NrdG is the activating enzyme (activase) of the class III anaerobic ribonucleotide reductase of Escherichia coli. It is a small cytoplasmic radical SAM protein of the organic radical-activating enzyme family that carries one oxygen-labile [4Fe-4S] cluster, ligated by the three cysteines of a CxxxCxxC motif with S-adenosyl-L-methionine (SAM) bound at the fourth iron. Using reduced flavodoxin as electron donor, the reduced cluster cleaves SAM to methionine and a 5'-deoxyadenosyl radical, which abstracts a hydrogen atom from Gly681 of the catalytic subunit NrdD and so installs the stable glycyl radical that NrdD needs to reduce ribonucleoside triphosphates to deoxyribonucleoside triphosphates. NrdG binds NrdD tightly in an alpha2beta2 assembly but acts catalytically, one NrdG activating several NrdD molecules. The nrdDG system supplies deoxyribonucleotides during strict anaerobic growth, and nrdG null mutants cannot grow without oxygen.
Definition: Catalyzes the activation of anaerobic (class III) ribonucleoside-triphosphate reductase by generating an organic free radical on a specific glycine residue of the reductase, via homolytic cleavage of S-adenosyl-L-methionine, in the reaction S-adenosyl-L-methionine + reduced flavodoxin + [anaerobic ribonucleoside-triphosphate reductase]-glycine = 5'-deoxyadenosine + L-methionine + flavodoxin semiquinone + [anaerobic ribonucleoside-triphosphate reductase]-glycin-2-yl radical + H+.
Justification: GO:0043364 glycyl-radical enzyme activating activity has substrate-specific children for the pyruvate formate-lyase activase (GO:0043365) and the choline trimethylamine-lyase activase (GO:0140111), but none for NrdG, the activase of the class III ribonucleotide reductase and the second founding member of the family (QuickGO children of GO:0043364, checked 2026-10-19). The missing sibling leaves NrdG annotated only to the generic parent and encourages InterPro2GO to map the NrdG family signature to the PFL activase term instead.
Parent term: glycyl-radical enzyme activating activity
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: Radical SAM domain InterPro2GO mapping to the root catalytic activity term. NrdG has a defined catalytic activity, SAM-dependent installation of the glycyl radical on NrdD. Reason: Per the RADICAL_SAM treatment rule, the root catalytic activity term is replaced by the specific reaction the gene carries. The most specific GO term for the NrdG reaction is GO:0043364 glycyl-radical enzyme activating activity, which the gene already carries by EXP (PMID:9305874) and IBA. Proposed replacements: glycyl-radical enzyme activating activity Supporting Evidence: PMID:9305874 Here, we demonstrate that AdoMet is directly reduced by the Fe-S center of beta2 during the activation of the enzyme, resulting in methionine and glycyl radical formation. |
| GO:0004748 ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor | IMP PMID:8954104 nrdD and nrdG genes are essential for strict anaerobic growt... | REMOVE | Summary: Contributes-to annotation of class I/II ribonucleoside-diphosphate reductase (thioredoxin) activity, based on the anaerobic growth defect of an nrdG knockout. Reason: This term is wrong for the class III system on two counts. First, NrdD is a ribonucleoside-triphosphate reductase, distinct from the aerobic ribonucleoside-diphosphate reductase that GO:0004748 describes (PMID:1460049). Second, NrdG does not contribute to the reduction chemistry. It is a catalytic activase that installs the glycyl radical on NrdD and can activate several NrdD molecules (PMID:10531327), so even the correct ribonucleoside-triphosphate reductase term would belong to NrdD. The knockout result (PMID:8954104) shows that nrdG is necessary for anaerobic growth, not which activity it has. NrdG's own activity is captured by GO:0043364. Supporting Evidence: PMID:1460049 an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) PMID:10531327 protein beta has the potential to activate several molecules of protein alpha, suggesting that protein beta is an activating enzyme rather than a component of an alpha(2)beta(2) complex as previously claimed PMID:8954104 We now show that knock-out mutants of either nrdD or nrdG cannot grow during strict anaerobiosis |
