YhcC is a 309-residue radical S-adenosyl-L-methionine (SAM) enzyme of the Elp3/MiaB/NifB-like clan and the E. coli orthologue of the Gram-positive tRNA-modifying enzyme MnmL. It binds one [4Fe-4S] cluster through three cysteines (Cys33, Cys45, Cys48) and reductively cleaves SAM to methionine and 5'-deoxyadenosine. YhcC acts in the biosynthesis of the wobble-base modification 5-methylaminomethyl-2-thiouridine (mnm5s2U) of tRNA: it binds tRNA and converts the MnmEG product 5-carboxymethylaminomethyl-2-thiouridine at position 34 (cmnm5s2U) into 5-aminomethyl-2-thiouridine (nm5s2U), which the MnmC2 methyltransferase domain then methylates to mnm5s2U. This route operates anaerobically: the flavin-dependent MnmC1 domain that performs the same demodification needs molecular oxygen, so cells grown without oxygen depend on YhcC, whose gene is activated by the anaerobic regulator FNR, while aerobically grown cells lacking YhcC are unaffected. The enzyme is cytoplasmic and its oxygen-sensitive cluster chemistry provides E. coli with an oxygen-independent counterpart to a flavoenzyme step, a pattern it also uses in heme and deoxyribonucleotide synthesis. On the strength of this assignment the gene has been proposed for renaming to mnmL.
Definition: Catalysis of the removal of the carboxymethyl group from 5-carboxymethylaminomethyl-2-thiouridine at position 34 of a tRNA, converting it to 5-aminomethyl-2-thiouridine(34). The reaction requires S-adenosyl-L-methionine and a [4Fe-4S] cluster: the cluster reductively cleaves S-adenosyl-L-methionine to L-methionine and a 5'-deoxyadenosyl radical, which initiates cleavage of the carbon-nitrogen bond of the substrate side chain. The full stoichiometry and the identity of the released carbon fragment have not been determined.
Justification: This is the reaction of E. coli YhcC and of its Gram-positive orthologue MnmL, the oxygen-independent route to the wobble modification mnm5s2U. GO has no matching term: QuickGO and OLS searches (2026-10-19) for "carboxymethylaminomethyl", "5-aminomethyl-2-thiouridine", "cmnm5" and "methylaminomethyl" return only GO:0004808, the subsequent MnmC2 methylation, and GO:0141102, an unrelated ribose methyltransferase. The enzyme that performs the same demodification aerobically, the MnmC1 domain of E. coli MnmC, is annotated only to the broad class GO:0016645 oxidoreductase activity, acting on the CH-NH group of donors, which does not fit a radical SAM enzyme using SAM rather than a flavin and an electron acceptor. The term should sit under GO:0140101 catalytic activity, acting on a tRNA.
Parent term: catalytic activity, acting on a tRNA
Mappings:
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: Root-level catalytic activity from the radical SAM InterPro signatures. The reaction of YhcC is now known: purified, [4Fe-4S]-reconstituted YhcC converts cmnm5s2U(34) of tRNA to nm5s2U in a SAM-dependent manner, and the conversion fails in vivo in a yhcC deletion or a cluster-ligand variant (PMID:42239373). Reason: Per the RADICAL_SAM project's treatment rule, GO:0003824 is replaced by the specific reaction the gene performs. GO has no term for this demodification (QuickGO and OLS checked 2026-10-19; the only neighbouring MF terms are GO:0004808 for the subsequent MnmC2 methylation step and GO:0016645, the generic class carried by the oxygen-dependent MnmC1 domain), so the replacement is the term requested in proposed_new_terms. Proposed replacements: tRNA 5-carboxymethylaminomethyl-2-thiouridine(34) demodifying activity (cmnm5s2U to nm5s2U, radical SAM-dependent) Supporting Evidence: PMID:42239373 providing direct in vitro evidence that YhcC catalyzes the cmnm5s2U to nm5s2U reaction on tRNA PMID:42239373 showing that the conversion of cmnn5s2U to nm5s2U relies on YhcC catalysis |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: Generic iron-sulfur cluster binding from the radical SAM and YhcC-like signatures. The cluster is specifically a [4Fe-4S] cluster, demonstrated on the protein itself (PMID:25117543) and reconstituted for the in vitro activity assays (PMID:42239373). Reason: Correct but general. Per the RADICAL_SAM project's treatment rule, replace with the descendant GO:0051539, which the gene already carries with experimental evidence. