LipA is the lipoyl synthase of Escherichia coli, the enzyme that completes biosynthesis of the lipoyl cofactor. Acting on an octanoyl chain that LipB has already transferred onto the lipoyl-domain lysine of a target protein, it inserts sulfur atoms at the unactivated C-6 and C-8 positions to give the dithiolane ring of lipoate. It is a radical SAM enzyme: a [4Fe-4S] cluster reductively cleaves S-adenosyl-L-methionine to a 5'-deoxyadenosyl radical that abstracts each hydrogen, and two SAM are consumed per lipoyl group. A second [4Fe-4S] cluster supplies both sulfur atoms and is destroyed in doing so, so continued turnover depends on the iron-sulfur carrier NfuA rebuilding it. Because free octanoate is not a substrate, the cofactor is assembled in place on its carrier; the physiological targets are the E2 subunits of the pyruvate and 2-oxoglutarate dehydrogenase complexes and the H protein of the glycine cleavage system, which cannot function without lipoylation. The protein is cytosolic, and cells lacking lipA cannot grow on glucose minimal medium unless lipoate is supplied.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: True of LipA but uninformative: the enzyme has a defined reaction. Reason: Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md): the root term is true, not an over-annotation, so it is refined rather than flagged. LipA has a defined activity, lipoate synthase (EC 2.8.1.8), which this entry already carries by IDA, so GO:0016992 replaces the root. Proposed replacements: lipoate synthase activity Supporting Evidence: file:ECOLI/lipA/lipA-uniprot.txt Catalyzes the radical-mediated insertion of two sulfur atoms into the C-6 and C-8 positions of the octanoyl moiety bound to the lipoyl domains of lipoate-dependent enzymes PMID:15157071 our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of ... H-protein |
| GO:0005515 protein binding | IPI PMID:21209092 Protein-protein interactions in assembly of lipoic acid on t... | REMOVE | Summary: Co-purification with the intact pyruvate and 2-oxoglutarate dehydrogenase complexes. Real, but bare protein binding says nothing about what LipA does to them. Reason: Removed under the repository's protein-binding policy: bare GO:0005515 is uninformative. The informative content of this interaction is already asserted better elsewhere in this review -- AceF and SucB carry the lipoyl domains that LipA modifies, so they are its substrates, which GO:0016992 states with the chemistry attached. Nothing is lost by dropping the term; what the experiment showed is recorded in the gene notes. Supporting Evidence: PMID:21209092 the intact pyruvate and 2-oxoglutarate dehydrogenase complexes specifically copurify with both LipB and LipA |
| GO:0005515 protein binding | IPI PMID:29051382 Destruction and reformation of an iron-sulfur cluster during... | REMOVE | Summary: Interaction with NfuA, the iron-sulfur cluster carrier that rebuilds LipA's auxiliary cluster between turnovers. Reason: Removed under the protein-binding policy, for the same reason as the other GO:0005515 row. The substance here is cluster regeneration, not adhesion: NfuA re-installs the auxiliary [4Fe-4S] cluster that LipA sacrifices each turnover, which is what makes the enzyme catalytic rather than single-turnover. That mechanism is recorded in the notes and in the core function's description; 'protein binding' captures none of it. Supporting Evidence: PMID:29051382 We found that the Escherichia coli iron-sulfur (Fe-S) cluster carrier protein NfuA efficiently reconstitutes the auxiliary cluster during LipA catalysis in a step that is not rate-limiting. |
| GO:0005737 cytoplasm | IDA PMID:15157071 Lipoyl synthase requires two equivalents of S-adenosyl-L-met... | MODIFY | Summary: Cytoplasmic, shown experimentally, but less specific than the cytosol row this entry also carries. Reason: Correct but general, so MODIFY rather than ACCEPT. LipA is a soluble protein with no transmembrane segment, and the entry already carries GO:0005829 cytosol by IDA from a cytosolic proteome measurement, which is the more specific descendant. GO:0005737 is not one of the five root organelle terms the ORGANELLES policy exempts, so the ordinary refinement rule applies. Proposed replacements: cytosol Supporting Evidence: PMID:18304323 Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. file:ECOLI/lipA/lipA-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | MODIFY | Summary: Electronic cytoplasm assignment; true, and refined by the gene's own cytosol row. Reason: Same refinement as the IDA cytoplasm row: the two rows share a term and must share an action. GO:0005829 is the descendant the gene carries by direct evidence. Proposed replacements: cytosol |