| GO:0005515 protein binding | IPI PMID:19402753 Global functional atlas of Escherichia coli encompassing pre... | REMOVE | Summary: High-throughput interaction of NrdG with NrdD (P28903) from a global E. coli functional atlas. Reason: The NrdD interaction is real and expected, since NrdD is the substrate protein that NrdG modifies, but protein binding is uninformative. The interaction is already captured by GO:0043364 glycyl-radical enzyme activating activity (NrdD is its substrate) and by GO:0031250 anaerobic ribonucleoside-triphosphate reductase complex. Supporting Evidence: PMID:7852304 The protein corresponding to this open reading frame is required for the generation of the glycyl radical of the anaerobic reductase and binds tightly to the reductase. |
| GO:0005515 protein binding | IPI PMID:24561554 The binary protein-protein interaction landscape of Escheric... | REMOVE | Summary: Binary yeast two-hybrid interaction of NrdG with NrdD (P28903). Reason: As for the other protein binding row, the NrdD interaction is the activase engaging its substrate protein. It is represented informatively by GO:0043364 and GO:0031250, and the protein binding term adds nothing. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location mapping to cytoplasm. Reason: NrdG is a soluble protein without signal peptide or transmembrane segment and acts on the soluble NrdD reductase, with which it co-purifies (PMID:8621608). No fractionation data specifically place it in the cytosol, so cytoplasm is as precise as the evidence allows. Supporting Evidence: file:ECOLI/nrdG/nrdG-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. |
| GO:0009265 2'-deoxyribonucleotide biosynthetic process | NAS PMID:8954104 nrdD and nrdG genes are essential for strict anaerobic growt... | MARK AS OVER ANNOTATED | Summary: Author statement that nrdDG encodes the anaerobic ribonucleotide reductase, used to place NrdG in deoxyribonucleotide synthesis. Reason: NrdG is necessary for anaerobic deoxyribonucleotide supply, but it does none of the work of the process. NrdD binds the ribonucleotides and reduces them, while NrdG installs the glycyl radical cofactor on NrdD and is not needed for each turnover. One NrdG activates several NrdD molecules (PMID:10531327). By the participation test, NrdG acts upstream, as a protein-modifying enzyme, of the process NrdD carries out. Its own process is the modification of NrdD (GO:0018201 peptidyl-glycine modification and GO:0051604 protein maturation, added as NEW rows). The comparator check agrees. PflA, the founding activase, carries no pyruvate fermentation term in GOA. NrdI, which activates the class Ib reductase cofactor, carries no deoxyribonucleotide biosynthesis term (QuickGO, checked 2026-10-19). The claim is not false at the level of the NrdDG system, so the row is marked as an over-annotation rather than removed. Supporting Evidence: PMID:10531327 The enzyme system consists of two proteins: protein alpha, on which ribonucleotides bind and are reduced, and protein beta, of which the function is to introduce a catalytically essential glycyl radical on protein alpha. PMID:8954104 The aerobic enzyme is coded by the nrdAB genes, the anaerobic enzyme by nrdDG. |
| GO:0015949 nucleobase-containing small molecule interconversion | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: PAINT inference placing the NrdG clade in nucleotide interconversion, seeded by the nrdG knockout annotation. Reason: NrdG lies inside the NrdG clade (PTN001576428), so the node placement is not the issue. The issue is the term. The node asserts that the activase takes part in converting ribonucleotides to deoxyribonucleotides, which is NrdD's chemistry. NrdG acts upstream of that conversion. It installs the glycyl radical on NrdD and can go on to activate further NrdD molecules (PMID:10531327), but it catalyses no step of the interconversion. The only descendant evidence is the nrdG knockout growth phenotype (PMID:8954104), which shows necessity, not participation. The action matches the other rows for this process. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: ROLE CONFLATION Sources checked: PANTHER:PTN001576428 SOURCE WEAK OR INFERRED The NrdG-clade node is correctly placed for clade membership, but its process assertion rests on the nrdG knockout (necessity) and attributes the reductase subunit's process to the activase that modifies it. UniProtKB:P0A9N8 SOURCE WEAK OR INFERRED The target's own descendant evidence is the IMP row from PMID:8954104, a growth phenotype that shows nrdG is required, not that NrdG performs a step of nucleotide interconversion. Supporting Evidence: PMID:10531327 The enzyme system consists of two proteins: protein alpha, on which ribonucleotides bind and are reduced, and protein beta, of which the function is to introduce a catalytically essential glycyl radical on protein alpha. |
