LipA (also known as yutB) is a lipoyl synthase belonging to the radical SAM superfamily that catalyzes the final step of de novo lipoate biosynthesis in Bacillus subtilis. The enzyme inserts two sulfur atoms into the C6 and C8 positions of protein-bound octanoyl groups (attached to lipoyl domains of acceptor proteins such as GcvH and E2 subunits of 2-oxoacid dehydrogenase complexes), converting them to lipoyl cofactors. LipA contains two [4Fe-4S] clusters: a radical-SAM cluster for generating 5'-deoxyadenosyl radicals from S-adenosyl-L-methionine, and an auxiliary cluster that serves as the sacrificial sulfur donor. In B. subtilis, LipA functions in a pathway where LipM first transfers octanoyl from ACP to GcvH, LipA then inserts sulfurs to form lipoyl-GcvH, and LipL transfers the lipoyl moiety to E2 domains. Disruption of lipA causes lipoate auxotrophy and severely impairs growth in minimal medium due to defective lipoate-dependent enzymes, leading to impaired branched-chain fatty acid biosynthesis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: This annotation from InterPro mapping assigns the very general term "catalytic activity" based on the presence of radical SAM domains. LipA is indeed a catalyst (lipoyl synthase, EC 2.8.1.8), but this term is far too general and uninformative. Reason: LipA has well-established lipoyl synthase activity (EC 2.8.1.8). The term "catalytic activity" is overly broad and should be replaced with the specific molecular function term GO:0016992 (lipoate synthase activity) which accurately describes LipA's enzymatic function. Proposed replacements: lipoate synthase activity |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: This annotation assigns cytoplasmic localization based on UniProt subcellular location annotation and UniRule. In B. subtilis (a Gram-positive bacterium lacking membrane-bound organelles), LipA and partner enzymes function in the cytosol where they act on soluble lipoyl domains of central metabolic complexes. Reason: Cytoplasmic localization is correct for B. subtilis LipA. The enzyme acts on cytosolic acceptor proteins including GcvH and E2 subunits of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and branched-chain 2-oxoacid dehydrogenase complexes (Cronan 2024; Christensen 2011). Supporting Evidence: PMID:19820084 The function of lipA was inferred from the results of genetic and physiological experiments file:BACSU/lipA/lipA-deep-research-falcon.md See deep research file for comprehensive analysis |
| GO:0009107 lipoate biosynthetic process | IEA GO_REF:0000120 | MODIFY | Summary: This IEA annotation assigns involvement in lipoate biosynthesis based on InterPro lipoyl synthase domain and UniRule mapping. LipA catalyzes the final step of de novo lipoate biosynthesis by inserting sulfur atoms into octanoyl groups. Reason: LipA is the key enzyme for de novo lipoate biosynthesis, catalyzing sulfur insertion into protein-bound octanoyl groups. This is the core biological process of the enzyme and is well supported by the literature (Cronan 2024; Christensen 2010). GO:0009107 was obsoleted by GO (2026-08-22, replaced_by GO:0009249 protein lipoylation, obsoleted because term usage was inconsistent), so the annotation should move to the replacement term. GOA already carries independent GO:0009249 rows for this gene, so the replacement merges onto an existing annotation rather than asserting a new one. Proposed replacements: protein lipoylation Supporting Evidence: PMID:19820084 We report the characterization of a Bacillus subtilis mutant obtained by disruption of the lipA (yutB) gene, which encodes lipoyl synthase (LipA), the enzyme that catalyzes the final step in the de novo biosynthesis of this cofactor. |
| GO:0009249 protein lipoylation | IEA GO_REF:0000104 | ACCEPT | Summary: This annotation assigns involvement in protein lipoylation based on UniRule transfer. LipA directly modifies protein-bound octanoyl groups on lipoyl domains, converting them to lipoyl groups covalently attached to lysine residues. Reason: LipA functions specifically on protein substrates (octanoyl-GcvH, octanoyl-E2), not free octanoate, and its product is a protein-bound lipoyl cofactor. This annotation accurately reflects the enzyme's role in the post-translational lipoylation of proteins (Douglas 2008; Cronan 2024). Supporting Evidence: PMID:19820084 lipoyl synthase (LipA), the enzyme that catalyzes the final step in the de novo biosynthesis of this cofactor |