| GO:0005829 cytosol | IDA PMID:18304323 Protein abundance profiling of the Escherichia coli cytosol. | ACCEPT | Summary: Cytosol, from a cytosolic-fraction proteome measurement. This is where the reaction happens. Reason: The most specific location the evidence supports, and the compartment in which the lipoyl cofactor is assembled: LipA's substrates are the lipoyl domains of the soluble 2-oxoacid dehydrogenase complexes and the glycine cleavage H protein. Retained as the core location. Supporting Evidence: PMID:18304323 Using a combination of LC-MS/MS approaches with protein and peptide fractionation steps we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100. file:ECOLI/lipA/lipA-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0009107 obsolete lipoate biosynthetic process | IEA GO_REF:0000120 | MODIFY | Summary: Obsolete term. GO replaced it with protein lipoylation, which this entry already carries. Reason: GO:0009107 is obsolete; its label in the fetched GOA data reads 'obsolete lipoate biosynthetic process' and GO's own replaced_by is GO:0009249 protein lipoylation. The replacement is also the biologically right one here, because the endogenous pathway never makes free lipoate: the sulfurs are inserted into an octanoyl chain already amide-linked to its carrier lysine. Same treatment as human LIAS. Proposed replacements: protein lipoylation Supporting Evidence: file:ECOLI/lipA/lipA-uniprot.txt Catalyzes the radical-mediated insertion of two sulfur atoms into the C-6 and C-8 positions of the octanoyl moiety bound to the lipoyl domains of lipoate-dependent enzymes file:ECOLI/lipA/lipA-uniprot.txt Free octanoate is not a substrate for LipA. file:ECOLI/lipA/lipA-uniprot.txt Protein modification; protein lipoylation via endogenous pathway; protein N(6)-(lipoyl)lysine from octanoyl-[acyl-carrier-protein]: step 2/2. |
| GO:0009107 obsolete lipoate biosynthetic process | IMP PMID:8444795 Lipoic acid metabolism in Escherichia coli: sequencing and f... | MODIFY | Summary: Mutant phenotype placing lipA in lipoate synthesis. The term is obsolete; the finding is not. Reason: Same obsolescence as the IEA row, and the two must be actioned consistently. The underlying genetics is sound and stands: lipA mutants cannot make lipoate. Only the term has moved, to GO:0009249. Note for the record that this paper's model of LipA acting on free octanoate was later superseded -- the substrate is the protein-bound octanoyl chain -- but that revises the mechanism, not this row's placement of lipA in the pathway. Proposed replacements: protein lipoylation Supporting Evidence: PMID:8444795 Cells grown under strictly anaerobic conditions required the lipA and lipB gene products for the synthesis of a functional glycine cleavage system. PMID:8444795 The lipA gene product failed to convert protein-bound octanoic acid moieties to lipoic acid moieties in vivo PMID:29051382 By contrast, E. coli lipA mutants cannot grow under these conditions. file:ECOLI/lipA/lipA-uniprot.txt Free octanoate is not a substrate for LipA. |
| GO:0009249 protein lipoylation | IEA GO_REF:0000104 | ACCEPT | Summary: Protein lipoylation: LipA catalyses the second and final step, converting the protein-bound octanoyl chain into the lipoyl cofactor. Reason: Core process, and the participation test is met directly rather than by necessity: LipA itself performs a step of the lipoylation, inserting both sulfurs into a chain that is already attached to the target lysine. UniProt places it as step 2/2 of the endogenous pathway. This is not a case of the gene merely being required for an outcome someone else produces. Supporting Evidence: file:ECOLI/lipA/lipA-uniprot.txt Protein modification; protein lipoylation via endogenous pathway; protein N(6)-(lipoyl)lysine from octanoyl-[acyl-carrier-protein]: step 2/2. PMID:15157071 the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA PMID:14700636 octanoyl domain and octanoyl-PDH were substrates for sulfur insertion in vitro |
| GO:0016783 sulfurtransferase activity | IEA GO_REF:0000104 | MODIFY | Summary: The right chemistry pitched without the substrate. Reason: Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md), extended to the adjacent generic MF rows: sulfurtransferase activity is true and is an ancestor of GO:0016992, which states the same sulfur insertion with the physiological substrate. Human LIAS MODIFYs this identical row to GO:0016992, and BACSU/lipA and PSEPK/lipA were brought to the same treatment in Phase 3. Proposed replacements: lipoate synthase activity Supporting Evidence: file:ECOLI/lipA/lipA-uniprot.txt Catalyzes the radical-mediated insertion of two sulfur atoms into the C-6 and C-8 positions of the octanoyl moiety bound to the lipoyl domains of lipoate-dependent enzymes |