| GO:0015949 nucleobase-containing small molecule interconversion | IMP PMID:8954104 nrdD and nrdG genes are essential for strict anaerobic growt... | MARK AS OVER ANNOTATED | Summary: nrdG knockout mutants cannot grow under strict anaerobiosis, used to infer a role in nucleotide interconversion. Reason: The knockout result is sound, but it shows that NrdG is necessary, not that it acts in the process. The interconversion (ribonucleotide reduction) is carried out by NrdD once NrdG has installed its glycyl radical. NrdG's own process is the protein modification (GO:0018201). The action matches the other rows for this term. Supporting Evidence: PMID:8954104 We now show that knock-out mutants of either nrdD or nrdG cannot grow during strict anaerobiosis, achieved by inclusion of sodium sulfide in the medium. |
| GO:0015949 nucleobase-containing small molecule interconversion | NAS PMID:8954104 nrdD and nrdG genes are essential for strict anaerobic growt... | MARK AS OVER ANNOTATED | Summary: Author statement that nrdDG encodes the anaerobic ribonucleotide reductase. Reason: Same claim as the IBA and IMP rows for this term. NrdG is the activating enzyme of the reductase, not a catalyst of nucleotide interconversion. Supporting Evidence: PMID:8954104 The aerobic enzyme is coded by the nrdAB genes, the anaerobic enzyme by nrdDG. |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping of the radical-activating enzyme conserved site to oxidoreductase activity. Reason: The term is correct but general. GO:0043364 glycyl-radical enzyme activating activity is an is_a descendant of GO:0016491, and NrdG already carries it by EXP and IBA. Proposed replacements: glycyl-radical enzyme activating activity Supporting Evidence: PMID:10821845 Its function is to mediate electron transfer from reduced flavodoxin to S-adenosylmethionine, required for the introduction of a glycyl radical in the large component, named protein alpha, which then becomes active for the reduction of ribonucleotides. |
| GO:0031250 anaerobic ribonucleoside-triphosphate reductase complex | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of membership in the NrdD-NrdG anaerobic reductase complex. Reason: The GO term is defined as two NrdD and two NrdG subunits, and NrdD and NrdG form a tight 1:1 complex (PMID:8621608). ComplexPortal (CPX-3821) records it as the nrdDG activation complex. NrdG acts catalytically and need not stay bound for NrdD turnover (PMID:10531327). Even so, the complex is the physical context in which it installs the radical, so membership is accepted. Supporting Evidence: PMID:8621608 The large and the small proteins form a tight complex. PMID:8621608 The anaerobic reductase thus has an alpha 2 beta 2 structure. |
| GO:0031250 anaerobic ribonucleoside-triphosphate reductase complex | IMP PMID:8954104 nrdD and nrdG genes are essential for strict anaerobic growt... | ACCEPT | Summary: Complex membership attributed to the knockout study. Reason: A growth phenotype does not by itself establish complex membership. The claim is nonetheless correct and well supported by co-purification and sucrose-gradient stoichiometry (PMID:8621608) and by the tight NrdD binding seen at discovery (PMID:7852304). The action matches the IBA row. Supporting Evidence: PMID:8621608 From sucrose gradient centrifugation, we determined a 1:1 stoichiometry of the two proteins in the complex. PMID:7852304 The protein corresponding to this open reading frame is required for the generation of the glycyl radical of the anaerobic reductase and binds tightly to the reductase. |
| GO:0043364 glycyl-radical enzyme activating activity | EXP PMID:9305874 Activation of the anaerobic ribonucleotide reductase from Es... | ACCEPT | Summary: NrdG reductively cleaves SAM through its [4Fe-4S] centre, forming methionine and the NrdD glycyl radical. Reason: This is the defining activity of NrdG and is directly demonstrated. GO:0043364 is the most specific existing term. Its only children are the PflB (GO:0043365) and choline trimethylamine-lyase (GO:0140111) activases, and neither applies to NrdD. A substrate-specific child term is proposed in proposed_new_terms. Supporting Evidence: PMID:9305874 Here, we demonstrate that AdoMet is directly reduced by the Fe-S center of beta2 during the activation of the enzyme, resulting in methionine and glycyl radical formation. PMID:7852304 The protein corresponding to this open reading frame is required for the generation of the glycyl radical of the anaerobic reductase and binds tightly to the reductase. |
