PflC (YijM) is a cytoplasmic radical S-adenosyl-L-methionine (SAM) enzyme of the glycyl-radical-enzyme activase family, a paralogue of the pyruvate formate-lyase activating enzyme PflA. Besides the radical SAM domain with its CX3CX2C [4Fe-4S] cluster-binding motif, it has one ferredoxin-like domain that is absent from PflA. pflC is encoded directly downstream of, and overlapping, pflD in the frw gene cluster. PflD is a glycyl radical enzyme long called "pyruvate formate-lyase 2", but it catalyses ring-opening C-O cleavage of 1,5-anhydromannitol 6-phosphate to 1-deoxyfructose 6-phosphate, the first step of an anaerobic "anhydroglycolysis" pathway. By analogy with PflA and the other members of its family, PflC is expected to install the glycyl radical on PflD, by reductive cleavage of SAM to a 5'-deoxyadenosyl radical that abstracts a hydrogen atom from the PflD Gly-loop glycine. PflC and its paralogue YbiY are each paired with one of the two paralogous anhydrohexitol-phosphate lyases, PflD and YbiW, which share a Gly-loop sequence distinct from that of pyruvate formate-lyase. No activity of PflC itself has yet been demonstrated, and there is no evidence that it activates pyruvate formate-lyase.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the radical SAM domain (IPR007197). True but uninformative. The gene already carries the specific activity it implies, glycyl-radical enzyme activating activity (GO:0043364). Reason: Project rule for generic radical SAM rows: MODIFY GO:0003824 to the specific reaction MF the gene carries. PflC carries GO:0043364 (IBA from the PflA/NrdG node; see that row). It is a member of the PFL-activating-enzyme family, has intact radical SAM ligands, and is translationally coupled to the GRE gene pflD (file:ECOLI/pflC/pflC-bioinformatics/RESULTS.md). Proposed replacements: glycyl-radical enzyme activating activity Supporting Evidence: file:ECOLI/pflC/pflC-uniprot.txt Belongs to the organic radical-activating enzymes family. file:ECOLI/pflC/pflC-bioinformatics/RESULTS.md Each of pflC, ybiY and yjjW sits in an apparent two-gene GRE + activase unit with pflD, ybiW and yjjI respectively, in the same arrangement as pflB-pflA and nrdD-nrdG. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping, by similarity to PflA. PflC has no signal peptide or transmembrane segment. Its partner PflD and the downstream anhydroglycolysis enzymes are soluble cytoplasmic enzymes. Reason: A soluble radical SAM activase that acts on a cytoplasmic GRE is cytoplasmic. Not experimentally shown for PflC, but there is nothing against it, and it is the location where the predicted activity would take place. Supporting Evidence: file:ECOLI/pflC/pflC-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000250}. PMID:39283600 YbiW and PflD catalyze ring-opening C-O cleavage of 1,5-anhydroglucitol-6-phosphate (AG6P) and 1,5-anhydromannitol-6-phosphate (AM6P), respectively |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the radical-activating-enzyme signatures (IPR001989, IPR012839). GO:0043364 glycyl-radical enzyme activating activity is a direct is_a child of oxidoreductase activity (checked in QuickGO), so this row is the general parent of the activity the gene already carries. Reason: The term is correct but general. Per the curation guidelines a correct but general term is MODIFY to the specific descendant the gene carries: GO:0043364. Proposed replacements: glycyl-radical enzyme activating activity Supporting Evidence: file:ECOLI/pflC/pflC-uniprot.txt Belongs to the organic radical-activating enzymes family. |
| GO:0043364 glycyl-radical enzyme activating activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PTN001251485 in PTHR30352 (pyruvate formate-lyase-activating enzyme family), grounded in E. coli PflA (P0A9N4) and NrdG (P0A9N8). Both have established activase activity, on PFL and on the anaerobic RNR respectively. The node asserts only the general activity, generation of a glycyl radical on a partner GRE, not a particular substrate GRE. Reason: The node placement is sound. Every characterised member of this family is a GRE activase, and PflC is inside the clade with no sign of loss: the radical SAM cysteine triad and SAM-binding residues are conserved. Genome context fits. pflC overlaps the 3' end of the GRE gene pflD on the same strand, the arrangement of pflB-pflA and nrdD-nrdG (file:ECOLI/pflC/pflC-bioinformatics/RESULTS.md). PflD is a bona fide GRE whose radical-dependent C-O lyase activity was demonstrated (PMID:39283600). The term does not name a substrate, so it is the right level for PflC. Activase activity of PflC itself has not been directly demonstrated, but this inference is as strong as sequence and context can make it. Supporting Evidence: file:ECOLI/pflC/pflC-bioinformatics/RESULTS.md pflC overlaps the 3' end of pflD by 35 bp file:ECOLI/pflC/pflC-uniprot.txt Activation of pyruvate formate-lyase 2 under anaerobic conditions by generation of an organic free radical PMID:42361324 radical is post-translationally installed by GRE activating enzymes (GRE-AEs), all of which are members of the radical S-adenosyl-L-methionine (RS) superfamily of enzymes. |
| GO:0043364 glycyl-radical enzyme activating activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea-to-GO mapping from the UniProt catalytic-activity line, RHEA:19225 (activation of glycyl-[formate C-acetyltransferase]). The Rhea reaction names PFL as the substrate, which is wrong for PflC (see the GO:0043365 row), but the mapped GO term is the general activase term. Reason: The GO term asserted is the general activase activity, which is accepted on the IBA row and is consistent with it. The RHEA:19225 assignment on the UniProt entry should instead be RHEA:61976 (glycyl-[protein] activation), as UniProt uses for the YjjW paralogue, because the partner GRE PflD is not a formate C-acetyltransferase (PMID:39283600). Supporting Evidence: PMID:39283600 YbiW and PflD catalyze ring-opening C-O cleavage of 1,5-anhydroglucitol-6-phosphate (AG6P) and 1,5-anhydromannitol-6-phosphate (AM6P), respectively |