| GO:0016992 lipoate synthase activity | IDA PMID:15157071 Lipoyl synthase requires two equivalents of S-adenosyl-L-met... | ACCEPT | Summary: Lipoate synthase activity, measured directly: two SAM are consumed per lipoyl group formed on the H protein. Reason: The core molecular function, on the strongest evidence in the file. This paper both establishes the reaction on a physiological protein substrate and fixes the stoichiometry at 2 SAM per lipoyl group, one for each C-H abstraction. EC 2.8.1.8, RHEA:16585. Supporting Evidence: PMID:15157071 our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of ... H-protein PMID:15157071 the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA file:ECOLI/lipA/lipA-uniprot.txt Catalyzes the radical-mediated insertion of two sulfur atoms into the C-6 and C-8 positions of the octanoyl moiety bound to the lipoyl domains of lipoate-dependent enzymes |
| GO:0016992 lipoate synthase activity | IDA PMID:29051382 Destruction and reformation of an iron-sulfur cluster during... | ACCEPT | Summary: Lipoate synthase activity assayed again, in the experiments that showed the auxiliary cluster is rebuilt by NfuA. Reason: Reinforces the same core function from a second direct experiment, and adds the reason the enzyme is catalytic at all: the auxiliary cluster is destroyed each turnover and re-installed by NfuA in a step that is not rate-limiting. Consistent with the other two GO:0016992 rows, as rows sharing a term must be. Supporting Evidence: PMID:29051382 We found that the Escherichia coli iron-sulfur (Fe-S) cluster carrier protein NfuA efficiently reconstitutes the auxiliary cluster during LipA catalysis in a step that is not rate-limiting. |
| GO:0016992 lipoate synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of the reaction that the two IDA rows establish directly. Reason: Correct, and consistent with the two IDA rows for the same term. An electronic row that agrees with direct experimental evidence on the same protein reinforces it; REMOVE is never the way to deduplicate rows that share a term. Supporting Evidence: file:ECOLI/lipA/lipA-uniprot.txt Belongs to the radical SAM superfamily. Lipoyl synthase family. |
| GO:0042803 protein homodimerization activity | IDA PMID:11106496 Escherichia coli LipA is a lipoyl synthase: in vitro biosynt... | MARK AS OVER ANNOTATED | Summary: Rests on LipA purifying as a mixture of monomeric and dimeric species. Not an established functional homodimer. Reason: Not false -- dimeric species were seen -- but an over-annotation of what was observed, which is a monomer/dimer mixture on purification rather than a defined oligomer. The earlier literature did read a homodimer into this: PMID:10747808 states flatly that 'Biotin synthase and lipoate synthase are homodimers' and reports cluster content per dimer, and PMID:15157071 found its AdoMet and sulfur stoichiometries 'consistent with a model in which two LipA proteins are required to synthesize one lipoyl group'. Those reports are superseded on this point, and the later work is what this row should be read against. PMID:15740115 showed that each lipoyl synthase polypeptide supplies both sulfur atoms of one lipoyl group, which retires the inter-subunit sulfur-sharing rationale that made the 1:2 LipA:product stoichiometry look like evidence for an obligate dimer -- though it does not by itself exclude a homodimer whose subunits each run a complete reaction. What the row rests on instead is that no defined homodimer has been established: UniProt still declines to choose, recording 'Monomer or homodimer', LipA alone runs near its monomer mass by molecular-sieve chromatography, and the one defined complex that experiment resolves is a 1:1 LipA-NfuA heterodimer rather than a LipA homodimer. Marked rather than REMOVEd because the IDA observation stands; it is the functional reading that overreaches. The oligomeric state is recorded as an open knowledge gap below. Supporting Evidence: PMID:11106496 The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. PMID:10747808 Biotin synthase and lipoate synthase are homodimers PMID:15157071 consistent with a model in which two LipA proteins are required to synthesize one lipoyl group PMID:15740115 each lipoyl synthase polypeptide contributes both of the sulfur atoms to the intact cofactor PMID:29051382 LipA alone (dotted trace) elutes at 59.6 mL by MSC, exhibiting an experimentally calculated mass of 44.1 kDa (theoretical mass, 38.2 