nrdJa (PA5497) encodes the large, catalytic polypeptide of the class II (adenosylcobalamin-dependent) ribonucleotide reductase of Pseudomonas aeruginosa. P. aeruginosa carries all three classes of ribonucleotide reductase, and its class II enzyme is unusual in being split: nrdJa and the adjacent nrdJb (PA5496) are separated by 16 bp and co-transcribed as the nrdJab operon, and the 734-residue NrdJa and the 229-residue NrdJb assemble into an (NrdJa-NrdJb)2 dimer of heterodimers. NrdJa holds the complete catalytic machinery: the adenosylcobalamin radical-generating site, the substrate site and the ATP allosteric specificity site; NrdJb contributes the C-terminal redox-active cysteine pair that performs thiol-disulfide exchange after each turnover, which an unsplit class II enzyme carries on its own C-terminal tail. Purified NrdJa plus NrdJb reduce ribonucleoside triphosphates (CTP) more than 60-fold faster than the corresponding diphosphate, so the enzyme is effectively a cobalamin-dependent ribonucleoside-triphosphate reductase that also reduces diphosphates. Like most class II enzymes it lacks the dATP overall-activity site. The enzyme supplies deoxyribonucleotides when the oxygen-dependent class I enzyme is unavailable: nrdJa transcript rises on entry to stationary phase, and adenosylcobalamin rescues growth of hydroxyurea-poisoned cells, in which class I activity is destroyed. The physiological thiol reductant has not been identified; both published assays used dithiothreitol.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO from the NrdJ/NrdZ signature (IPR013344). Reason: True but uninformative. NrdJa binds nucleotides as substrate (CTP/CDP) and as allosteric effector (ATP, dNTPs); the specific term ATP binding (GO:0005524) is an is_a descendant already carried by the gene, and is the better level. Proposed replacements: ATP binding Supporting Evidence: PMID:26225432 50 mM Tris-HCl buffer at pH 7.5, 30 mM DTT, 10 mM MgCl2, 100 ΞΌM AdoCbl, 1 mM ATP, 1 mM CTP, and 2 ΞΌM of NrdJa and NrdJb |
| GO:0004748 ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor | IBA GO_REF:0000033 | MODIFY | Summary: IBA from PTN000171476, a class II RNR node of PTHR43371, whose IBD rests only on this gene's PseudoCAP IDA row (39 leaf IBAs over 33 taxa). This gene appearing in its own WITH/FROM reflects that experimental grounding exists on the target itself, and is expected rather than circular. Reason: The clade assignment is right and NrdJa is a genuine cobalamin-dependent ribonucleotide reductase, so the node is correctly placed; but the propagated term over-specifies on both of its qualifying clauses. The purified P. aeruginosa enzyme reduces CTP more than 60-fold faster than CDP, so the triphosphate term GO:0008998 describes its main reaction, and the acceptor was not thioredoxin in the purified-enzyme assays (DTT and TCEP were used; the reductant of the 2005 extract assays is unchecked, abstract only). PTHR43371 mixes NDP-specific and NTP-specific members, including several rtpR adenosylcobalamin-dependent ribonucleoside-triphosphate reductases, so propagating only the diphosphate term asserts a substrate specificity the node cannot support. GO offers no cobalamin-dependent RNR term that is neutral between NDP and NTP below GO:0016728, so the node should carry both terms. The correction is lateral, not a refinement, and the diphosphate reaction is real for this enzyme, just minor, so the row is retained with the triphosphate term proposed alongside rather than removed. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000171476 Β· PTN000171476 SUPPORTS TRANSFER Class II (vitamin B12-dependent) RNR node of PTHR43371; 39 leaf IBAs over 33 taxa, with this gene the only seed. Membership in the clade is not in doubt: PA5497 has both RNR large-subunit domains, TIGR02504 (NrdJ_Z) and demonstrated cobalamin-dependent RNR activity. The node term is the problem: the family's own member list mixes NDP-specific enzymes with rtpR adenosylcobalamin-dependent ribonucleoside-triphosphate reductases, so a single substrate-specific term cannot be right for all 39 leaves. UniProtKB:Q9HT76 Β· PA5497 nrdJa SUPPORTS TRANSFER The target's own PseudoCAP IDA row (PMID:15722359) is the IBD seed, so it appears in its own WITH/FROM. That is the expected marker of experimental grounding on the target, not circularity. The later purified-enzyme study of the same protein (PMID:26225432) is what shows the seeded term's substrate clause to be the minor reaction. Proposed replacements: ribonucleoside-triphosphate reductase (thioredoxin) activity Supporting Evidence: PMID:26225432 The results revealed that CTP is the preferred substrate with a >60-fold higher activity compared to CDP PMID:26225432 For NrdJa-NrdJb from PA we found that NTP was the favored substrate. file:PSEAE/nrdJa/nrdJa-notes.md its own member list includes several `rtpR` adenosylcobalamin-dependent ribonucleoside-*tri*phosphate reductases |