| GO:0043365 [formate-C-acetyltransferase]-activating enzyme activity | IEA GO_REF:0000003 | REMOVE | Summary: EC-to-GO mapping from UniProt's EC 1.97.1.4 on this entry. That EC rests on the historical name "pyruvate formate-lyase 2" for the partner GRE PflD, and the function line is by similarity only (ECO:0000250). GO:0043365 is the PFL-specific child of GO:0043364; its definition names [formate C-acetyltransferase]-glycine as the substrate. Reason: Wrong substrate: this is a demonstrably incorrect EC/IEA mapping. The partner GRE PflD is a C-O lyase of 1,5-anhydromannitol 6-phosphate, not a formate C-acetyltransferase (PMID:39283600); its Gly loop (RVAGYSA) differs from the PFL/TdcE loop (RVSGYAV), and activase specificity follows the Gly-loop sequence (PMID:42361324; file:ECOLI/pflC/pflC-bioinformatics/RESULTS.md). No experiment shows PflC activating PFL, and PFL is served by PflA. Aligned with the pflA/nrdG reviews (GO:0043365 is correct only when the substrate is PflB): the PflB-specific child is removed here, and the correct general activase activity GO:0043364 is retained on the IBA and Rhea rows. Supporting Evidence: PMID:39283600 The model bacterium Escherichia coli MG1655 contains two GREs of unknown function, YbiW and PflD PMID:42361324 These results suggest that GRE-AE specificity for its partner GRE is dependent upon Gly loop sequence. file:ECOLI/pflC/pflC-bioinformatics/RESULTS.md PflD and YbiW share one Gly-loop heptamer, RVAGYSA, which differs from the PFL/TdcE loop (RVSGYAV) at three of seven positions. file:ECOLI/pflC/pflC-uniprot.txt FUNCTION: Activation of pyruvate formate-lyase 2 under anaerobic conditions by generation of an organic free radical |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the radical SAM domain. PflC keeps the CX3CX2C triad (C47, C51, C54) that binds the radical SAM [4Fe-4S] cluster. Reason: Project rule for generic radical SAM rows: MODIFY GO:0051536 to its descendant GO:0051539, which the gene already carries. The radical SAM cluster is a [4Fe-4S] cluster. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:ECOLI/pflC/pflC-uniprot.txt Binds 1 [4Fe-4S] cluster. The cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: InterPro2GO mapping from the radical-activating-enzyme signature. PflC keeps the radical SAM cysteine triad. It also has a 4Fe-4S ferredoxin-type domain (62-96), which may hold an auxiliary cluster as in other GRE activases. Reason: Project rule: GO:0051539 is ACCEPT for radical SAM enzymes with established catalysis and KEEP_AS_NON_CORE where catalysis is not established. No activity or cluster content has been measured for PflC. Cluster binding is well supported by the conserved ligands, but it is kept as non-core. Supporting Evidence: file:ECOLI/pflC/pflC-uniprot.txt /ligand_note="4Fe-4S-S-AdoMet" PMID:42361324 The vast majority of identified GRE-AEs contain the RS domain and an auxiliary ferredoxin-like domain |
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Download this section (compressed HTML)Q: Which activase installed the glycyl radical on PflD in the anhydroglycolysis study (PMID:39283600): PflC, YbiY, or a non-native activase? The low in vitro radical loading (0.06 per monomer) may reflect use of a non-cognate activase.
Q: Can PflC and YbiY cross-activate PflD and YbiW, given that the two lyases share the Gly-loop heptamer RVAGYSA?
Experiment: Co-express or mix anaerobically reconstituted PflC with PflD plus SAM, reduced flavodoxin and a reductant. Detect the PflD glycyl radical by EPR and assay 1,5-anhydromannitol-6-phosphate C-O lyase activity, comparing PflC, YbiY and PflA as the activase.
Hypothesis: PflC is the cognate activase of PflD and installs its glycyl radical.
Type: in vitro enzymology
Experiment: Construct pflC, ybiY and pflC ybiY deletions and test anaerobic growth on 1,5-anhydromannitol and 1,5-anhydroglucitol as sole carbon source.
Hypothesis: PflC is required for anaerobic growth on 1,5-anhydromannitol.
Type: genetic
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: It is unknown whether PflC activates PflD in vivo, and whether it is the only activase able to do so: YbiY, which serves the PflD paralogue YbiW, could also act on PflD, since the two lyases share an identical Gly loop.
OPEN BIOLOGY MF_DARK
What is known: PflD is an established glycyl radical C-O lyase of 1,5-anhydromannitol 6-phosphate. pflC is translationally coupled to pflD. PflC and YbiY are paralogous activases (37% identity), and PflD and YbiW share the Gly-loop heptamer RVAGYSA. PflC has not been purified, its cluster content has not been measured, and no deletion phenotype has been reported.
Significance: This decides whether PflD activation in vivo depends on PflC specifically, on YbiY, or on redundancy between them, which matters for engineering the anhydroglycolysis pathway and for interpreting pflC deletions.
What would resolve it: Purify PflC anaerobically, reconstitute its [4Fe-4S] cluster, and assay SAM-dependent glycyl-radical installation on PflD by EPR and activity. Test pflC and ybiY single and double deletions for anaerobic growth on 1,5-anhydromannitol and 1,5-anhydroglucitol.
Provenance (the field's own admissions):
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