The Escherichia coli Radical SAM Enzyme YhcC Replaces MnmC in mnm 5 s 2 U tRNA Modification During Anaerobic Growth

Abstract tRNA wobble uridines are heavily modified to influence anticodon-codon pairing and tune anticodon stem-loop structure for efficient, accurate translation. Many bacteria and some archaea modify wobble uridines with either a 5-carboxymethylaminomethyl (cmnm 5 ) or 5-methylaminomethyl (mnm 5 ) group, often together with a 2-thio (s 2 ) moiety. Bacteria utilize the conserved MnmEG complex to produce cmnm 5 U, which is further converted to mnm 5 U by non-orthologous enzymes in different lineages. Escherichia coli uses the bifunctional enzyme MnmC to demodify cmnm 5 U to nm 5 U and subsequently methylate nm 5 U to mnm 5 U, whereas Bacillus subtilis r elies on the radical SAM (rSAM) enzyme MnmL and the stand-alone methylase MnmM. Although E. coli and related bacteria encode homologs of MnmL, the function of the E. coli homolog, YhcC, remained unknown. Here, we show that YhcC is required for cmnm 5 s 2 U demodification in vivo during anaerobic growth, whereas the equivalent MnmC-dependent reaction requires O 2 and occurs only aerobically. In vitro, purified [4Fe-4S]-reconstituted YhcC binds tRNA and catalyzes nm 5 s 2 U-tRNA synthesis from cmnm 5 s 2 U-tRNA. Together, these results define the previously unknown function of the E. coli rSAM enzyme YhcC and demonstrate that it replaces MnmC under anaerobic conditions to generate nm 5 s 2 U. These parallel pathways reveal how E. coli maintains synthesis of a critical wobble-base modification under both aerobic and anaerobic growth conditions. General audience summary Transfer RNA (tRNA) molecules serve as critical components in protein synthesis through their direct interaction with the ribosome and messenger RNA (mRNA). During protein synthesis, tRNA molecules utilize an anticodon composed of three bases that recognize a complementary mRNA codon. At the 3’CCA end of tRNA, the amino acid corresponding to the anticodon sequence is incorporated into the growing polypeptide chain. While canonical Watson-Crick base pairing, pairing between U:A and G:C, occurs between the second and third bases of the anticodon and the corresponding bases of the codon, there is increased flexibility in the ribosome at the first position of the anticodon. This results in non-canonical base pairing and allows a single tRNA molecule to recognize multiple codons. Modification at this site restricts or enhances this “wobble” base pairing. Bacteria often incorporate mnm 5 s 2 U34 into various tRNAs to facilitate proper and efficient translation. In E. coli , three proteins are responsible for this hypermodification pathway: MnmE and MnmG convert s 2 U to cmnm 5 s 2 U, while MnmC subsequently removes the carboxymethyl group to generate nm 5 s 2 U and methylates this intermediate into the ultimate product, mnm 5 s 2 U. We found that an additional enzyme, YhcC, is required for mnm 5 s 2 U synthesis in anaerobic conditions, specifically at the cmnm 5 s 2 U demodification step. Previous studies showed that MnmC-catalyzed demodification is dependent on FAD for the initial oxidation of tRNA substrate, generating FADH 2 . We propose that FADH 2 recycling is dependent on O 2 to regenerate FAD, allowing multiple catalytic turnovers. We show that, in vitro, O 2 strongly promotes cmnm 5 s 2 U demodification by MnmC, supporting a new model of mnm 5 s 2 U synthesis with alternative aerobic and anaerobic routes. Conservation of multiple enzymes that perform similar chemistry, albeit under differing environmental conditions, highlights the importance of maintaining wobble base modifications as well as the ability of bacteria to adapt to their surroundings.