| GO:0004748 ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor | IDA PMID:15722359 Two proteins mediate class II ribonucleotide reductase activ... | MODIFY | Summary: The PseudoCAP row, and the sole seed of the PTN000171476 IBD. Torrents et al. 2005 show that both NrdJa and NrdJb are needed for class II RNR activity in P. aeruginosa extracts and that adenosylcobalamin rescues hydroxyurea-poisoned cells; it establishes cobalamin-dependent ribonucleotide reductase activity for this gene product. Reason: The activity claim is sound, and NrdJa is the catalytic subunit that carries the cobalamin, substrate and effector sites, so enables is the right relation even though efficient turnover needs NrdJb. The substrate clause is the problem: with purified subunits the enzyme prefers ribonucleoside triphosphates by more than 60-fold, so GO:0008998 is the better term for the main reaction. This is a lateral correction, not a refinement; diphosphate reduction does occur, so the ideal record carries both terms, and the proposed replacement is the one the row is missing. The acceptor clause is untested in the purified-enzyme study (DTT, TCEP), and unchecked for PMID:15722359, whose full text was not read; this is a caveat on both terms. Proposed replacements: ribonucleoside-triphosphate reductase (thioredoxin) activity Supporting Evidence: PMID:15722359 showed for the first time that presence of both NrdJa and NrdJb subunits were PMID:26225432 the full catalytic machinery for NTP reduction, including AdoCbl cofactor binding, resides within the NrdJa subunit, and that the main role of NrdJb is to provide thiol-disulfide exchange PMID:26225432 The results revealed that CTP is the preferred substrate with a >60-fold higher activity compared to CDP |
| GO:0004748 ribonucleoside-diphosphate reductase activity, thioredoxin disulfide as acceptor | IEA GO_REF:0000120 | MODIFY | Summary: RuleBase/InterPro/EC/Rhea mapping of the class II RNR family to EC 1.17.4.1 (the diphosphate reaction). Reason: Same claim and same over-specification as the IDA and IBA rows: the reaction the gene mostly catalyses is the triphosphate one. Consistent with those rows, the better term is GO:0008998, with the diphosphate reaction retained as the minor activity. Proposed replacements: ribonucleoside-triphosphate reductase (thioredoxin) activity Supporting Evidence: file:PSEAE/nrdJa/nrdJa-uniprot.txt Belongs to the ribonucleoside diphosphate reductase class-2 PMID:26225432 For NrdJa-NrdJb from PA we found that NTP was the favored substrate. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO from the RNR large-subunit N-terminal domain (IPR013509), which is the allosteric specificity site. Reason: Directly supported: ATP is the allosteric effector in the standard assay of the purified P. aeruginosa enzyme, activity responds to ATP concentration, and the effector site is in NrdJa. Non-catalytic but integral to the core activity. Supporting Evidence: PMID:26225432 50 mM Tris-HCl buffer at pH 7.5, 30 mM DTT, 10 mM MgCl2, 100 ΞΌM AdoCbl, 1 mM ATP, 1 mM CTP, and 2 ΞΌM of NrdJa and NrdJb PMID:26225432 the full catalytic machinery for NTP reduction, including AdoCbl cofactor binding, resides within the NrdJa subunit, and that the main role of NrdJb is to provide thiol-disulfide exchange |
| GO:0009263 deoxyribonucleotide biosynthetic process | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO process term for the RNR large subunit. Reason: NrdJa itself catalyses the reduction that makes a deoxyribonucleotide, so it does the work of this process. In vivo relevance is shown by adenosylcobalamin rescue of hydroxyurea-poisoned cells, where class I RNR is inactivated and the class II enzyme must supply the dNTP pool. Supporting Evidence: PMID:15722359 Hydroxyurea treatment known to knock out the activity PMID:15722359 class II RNRs, was added to the rich medium. PMID:26225432 the specific activity of the enzyme was determined to be 1.5 nmol/min/mg (with respect to the NrdJa subunit) |
| GO:0031419 cobalamin binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO from the NrdJ/NrdZ signature; class II RNRs use adenosylcobalamin. Reason: Experimentally localised to this subunit: adenosylcobalamin binding resides in NrdJa, and the activity of NrdJa alone, as well as of the complex, saturates with AdoCbl. This cofactor binding is part of the core catalytic mechanism (radical generation). Supporting Evidence: PMID:26225432 the full catalytic machinery for NTP reduction, including AdoCbl cofactor binding, resides within the NrdJa subunit, and that the main role of NrdJb is to provide thiol-disulfide exchange PMID:26225432 Class II RNRs utilize the B12 coenzyme adenosylcobalamin (AdoCbl) as a radical generator. |
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Download this section (compressed HTML)Q: Which thiol redox protein (thioredoxin, glutaredoxin or an NrdH-like protein) is the physiological electron donor to the P. aeruginosa NrdJa-NrdJb enzyme? Both published assays used DTT, so the "thioredoxin disulfide as acceptor" clause in GO:0004748 and GO:0008998 is an inference rather than a measurement.
Q: Should GO provide a cobalamin-dependent ribonucleotide reductase term that is neutral with respect to the phosphorylation state of the substrate, so that PAINT nodes spanning NDP- and NTP-specific class II enzymes (such as PTN000171476 in PTHR43371) need not commit to one?
Q: Is the split architecture a regulatory device, i.e. is NrdJb limiting in vivo under any condition, so that the cell can tune class II RNR activity by subunit availability?
Experiment: Assay purified NrdJa-NrdJb with the P. aeruginosa thioredoxin and glutaredoxin systems (plus NADPH and the corresponding reductases) in place of DTT, with CTP and CDP in parallel, to identify the physiological reductant and to quantify the NTP/NDP preference under physiological reducing conditions.
Experiment: Measure dNTP pools and mutation rates in nrdJa, nrdJb and nrdJab deletion strains under aerobic, microaerobic and hydroxyurea-stressed growth, with and without cobalamin supplementation, to test the proposed DNA-repair and low-oxygen replication role.
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