The Escherichia coli Radical SAM Enzyme YhcC Substitutes for the FAD-Dependent Oxidase Activity of MnmC in 5-Methylaminomethyl-2-Thiouridine tRNA Modification Under Anaerobic Conditions

tRNA wobble base uridines are heavily modified to influence anticodon:codon base pairing and tune the structure of the anticodon stem loop for efficient and accurate translation. Both Gram-positive and negative bacteria, as well as certain archaea, modify wobble uridines to contain either a 5-carboxymethylaminomethyl (cmnm5) or a 5-methylaminomethyl (mnm5) moiety, as well as in some instances a 2-thio (s2) moiety. Bacteria utilize the conserved MnmEG complex to produce cmnm5U, which is further modified in some tRNAs to mnm5U. The steps to synthesize the latter are catalyzed by non-orthologous enzymes in distantly related bacteria. Escherichia coli utilizes a single bifunctional enzyme, MnmC, to both demodify cmnm5U to nm5U and subsequently methylate nm5U to mnm5U, while Bacillus subtilis relies on the radical SAM (rSAM) enzyme MnmL, followed by the stand-alone MnmM methylase for mnm5U production. It was previously noted that E. coli and related bacteria that encode MnmC can also contain homologs of MnmL. As an E. coli mnmC mutant accumulates cmnm5U, the function of the MnmL homolog in this organism, YhcC, was unknown. Here, we find YhcC is necessary for cmnm5s2U demodification in vivo under anaerobic growth, and that the same MnmC-mediated demodification activity requires O2 and only occurs under aerobic growth. In vitro reaction experiments demonstrate that purified YhcC, reconstituted to its [4Fe-4S] form, is able to bind tRNA and catalyze the nm5s2U-tRNA synthesis from cmnm5s2U-tRNA. Together, these results define the heretofore unknown biochemistry of the E. coli rSAM enzyme YhcC and show this enzyme replaces MnmC under anaerobic conditions to carry out the synthesis of nm5s2U. These parallel tRNA modification pathways highlight how E. coli has adapted to maintain biosynthesis of a critical wobble base modification under both aerobic and anaerobic growth. General audience summaryTransfer 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 3CCA 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 install mnm5s2U34 to various tRNAs to facilitate proper and efficient translation. In E. coli, three proteins are responsible for this hypermodification pathway: MnmE and MnmG convert s2U to cmnm5s2U, while MnmC subsequently removes the carboxymethyl group to generate nm5s2U and methylates this intermediate into the ultimate product, mnm5s2U. We found that an additional enzyme, YhcC, is required for mnm5s2U synthesis in anaerobic conditions, specifically at the cmnm5s2U demodification step. Previous studies showed that MnmC-catalyzed demodification is dependent on FAD for the initial oxidation of tRNA substrate, generating FADH2. We propose that FADH2 recycling is dependent on O2 to regenerate FAD, allowing multiple catalytic turnovers. We show that, in vitro, O2 is required for cmnm5s2U demodification by MnmC, supporting a new model of mnm5s2U 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.