| GO:0043364 glycyl-radical enzyme activating activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of glycyl-radical activase activity at the organic radical-activating enzyme node shared with PflA. Reason: The node (PTN001251485) is seeded by experimental activase annotations of PflA and of NrdG itself. That NrdG appears among its own sources marks experimental grounding on the target. Activase activity is the ancestral function of this radical SAM clade, and NrdG retains the CxxxCxxC cluster motif and the activity. Supporting Evidence: PMID:10821845 Such a sequence is also present in the activase of the pyruvate formate-lyase and in the biotin synthase, both carrying an iron-sulfur center involved in reductive activation of S-adenosylmethionine. |
| GO:0043364 glycyl-radical enzyme activating activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea mapping of the generic glycyl-[protein] radical-forming reaction. Reason: The UniProt catalytic activity, supported experimentally by PMID:9305874, maps to this term, and it agrees with the EXP row. Supporting Evidence: file:ECOLI/nrdG/nrdG-uniprot.txt Reaction=glycyl-[protein] + reduced [flavodoxin] + S-adenosyl-L-methionine = glycin-2-yl radical-[protein] + semiquinone [flavodoxin] + 5'-deoxyadenosine + L-methionine + H(+) |
| GO:0043365 [formate-C-acetyltransferase]-activating enzyme activity | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping of the NrdG family entry (IPR012837) to the pyruvate formate-lyase activase activity. Reason: The mapping is demonstrably wrong. GO:0043365 is defined by the reaction on [formate C-acetyltransferase]-glycine, the PflB/G734 substrate of PflA. NrdG's substrate is NrdD (Gly681), not pyruvate formate-lyase (PMID:7852304, PMID:8621608). The InterPro entry that triggers the row is the NrdG family signature itself, so the mapping assigns a paralog's substrate specificity to the whole NrdG family. The correct parent activity, GO:0043364, is retained. Supporting Evidence: PMID:7852304 Immediately downstream, we now find an open reading frame with the potential to code for a 17.5-kDa protein with sequence homology to a protein required for the generation of the glycyl radical of pyruvate formate lyase. PMID:8621608 with a stable, oxygen-sensitive radical located at Gly-681 of the 80-kDa polypeptide chain |
| GO:0051536 iron-sulfur cluster binding | IDA PMID:8381402 An iron-sulfur center and a free radical in the active anaer... | MODIFY | Summary: Early detection of an Fe-S centre in active anaerobic reductase preparations. Reason: Per the RADICAL_SAM treatment rule, GO:0051536 is refined to GO:0051539. In the 1993 study the centre was attributed to the reductase and read as a 3Fe-4S cluster. Later work showed that the signal came from co-purifying NrdG and that its intact form is a [4Fe-4S] cluster (PMID:8621608, PMID:10531327). Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:8381402 The reductase contained roughly equimolar amounts of iron and sulfide, suggesting the presence of an Fe-S complex. PMID:8621608 A degraded form of this cluster may have been responsible for an EPR signal observed earlier in preparations of the large 160-kDa subunit that suggested the presence of a 3Fe-4S cluster in the reductase. |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: Radical SAM domain InterPro2GO mapping to iron-sulfur cluster binding. Reason: Per the RADICAL_SAM treatment rule, refined to GO:0051539, which the gene carries by IDA (PMID:10531327). Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:10531327 Protein beta can assemble one [4Fe-4S] center per polypeptide |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:10531327 The anaerobic ribonucleotide reductase from Escherichia coli... | ACCEPT | Summary: Iron and sulfide analysis, Mossbauer and EPR show one [4Fe-4S] centre per NrdG polypeptide, in the 2+ and 1+ states. Reason: The radical SAM [4Fe-4S] cluster is the catalytic cofactor that cleaves SAM. Its three cysteine ligands of the CxxxCxxC motif are essential for activity (PMID:10821845). Supporting Evidence: PMID:10531327 Protein beta can assemble one [4Fe-4S] center per polypeptide PMID:10821845 By site-directed mutagenesis we demonstrate that the three cysteines of the conserved CXXXCXXC sequence are involved in iron chelation. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping to [4Fe-4S] cluster binding. Reason: Agrees with the IDA evidence (PMID:10531327) and the RADICAL_SAM treatment rule. Supporting Evidence: PMID:10531327 Protein beta can assemble one [4Fe-4S] center per polypeptide |