| GO:0016740 transferase activity | IEA GO_REF:0000043 | MODIFY | Summary: This annotation assigns transferase activity based on UniProt keyword mapping (Transferase KW-0808). LipA is classified as EC 2.8.1.8, which is a sulfurtransferase that transfers sulfur atoms to octanoyl groups. Reason: Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md): GO:0016740 is true of LipA but two levels above the reaction. LipA has a defined activity, lipoate synthase (EC 2.8.1.8), which the entry already carries, so the general transferase parent is replaced by it rather than retained alongside. Matches the treatment of the same row in STAWA/cfr and of GO:0016783 in human LIAS. Proposed replacements: lipoate synthase activity |
| GO:0016783 sulfurtransferase activity | IEA GO_REF:0000104 | MODIFY | Summary: This annotation assigns sulfurtransferase activity based on UniRule mapping. LipA inserts sulfur atoms from its auxiliary [4Fe-4S] cluster into octanoyl substrates at C6 and C8 positions, making it a sulfurtransferase by definition. Reason: Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md): sulfurtransferase activity is the correct chemistry but omits the substrate. GO:0016992 lipoate synthase activity, which the entry already carries, states the same sulfur insertion on the protein-bound octanoyl group. Human LIAS MODIFYs this identical row to GO:0016992. Proposed replacements: lipoate synthase activity |
| GO:0016992 lipoate synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: This IEA annotation assigns lipoate synthase activity based on InterPro domain mapping and UniRule. This is the correct, specific molecular function term for LipA. Reason: GO:0016992 (lipoate synthase activity) is the most specific and accurate molecular function term for LipA. The enzyme catalyzes sulfur insertion into octanoyl groups to form lipoyl cofactors (EC 2.8.1.8). This is a core function annotation (Cronan 2024). Supporting Evidence: PMID:19820084 lipoyl synthase (LipA), the enzyme that catalyzes the final step in the de novo biosynthesis of this cofactor |
| GO:0046872 metal ion binding | IEA GO_REF:0000043 | MODIFY | Summary: This annotation assigns metal ion binding based on UniProt keyword mapping. LipA binds two [4Fe-4S] clusters which contain iron ions. Reason: Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md): a lateral correction, because LipA binds its iron only as two [4Fe-4S] clusters, not as a free metal ion. UniProt's HAMAP-curated cofactor note records both clusters and no mononuclear metal site, so GO:0051539 (already carried by this entry) replaces the generic parent. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:BACSU/lipA/lipA-uniprot.txt Note=Binds 2 [4Fe-4S] clusters per subunit. One cluster is coordinated file:BACSU/lipA/lipA-uniprot.txt Name=[4Fe-4S] cluster; Xref=ChEBI:CHEBI:49883; |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000120 | MODIFY | Summary: This annotation assigns iron-sulfur cluster binding based on InterPro radical SAM domains and UniProt keyword. LipA contains two distinct [4Fe-4S] clusters essential for its catalytic mechanism. Reason: Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md): correct but general. Both of LipA's clusters are [4Fe-4S] (the radical SAM cluster and the sacrificial sulfur-donor cluster), so GO:0051539 is the descendant the protein always satisfies and entails this parent. Human LIAS and ECOLI/hemN make the same replacement. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:BACSU/lipA/lipA-uniprot.txt Note=Binds 2 [4Fe-4S] clusters per subunit. One cluster is coordinated |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000120 | ACCEPT | Summary: This annotation specifically assigns [4Fe-4S] cluster binding based on InterPro lipoyl synthase domain and UniProt keywords. LipA contains two [4Fe-4S] clusters. Reason: LipA specifically binds [4Fe-4S] clusters, not other iron-sulfur cluster types. One cluster is the radical-SAM cluster (coordinated with 3 cysteines and exchangeable SAM), and the other is the auxiliary cluster that is consumed during catalysis as the sulfur source. Both are [4Fe-4S] clusters (Lee et al. 2008; Cronan 2024). Supporting Evidence: UniProt:O32129 Binds 2 [4Fe-4S] clusters per subunit. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:0009107 lipoate biosynthetic process | IGI PMID:19820084 A lipA (yutB) mutant, encoding lipoic acid synthase, provide... | MODIFY | Summary: This experimental annotation (IGI - Inferred from Genetic Interaction) assigns involvement in lipoate biosynthesis based on the lipA mutant study by Martin et al. (2009). Disruption of lipA interrupted lipoate-dependent reactions in B. subtilis. Reason: The Martin et al. (2009) study directly demonstrated that disruption of lipA eliminates de novo lipoate biosynthesis in B. subtilis, causing lipoate auxotrophy. The IGI evidence is strong experimental support for this core biological process. GO:0009107 was obsoleted by GO (2026-08-22, replaced_by GO:0009249 protein lipoylation, obsoleted because term usage was inconsistent), so the annotation should move to the replacement term. GOA already carries independent GO:0009249 rows for this gene, so the replacement merges onto an existing annotation rather than asserting a new one. Proposed replacements: protein lipoylation Supporting Evidence: PMID:19820084 We report the characterization of a Bacillus subtilis mutant obtained by disruption of the lipA (yutB) gene, which encodes lipoyl synthase (LipA), the enzyme that catalyzes the final step in the de novo biosynthesis of this cofactor. |
| GO:0009249 protein lipoylation | IGI PMID:19820084 A lipA (yutB) mutant, encoding lipoic acid synthase, provide... | ACCEPT | Summary: This experimental annotation assigns involvement in protein lipoylation based on the lipA mutant study. The loss of LipA function led to interruption of lipoate-dependent reactions, which require lipoylated proteins. Reason: The Martin et al. (2009) study showed that lipA disruption interrupts lipoate-dependent reactions, which implies loss of protein lipoylation. LipA acts directly on protein-bound octanoyl substrates to generate protein-bound lipoyl cofactors. Supporting Evidence: PMID:19820084 Interrupting lipoate-dependent reactions strongly inhibits growth in minimal medium |
| GO:0016992 lipoate synthase activity | IGI PMID:19820084 A lipA (yutB) mutant, encoding lipoic acid synthase, provide... | ACCEPT | Summary: This experimental annotation assigns lipoate synthase activity based on the lipA mutant study. The gene was identified as encoding the lipoyl synthase responsible for the final step in lipoate biosynthesis. Reason: The Martin et al. (2009) study identified lipA (yutB) as encoding lipoyl synthase and demonstrated through mutant analysis that the gene product is required for lipoate biosynthesis, consistent with lipoate synthase activity. Supporting Evidence: PMID:19820084 the lipA (yutB) gene, which encodes lipoyl synthase (LipA), the enzyme that catalyzes the final step in the de novo biosynthesis of this cofactor |
| GO:1904047 S-adenosyl-L-methionine binding | IC file:BACSU/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 SAM are consumed per lipoyl group formed. 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 curated cofactor note records the exchangeable S-adenosyl-L-methionine on the radical SAM cluster and the entry's binding features name the 4Fe-4S-S-AdoMet ligand, so the claim rests on curated record content rather than on an electronic GO line. Coded IC because the inference is the reviewer's, drawn from that curated content and the established lipoate synthase activity rather than from a SAM-binding assay on this protein. 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. That is also why those five genes have an existing row to ACCEPT or MODIFY while this one needs a NEW row. Supporting Evidence: file:BACSU/lipA/lipA-uniprot.txt Note=Binds 2 [4Fe-4S] clusters per subunit. One cluster is coordinated file:BACSU/lipA/lipA-uniprot.txt cysteines and an exchangeable S-adenosyl-L-methionine file:BACSU/lipA/lipA-uniprot.txt /ligand_note="4Fe-4S-S-AdoMet" |
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Q: What is the order of sulfur insertion (C6 first or C8 first) for B. subtilis LipA?
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