YbiY 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 and of PflC. It has a radical SAM domain with the CX3CX2C [4Fe-4S] cluster-binding motif. ybiY is encoded immediately downstream of, and on the same strand as, ybiW in a two-gene unit. YbiW is a glycyl radical enzyme that catalyses ring-opening C-O cleavage of 1,5-anhydroglucitol 6-phosphate to 1-deoxyfructose 6-phosphate, the first step of an anaerobic "anhydroglycolysis" pathway that lets E. coli grow on 1,5-anhydroglucitol. By analogy with PflA and its family, YbiY is expected to install the glycyl radical on YbiW, by reductive cleavage of SAM to a 5'-deoxyadenosyl radical that abstracts a hydrogen atom from the YbiW Gly-loop glycine. YbiY and PflC are the paralogous activases of the two paralogous anhydrohexitol-phosphate lyases YbiW and PflD, which share a Gly-loop sequence distinct from that of pyruvate formate-lyase. No activity of YbiY itself has 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. YbiY carries GO:0043364 (IBA from the PflA/NrdG node). It is a member of the PFL-activating-enzyme family, keeps the radical SAM ligands, and is translationally coupled to the GRE gene ybiW (file:ECOLI/pflC/pflC-bioinformatics/RESULTS.md). Proposed replacements: glycyl-radical enzyme activating activity Supporting Evidence: file:ECOLI/ybiY/ybiY-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. YbiY has no signal peptide or transmembrane segment. Its partner YbiW and the downstream anhydroglycolysis enzymes are soluble cytoplasmic proteins. Reason: A soluble radical SAM activase acting on a cytoplasmic GRE is cytoplasmic. Not experimentally shown for YbiY, but nothing argues against it and it is where the predicted activity occurs. Supporting Evidence: file:ECOLI/ybiY/ybiY-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. GO:0043364 is a direct is_a child of oxidoreductase activity (QuickGO), so this is the general parent of the activity the gene already carries. Reason: Correct but general. Per the curation guidelines, MODIFY a correct but general term to the specific descendant the gene carries: GO:0043364. Proposed replacements: glycyl-radical enzyme activating activity Supporting Evidence: file:ECOLI/ybiY/ybiY-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, grounded in E. coli PflA (P0A9N4) and NrdG (P0A9N8), both with established activase activity. 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 YbiY is inside the clade with no sign of loss: the radical SAM cysteine triad and SAM-binding residues are conserved. Genome context fits: ybiY is directly downstream of the GRE gene ybiW on the same strand, the arrangement of pflB-pflA and nrdD-nrdG (file:ECOLI/pflC/pflC-bioinformatics/RESULTS.md). YbiW is a bona fide GRE whose radical-dependent C-O lyase activity was demonstrated (PMID:39283600). The term names no substrate, so it is the right level. YbiY's activase activity has not been directly shown, but this inference is as strong as sequence and context can make it. Supporting Evidence: file:ECOLI/pflC/pflC-bioinformatics/RESULTS.md ybiY starts 5 bp after ybiW file:ECOLI/ybiY/ybiY-uniprot.txt /evidence="ECO:0000250|UniProtKB:P0A9N4" 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 substrate, which is wrong for YbiY (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, consistent with the IBA row. 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 YbiW 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. That EC rests on the historical name "pyruvate formate-lyase 3" for the partner, and the function line is by similarity only. GO:0043365 is the PFL-specific child of GO:0043364 and names [formate C-acetyltransferase]-glycine as its substrate. Reason: Wrong substrate: a demonstrably incorrect EC/IEA mapping. The partner GRE YbiW is a C-O lyase of 1,5-anhydroglucitol 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 (PMID:42361324). No experiment shows YbiY activating PFL, which 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, 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/ybiY/ybiY-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. YbiY keeps the CX3CX2C triad (C25, C29, C32) 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/ybiY/ybiY-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. YbiY keeps the radical SAM cysteine triad that binds the [4Fe-4S] cluster. 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 YbiY. Cluster binding is well supported by the conserved ligands, but is kept as non-core. Supporting Evidence: file:ECOLI/ybiY/ybiY-uniprot.txt /ligand_note="4Fe-4S-S-AdoMet" 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. |
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Download this section (compressed HTML)Q: Which activase installed the glycyl radical on YbiW in the anhydroglycolysis study (PMID:39283600): YbiY, PflC, or a non-native activase? The very low in vitro radical loading (0.02 per monomer) may reflect use of a non-cognate activase.
Q: Can YbiY and PflC cross-activate YbiW and PflD, given that the two lyases share the Gly-loop heptamer RVAGYSA?
Experiment: Mix anaerobically reconstituted YbiY with YbiW plus SAM, reduced flavodoxin and a reductant. Detect the YbiW glycyl radical by EPR and assay 1,5-anhydroglucitol-6-phosphate C-O lyase activity, comparing YbiY, PflC and PflA as the activase.
Hypothesis: YbiY is the cognate activase of YbiW and installs its glycyl radical.
Type: in vitro enzymology
Experiment: Construct ybiY, pflC and ybiY pflC deletions and test anaerobic growth on 1,5-anhydroglucitol and 1,5-anhydromannitol as sole carbon source.
Hypothesis: YbiY is required for anaerobic growth on 1,5-anhydroglucitol.
Type: genetic
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: It is unknown whether YbiY activates YbiW in vivo, and whether it is the only activase able to do so: PflC, which serves the YbiW paralogue PflD, could also act on YbiW, since the two lyases share an identical Gly loop.
OPEN BIOLOGY MF_DARK
What is known: YbiW is an established glycyl radical C-O lyase of 1,5-anhydroglucitol 6-phosphate. ybiY is directly downstream of ybiW. YbiY and PflC are paralogous activases (37% identity), and YbiW and PflD share the Gly-loop heptamer RVAGYSA. YbiY has not been purified, its cluster content has not been measured, and no deletion phenotype has been reported.
Significance: This decides whether YbiW activation in vivo depends on YbiY specifically, on PflC, or on redundancy between them, which matters for engineering anhydroglycolysis and for interpreting ybiY deletions.
What would resolve it: Purify YbiY anaerobically, reconstitute its [4Fe-4S] cluster, and assay SAM-dependent glycyl-radical installation on YbiW by EPR and activity. Test ybiY and pflC single and double deletions for anaerobic growth on 1,5-anhydroglucitol and 1,5-anhydromannitol.
Provenance (the field's own admissions):
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