| GO:1904047 S-adenosyl-L-methionine binding | IDA PMID:9305874 Activation of the anaerobic ribonucleotide reductase from Es... | NEW | Summary: NrdG binds SAM with a Kd of 10 uM and 1:1 stoichiometry, forming an EPR-detectable complex with its [4Fe-4S] centre. Reason: GOA has no SAM-binding row for NrdG. Following the RADICAL_SAM Phase 1 convention, GO:1904047 is added for every member whose radical SAM catalysis is established. For NrdG both binding and reductive cleavage of SAM are directly measured. This is a molecular function of NrdG itself, so the participation test is met. Supporting Evidence: PMID:9305874 Direct binding experiments showed that AdoMet binds to beta2 with a Kd of 10 microM and a 1:1 stoichiometry. PMID:9305874 Binding was confirmed by EPR spectroscopy that demonstrated the formation of a complex between AdoMet and the [4Fe-4S] center of beta2. |
| GO:0018201 peptidyl-glycine modification | IDA PMID:9305874 Activation of the anaerobic ribonucleotide reductase from Es... | NEW | Summary: NrdG converts Gly681 of NrdD into a glycyl radical, a post-translational modification of a peptidyl-glycine. Reason: GO has no glycyl radical formation term, and the modification is represented by GO:0018201 peptidyl-glycine modification. PAINT places this term on the PflA activase node (PTN002018048) and PflA carries it by IDA, but NrdG has no row for it. Participation test. NrdG is the enzyme that performs the modification, abstracting the Gly681 hydrogen via the 5'-deoxyadenosyl radical it generates, so it does the work of the process rather than being its substrate. Comparator check. The paralogous activase PflA, in the same role toward PflB Gly734, carries this term (IBA and IDA), and the substrate proteins (NrdD, PflB) would not qualify. The term is neither an ancestor nor a descendant of GO:0051604. Supporting Evidence: PMID:9305874 Here, we demonstrate that AdoMet is directly reduced by the Fe-S center of beta2 during the activation of the enzyme, resulting in methionine and glycyl radical formation. PMID:8621608 with a stable, oxygen-sensitive radical located at Gly-681 of the 80-kDa polypeptide chain |
| GO:0051604 protein maturation | IDA PMID:7852304 Generation of the glycyl radical of the anaerobic Escherichi... | NEW | Summary: NrdG converts inactive NrdD into its catalytically active glycyl-radical form. Reason: Radical installation is the step by which NrdD attains its full functional capacity, which matches the definition of GO:0051604. PAINT places protein maturation on the PflA activase node and PflA carries it by IDA, so adding it here keeps the two founding activases consistent. NrdG is the catalyst of this maturation step, so it passes the participation test. The comparator check is the same as for GO:0018201. Supporting Evidence: PMID:7852304 The protein corresponding to this open reading frame is required for the generation of the glycyl radical of the anaerobic reductase and binds tightly to the reductase. PMID:10821845 Its function is to mediate electron transfer from reduced flavodoxin to S-adenosylmethionine, required for the introduction of a glycyl radical in the large component, named protein alpha, which then becomes active for the reduction of ribonucleotides. |
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Download this section (compressed HTML)Q: Does NrdG remain bound to NrdD during ribonucleotide reduction in vivo, or does it dissociate after radical installation as the catalytic activation in vitro suggests?
Q: How does NrdG distinguish NrdD Gly681 from the glycyl radical sites of PflB and the other E. coli glycyl radical enzymes, given its homology to the PflA activase?
Experiment: Quantify NrdD and NrdG copy numbers in anaerobic cells by targeted proteomics, and test by in-cell crosslinking or co-immunoprecipitation whether NrdG stays bound to glycyl radical-bearing NrdD.
Hypothesis: NrdG acts catalytically in vivo, so cellular NrdG is substoichiometric to NrdD and the two dissociate after activation.
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