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:ECOLI/yhcC/yhcC-uniprot.txt Binds 1 [4Fe-4S] cluster. The cluster is coordinated with 3 PMID:42239373 YhcC has also been confirmed to bind a [4Fe-4S] cluster and is capable of cleaving SAM to methionine and 5β-deoxyadenosine in the presence of a chemical reductant |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:25117543 An integrative computational model for large-scale identific... | ACCEPT | Summary: Direct demonstration that YhcC is a genuine iron-sulfur protein (PMID:25117543), with UniProt recording one [4Fe-4S] cluster coordinated by three cysteines and an exchangeable SAM. The cluster was reconstituted for the catalytic assays, and substituting the ligand Cys33 abolishes function in vivo (PMID:42239373). Reason: Experimentally established and appropriately specific. Per the RADICAL_SAM project's treatment rule, GO:0051539 is a core function wherever catalysis is established, and for YhcC it now is: the cluster is the catalytic cofactor that cleaves SAM to the 5'-deoxyadenosyl radical. Supporting Evidence: PMID:25117543 Two newly-identified proteins, YhcC and YdiJ, were functionally validated as genuine iron-sulfur proteins file:ECOLI/yhcC/yhcC-uniprot.txt Binds 1 [4Fe-4S] cluster. The cluster is coordinated with 3 PMID:42239373 Expression of this variant did not recover the ΞyhcC phenotype, as it accumulated only cmnm5s2U |
| GO:1904047 S-adenosyl-L-methionine binding | IC PMID:42239373 The Escherichia coli Radical SAM Enzyme YhcC Substitutes for... | NEW | Summary: YhcC binds SAM as the radical-generating cosubstrate at the unique iron of its [4Fe-4S] cluster: it cleaves SAM to methionine and 5'-deoxyadenosine with a chemical reductant (PMID:25117543, restated in PMID:42239373), and its tRNA demodification reaction in vitro is SAM-dependent. Reason: GOA has no SAM-binding row for yhcC, although the RADICAL_SAM project treats GO:1904047 as a core cosubstrate-binding function of every radical SAM enzyme with established catalysis. This is a molecular function, so the participation test is met directly: YhcC itself binds and cleaves SAM. Comparator check, against GOA rather than against this project's own proposals: GOA does annotate GO:1904047 to radical SAM enzymes in exactly this role, by UniRule -- ECOLI/queE (UR000100349), ECOLI/moaA and PSEPK/moaA (UR000101305), METEA/pqqE and PSEPK/pqqE (UR000098303). So the absence here is patchy UniRule coverage, which rules happen to carry the term, not a GO convention against annotating a radical SAM enzyme's own cosubstrate. That is also why those five genes have an existing row to ACCEPT or MODIFY while this one needs a NEW row. Supporting Evidence: PMID:42239373 YhcC has also been confirmed to bind a [4Fe-4S] cluster and is capable of cleaving SAM to methionine and 5β-deoxyadenosine in the presence of a chemical reductant PMID:42239373 These reactions showed a clear YhcC- and SAM-dependent consumption of substrate cmnm5s2U over time file:ECOLI/yhcC/yhcC-uniprot.txt can cleave S-adenosyl-L-methionine into methionine |
| GO:0000049 tRNA binding | IDA PMID:42239373 The Escherichia coli Radical SAM Enzyme YhcC Substitutes for... | NEW | Summary: Electrophoretic mobility shift assays with purified YhcC and unmodified E. coli tRNA-Lys(UUU) made by in vitro transcription give a dissociation constant of about 5 micromolar (PMID:42239373); the predicted structure has a positively charged cleft next to the active site. Reason: A directly measured binding activity of the gene product, absent from GOA, and the substrate-recognition half of its catalytic function. Molecular function, so the participation test applies directly and is met. Supporting Evidence: PMID:42239373 found that YhcC binds tRNA with a KD of approximately 5 ΞΌM |
| GO:0002098 tRNA wobble uridine modification | IMP PMID:42239373 The Escherichia coli Radical SAM Enzyme YhcC Substitutes for... | NEW | Summary: Deleting yhcC abolishes mnm5s2U and causes accumulation of the cmnm5s2U precursor in tRNA from anaerobically grown cells even though mnmC is intact; complementation with wild-type yhcC restores mnm5s2U, while a Cys33 cluster-ligand variant does not (PMID:42239373). The missing step, cmnm5s2U to nm5s2U at wobble position 34, is catalysed by YhcC itself in vitro. Reason: GOA has no process row for this gene. Participation test: YhcC performs one step of the pathway, the demodification of the wobble uridine, shown with purified enzyme, so it does the work of the process rather than merely being required for it. Comparator check: the enzymes of this pathway carry the term, including E. coli MnmC (P77182), which performs the same step aerobically and holds GO:0002098 by IMP, and B. subtilis MnmL is the orthologous enzyme in Gram-positive bacteria (PMID:38551342). This term is the appropriate level: no GO term exists for mnm5s2U biosynthesis specifically (QuickGO and OLS, 2026-10-19). Supporting Evidence: PMID:42239373 Additionally, cmnm5s2U accumulated in tRNAs extracted from the ΞyhcC mutant grown under anoxic conditions despite the presence of mnmC. PMID:42239373 The complemented E. coli ΞyhcC strain was found to only produce mnm5s2U PMID:42239373 we find YhcC is necessary for cmnm5s2U demodification in vivo under anaerobic growth |
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Download this section (compressed HTML)Q: What is the fate of the carbon fragment removed from cmnm5s2U by YhcC, and how many equivalents of S-adenosyl-L-methionine are consumed per modified tRNA?