kDa) PMID:29051382 The sample containing both LipA and NfuA (solid trace) shows an elution at 54.6 mL, corresponding to an experimentally calculated mass of 67.2 kDa, suggesting a 1:1 heterodimer of LipA and NfuA file:ECOLI/lipA/lipA-uniprot.txt Monomer or homodimer. |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: Correct but general: both of LipA's clusters are [4Fe-4S]. Reason: Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md): correct but general. LipA binds two [4Fe-4S] clusters per subunit -- the radical SAM cluster and the sacrificial sulfur-donor cluster -- and no [2Fe-2S] cofactor is recorded for the active enzyme, so GO:0051539 is the descendant the protein always satisfies and the one this entry already carries by IDA. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:ECOLI/lipA/lipA-uniprot.txt Note=Binds 2 [4Fe-4S] clusters per subunit. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:15362861 We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:10747808 Iron-sulfur center of biotin synthase and lipoate synthase. | ACCEPT | Summary: Spectroscopic demonstration of a [4Fe-4S] cluster under strict anaerobiosis. Reason: Core cofactor binding. This is the earlier of the two direct cluster studies and reports one [4Fe-4S] per subunit, which the later work refined to two distinct clusters; the term asserted here, [4Fe-4S] cluster binding, is what both show and is unaffected by the revision in count. Supporting Evidence: PMID:10747808 under strict anaerobiosis, lipoate synthase can accommodate a (4Fe-4S) cluster per subunit |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:15362861 Escherichia coli lipoyl synthase binds two distinct [4Fe-4S]... | ACCEPT | Summary: Two distinct [4Fe-4S] clusters per polypeptide, in the form relevant to turnover. Reason: Core cofactor binding, and the definitive measurement for this gene. One cluster reductively cleaves SAM to the 5'-deoxyadenosyl radical; the second is the source of the two sulfur atoms that end up in the lipoyl ring and is consumed doing so. Neither is ancillary to lipoate synthase activity -- the second cluster is part of the product. Supporting Evidence: PMID:15362861 We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. file:ECOLI/lipA/lipA-uniprot.txt Note=Binds 2 [4Fe-4S] clusters per subunit. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic assignment of the cluster the two IDA rows establish. Reason: Correct, and consistent with the two IDA rows for the same term. Supporting Evidence: file:ECOLI/lipA/lipA-uniprot.txt Note=Binds 2 [4Fe-4S] clusters per subunit. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:1904047 S-adenosyl-L-methionine binding | IC file:ECOLI/lipA/lipA-uniprot.txt | NEW | Summary: LipA binds S-adenosyl-L-methionine at the unique iron of its radical SAM [4Fe-4S] cluster and cleaves it to the 5'-deoxyadenosyl radical; two are consumed per lipoyl group, one for each of the C-6 and C-8 hydrogen abstractions. Reason: Not in GOA. Added under the RADICAL_SAM treatment rule, which treats GO:1904047 as a core cosubstrate-binding function of every radical SAM enzyme whose catalysis is established. This is a molecular function the protein itself performs, so the participation test is met directly: LipA binds SAM and cleaves it, rather than merely being required for a process that consumes it. UniProt's HAMAP-curated cofactor note records the exchangeable S-adenosyl-L-methionine on the radical SAM cluster, and the measured stoichiometry is two SAM cleaved irreversibly per lipoyl group. Coded IC because the inference is the reviewer's, drawn from that curated content and the established reaction rather than from a SAM-binding assay on this protein; the independent physiological evidence that LipA is inhibited by accumulating 5'-deoxyadenosine, the product of that cleavage, points the same way. 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. Supporting Evidence: file:ECOLI/lipA/lipA-uniprot.txt Note=Binds 2 [4Fe-4S] clusters per subunit. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:15157071 our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of ... H-protein PMID:15911379 Physiological experiments indicate that lipoic acid synthase, another SAM radical enzyme, is also inhibited by 5'-deoxyadenosine accumulation. |
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Download this section (compressed HTML)Q: Which species of LipA carries out catalysis in the cell -- a monomer, a homodimer, or a complex with an iron-sulfur carrier -- and does the oligomeric state change over the course of a turnover cycle as the auxiliary cluster is consumed and rebuilt?
