The Escherichia coli Radical SAM Enzyme YhcC Replaces MnmC in mnm5s2U tRNA Modification During Anaerobic Growth
- 1University of Florida
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 (cmnm5) or 5-methylaminomethyl (mnm5) group, often together with a 2-thio (s2) moiety. Bacteria utilize the conserved MnmEG complex to produce cmnm5U, which is further converted to mnm5U by non-orthologous enzymes in different lineages. Escherichia coli uses the bifunctional enzyme MnmC to demodify cmnm5U to nm5U and subsequently methylate nm5U to mnm5U whereas Bacillus subtilis relies 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 cmnm5s2 U demodification in vivo during anaerobic growth, whereas the equivalent MnmC-dependent reaction requires O2 and occurs only aerobically. In vitro, purified [4Fe-4S]-reconstituted YhcC binds tRNA and catalyzes nm5s2U-tRNA synthesis from cmnm5s2U-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 nm5s2U. These parallel pathways reveal how E. coli maintains synthesis of a critical wobble-base modification under both aerobic and anaerobic growth conditions.
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BibTeX
@misc{boswinkle2026escherichia,
title = {{The Escherichia coli Radical SAM Enzyme YhcC Replaces MnmC in mnm5s2U tRNA Modification During Anaerobic Growth}},
author = {K. Boswinkle and P. N. Roehling and C. J. Reed and J. Marakorn and T. Carell and A. Dziergowska and P. C. Dos Santos and J. S. Mugridge and V. de Crecy-Lagard},
howpublished = {bioRxiv},
year = {2026},
month = may,
doi = {10.64898/2026.05.18.725915},
url = {https://www.biorxiv.org/content/10.64898/2026.05.18.725915v2},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - The Escherichia coli Radical SAM Enzyme YhcC Replaces MnmC in mnm5s2U tRNA Modification During Anaerobic Growth
AU - K. Boswinkle
AU - P. N. Roehling
AU - C. J. Reed
AU - J. Marakorn
AU - T. Carell
AU - A. Dziergowska
AU - P. C. Dos Santos
AU - J. S. Mugridge
AU - V. de Crecy-Lagard
PY - 2026
DA - 2026/05/18
PB - bioRxiv
DO - 10.64898/2026.05.18.725915
UR - https://www.biorxiv.org/content/10.64898/2026.05.18.725915v2
AB - 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 (cmnm5) or 5-methylaminomethyl (mnm5) group, often together with a 2-thio (s2) moiety. Bacteria utilize the conserved MnmEG complex to produce cmnm5U, which is further converted to mnm5U by non-orthologous enzymes in different lineages. Escherichia coli uses the bifunctional enzyme MnmC to demodify cmnm5U to nm5U and subsequently methylate nm5U to mnm5U whereas Bacillus subtilis relies 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 cmnm5s2 U demodification in vivo during anaerobic growth, whereas the equivalent MnmC-dependent reaction requires O2 and occurs only aerobically. In vitro, purified [4Fe-4S]-reconstituted YhcC binds tRNA and catalyzes nm5s2U-tRNA synthesis from cmnm5s2U-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 nm5s2U. These parallel pathways reveal how E. coli maintains synthesis of a critical wobble-base modification under both aerobic and anaerobic growth conditions.
ER -