 [medline] PHST- 2023/12/05 17:44 [pubmed] PHST- 2023/12/05 08:23 [entrez] AID - 10.1021/jacs.3c10182 [doi] PST - ppublish SO - J Am Chem Soc. 2023 Dec 13;145(49):26947-26961. doi: 10.1021/jacs.3c10182. Epub 2023 Dec 5.