Suggested experts: Patricia C Dos Santos, Jeffrey S Mugridge, Squire J Booker
Q: Does YhcC act on a defined subset of tRNAs, and does it require the 2-thio group of the substrate, given that only thiolated species were detected in vivo?
Suggested experts: Valerie de Crecy-Lagard, Jeffrey S Mugridge
Q: Should the gene be renamed mnmL, as the authors propose, and is the E. coli enzyme functionally interchangeable with B. subtilis MnmL in vivo?
Suggested experts: Valerie de Crecy-Lagard
Experiment: React reconstituted YhcC with tRNA carrying cmnm5s2U labelled in the carboxymethyl group, and quantify 5'-deoxyadenosine, methionine and labelled small molecules by LC-MS and NMR, establishing stoichiometry and the identity of the released fragment.
Hypothesis: YhcC catalyses carbon-nitrogen bond cleavage of the cmnm5s2U side chain with release of a two-carbon fragment.
Type: anaerobic enzymology with isotope labelling
Experiment: Grow wild-type, yhcC, mnmC and double mutants anaerobically in competition and in defined media, and measure growth rate, codon-specific translational fidelity with reporter assays and ribosome profiling.
Hypothesis: The anaerobic route via YhcC is needed for fitness and accurate decoding without oxygen.
Type: competitive fitness and translational fidelity assays
Experiment: Measure binding and turnover of reconstituted YhcC with individual in vitro transcribed tRNAs bearing cmnm5U or cmnm5s2U and with anticodon-loop variants, to define substrate specificity.
Hypothesis: YhcC selects substrate tRNAs by recognising the anticodon stem loop and the 2-thio group.
Type: in vitro substrate specificity profiling
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The chemical mechanism of the YhcC reaction is not established: what happens to the carbon fragment removed from the cmnm5s2U side chain, how many equivalents of SAM are consumed per turnover, and whether the released product is glyoxylate as in the flavin-dependent MnmC1 route are all unknown.
OPEN BIOLOGY MF_DARK
What is known: Established: purified, [4Fe-4S]-reconstituted YhcC converts cmnm5s2U(34) to nm5s2U on tRNA in a SAM-dependent reaction, binds tRNA with a dissociation constant near 5 micromolar, and cleaves SAM to methionine and 5'-deoxyadenosine with a chemical reductant. No product other than the modified tRNA has been identified and no kinetic or isotope-labelling analysis has been reported.
Significance: The stoichiometry decides whether YhcC is a true catalyst or consumes SAM per turnover, and identifying the released fragment would show how a radical enzyme accomplishes a transformation that the aerobic route performs by flavin-dependent oxidation.
What would resolve it: Quantify 5'-deoxyadenosine, methionine and candidate small products such as glyoxylate or formaldehyde per nm5s2U formed, using isotopically labelled substrate tRNA and the reconstituted enzyme, and determine steady-state kinetics.
Provenance (the field's own admissions):
Gap: The physiological consequence of losing the anaerobic route is not defined: no growth or translational-fidelity phenotype of a yhcC single mutant has been reported, so it is unknown what the modification contributes to anaerobic life beyond its presence.
OPEN BIOLOGY BP_DARK
What is known: Established: yhcC is FNR-activated and is required for mnm5s2U synthesis under anoxic growth, while aerobically grown yhcC mutants make mnm5s2U normally. Reported phenotypes concern the modification profile, not fitness or decoding.
Significance: Wobble modifications tune decoding of split codon boxes; whether their loss specifically impairs anaerobic growth, motility or stress survival would show why E. coli maintains a second, oxygen-independent enzyme for this step.
What would resolve it: Compete yhcC mutants against wild type under anaerobic and fermentative conditions, and measure frameshifting, missense suppression and proteome-wide translation rates in the mutant grown anaerobically.
Provenance (the field's own admissions):
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