Q: Which carrier is the second route that rebuilds the LipA auxiliary cluster in vivo, given that nfuA deletion is tolerated -- IscU-HscA/HscB, ErpA or SUF components -- and does the identity of the donor set how many lipoyl groups one LipA molecule can make?
Q: Does LipA discriminate between the lipoyl domains of its several substrate proteins (the 2-oxoacid dehydrogenase E2 subunits and the glycine cleavage H protein), and if so, is the selectivity in the domain fold or in the surrounding sequence?
Experiment: Follow the solution mass of LipA during single-turnover and multiple-turnover reactions with octanoyl-H protein, with and without NfuA, by native mass spectrometry and mass photometry, and compare against the resting enzyme. Test whether a covalently enforced LipA dimer is active and whether a monomer tethered away from any partner is.
Hypothesis: The catalytically relevant form of LipA is a 1:1 complex with an iron-sulfur carrier rather than a LipA homo-oligomer.
Type: biophysical analysis
Experiment: Quantify lipoylated H protein and E2 subunits per LipA molecule in vivo, in strains expressing tagged LipA at defined levels, across nfuA, iscU, hscA, erpA and suf deletion backgrounds and their combinations, and follow 34S from labelled cluster donors into the lipoyl group by mass spectrometry.
Hypothesis: NfuA is the physiological auxiliary-cluster donor, with a redundant pathway able to substitute.
Type: genetics and isotope labelling
Experiment: Measure lipoylation rates on isolated octanoylated lipoyl domains from each E. coli substrate protein and on chimeras that swap the domain core against its flanking sequence.
Hypothesis: LipA selects its substrate lipoyl domains by recognising features outside the conserved domain fold.
Type: biochemical analysis
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The oligomeric state in which LipA turns over is undetermined. Purification gives a monomer/dimer mixture, the early stoichiometric work was read as an obligate homodimer, and the one defined complex molecular-sieve chromatography resolves is a 1:1 LipA-NfuA heterodimer; which of these is the species that converts octanoyl-H protein in the cell is not known.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: The chemistry is settled: both sulfur atoms of one lipoyl group come from the auxiliary [4Fe-4S] cluster of a single LipA polypeptide, and two AdoMet are cleaved irreversibly per lipoyl group. The unknown is the quaternary structure, not the reaction.
Significance: It decides how the GO:0042803 row should be read, and whether the catalytically relevant partner-bound form is a heterodimer with a cluster carrier rather than any homo-oligomer. It also bears on whether the destroyed auxiliary cluster is rebuilt in place within one LipA molecule or requires exchange between subunits.
What would resolve it: Determine the mass and stoichiometry of LipA under turnover conditions, with and without NfuA, by a method that reports on the solution species rather than the purified protein.
Provenance (the field's own admissions):
Gap: Which iron-sulfur carrier rebuilds the LipA auxiliary [4Fe-4S] cluster in vivo is undetermined. Deleting nfuA does not give a severe lipoate-dependent growth defect, so a second route must exist in the cell; which carrier provides it, and whether NfuA is the physiological donor at all rather than one of several competent in vitro, is open.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: That the auxiliary cluster is sacrificed as the sulfur source, and so must be rebuilt for each turnover, is firmly established. So is in vitro competence: NfuA rebuilds it in a step that is not rate-limiting, and [4Fe-4S]-loaded IscU in excess also supports further turnover. What is open is which of them does the job in the cell.
Significance: This is the same question recorded for ECOLI/bioB, whose [2Fe-2S] cluster is consumed in the analogous way, and it governs how many lipoyl groups one LipA molecule can make. A dedicated carrier would make cluster delivery part of the lipoylation mechanism rather than general Fe-S housekeeping.
What would resolve it: Measure lipoylation output per LipA molecule in vivo across carrier deletion backgrounds and follow labelled sulfur from defined cluster donors into the lipoyl group.
Provenance (the field's own admissions):
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