BioB is biotin synthase (EC 2.8.1.6), the radical S-adenosyl-L-methionine (SAM) enzyme that catalyses the last step of biotin biosynthesis: insertion of a sulfur atom between the C6 and C9 carbons of dethiobiotin to close the thiophane ring of biotin. It is a soluble homodimer of 346-residue subunits, each carrying two iron-sulfur clusters. A radical SAM [4Fe-4S] cluster (Cys53, Cys57, Cys60) binds SAM at its unique iron and, after one-electron reduction by the flavodoxin system, cleaves it to methionine and a 5'-deoxyadenosyl radical; two such radicals are used per biotin, abstracting hydrogen atoms first from C9 and then from C6. An auxiliary [2Fe-2S] cluster (Cys97, Cys128, Cys188, Arg260), buried next to the dethiobiotin site, supplies the sulfur atom and is destroyed in the reaction, passing through a 9-mercaptodethiobiotin intermediate. In vitro the enzyme therefore makes less than one biotin per polypeptide, but in cells it is catalytic, relying on the ISC iron-sulfur assembly machinery (the HscA chaperone and IscU scaffold) to rebuild the [2Fe-2S] cluster, and on the Pfs nucleosidase to clear the inhibitory 5'-deoxyadenosine product. Turnover also exposes the protein to proteolytic destruction.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO root catalytic activity from the radical SAM signatures (IPR006638, IPR007197). True, but it names no reaction. Reason: Following the RADICAL_SAM project rule for generic rows: BioB has a defined reaction, biotin synthase activity (EC 2.8.1.6), shown directly for the E. coli enzyme (IDA, PMID:17014080, PMID:8142361), so the root term is replaced by the specific reaction term that the gene already carries. Proposed replacements: biotin synthase activity Supporting Evidence: file:ECOLI/bioB/bioB-uniprot.txt Catalyzes the conversion of dethiobiotin (DTB) to biotin by the insertion of a sulfur atom into dethiobiotin via a radical-based mechanism. PMID:8142361 The bioB gene product we have isolated is active in the conversion of dethiobiotin to biotin in vitro |
| GO:0004076 biotin synthase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA for biotin synthase activity from the biotin synthase node PTN000540961, with E. coli BioB itself, yeast BIO2, S. pombe bio2 and M. tuberculosis BioB among the experimental descendants. Reason: Biotin synthase activity is the defining function of the BioB family. E. coli BioB sits inside the clade, keeps the CxxxCxxC radical SAM motif and all three cysteines plus Arg260 of the [2Fe-2S] site, and has its own direct biochemical evidence (PMID:8142361, PMID:17014080). Its appearance among the IBA sources reflects that experimental grounding. Supporting Evidence: file:ECOLI/bioB/bioB-uniprot.txt Belongs to the radical SAM superfamily. Biotin synthase family. PMID:14704425 Biotin synthase catalyzes the radical-mediated insertion of sulfur into dethiobiotin to form biotin. |
| GO:0004076 biotin synthase activity | IDA PMID:17014080 Biotin synthase mechanism: mutagenesis of the YNHNLD conserv... | ACCEPT | Summary: Lotierzo et al. 2006 assayed wild-type and YNHNLD-motif variants of E. coli BioB for conversion of dethiobiotin to biotin and for SAM cleavage; wild type gave biotin with a 5'-deoxyadenosine to biotin ratio of about 2.8. Reason: Direct in vitro assay of the core reaction with purified E. coli BioB. The paper also shows that the six cluster-ligating cysteines and the YNHNLD motif are required, tying the activity to the radical SAM mechanism. Supporting Evidence: PMID:17014080 Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin (DTB). PMID:17014080 Single-point mutation of each of these six conserved cysteines produced inactive variants. |
| GO:0004076 biotin synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Biotin synthase activity from combined automated methods (InterPro IPR024177, Rhea 22060, EC 2.8.1.6, UniRule). Reason: Consistent with the direct biochemical evidence for E. coli BioB and with the HAMAP family rule. Supporting Evidence: file:ECOLI/bioB/bioB-uniprot.txt Catalyzes the conversion of dethiobiotin (DTB) to biotin by the insertion of a sulfur atom into dethiobiotin via a radical-based mechanism. |
| GO:0005506 iron ion binding | IEA GO_REF:0000104 | MODIFY | Summary: Iron ion binding from a UniRule transfer. Reason: Treated as the RADICAL_SAM rule treats GO:0046872 metal ion binding: a lateral correction, because BioB binds its iron only as part of two iron-sulfur clusters, a [4Fe-4S] radical SAM cluster and a [2Fe-2S] auxiliary cluster (PMID:14967041, PMID:14704425), not as a free iron ion. No separate mononuclear iron site is known. Both cluster terms are listed because both are supported and the gene already carries both. Proposed replacements: 4 iron, 4 sulfur cluster binding 2 iron, 2 sulfur cluster binding Supporting Evidence: PMID:14967041 The results confirm that BioB contains two distinct Fe-S cluster binding sites. |
| GO:0005515 protein binding | IPI PMID:19821612 A complex between biotin synthase and the iron-sulfur cluste... | REMOVE | Summary: Reyda et al. 2009 showed that the ISC Hsp70-type chaperone HscA (P0A6Z1) binds BioB, preferentially when BioB lacks one or both clusters, and improves in vivo assembly of the BioB [2Fe-2S] cluster. Reason: The generic protein binding term is uninformative. The interaction is real and biologically meaningful, but in it BioB is the client apoprotein receiving a cluster; the paper does not establish a BioB molecular function beyond being bound. Removal does not dispute the interaction. The relevant BioB function, binding the [2Fe-2S] cluster that this machinery rebuilds, is captured by GO:0051537. Supporting Evidence: PMID:19821612 In vitro, we show that HscA binds with increased affinity to BioB missing one or both FeS clusters, with a maximum of two HscA molecules per BioB dimer. PMID:19821612 we show that in Escherichia coli, HscA significantly improves the efficiency of the in vivo assembly of the [2Fe-2S](2+) cluster on BioB under conditions of low to moderate iron |
| GO:0005515 protein binding | IPI PMID:24561554 The binary protein-protein interaction landscape of Escheric... | REMOVE | Summary: Yeast two-hybrid interaction with YafJ (Q47147, putative glutamine amidotransferase) from the genome-wide E. coli binary interactome screen. Reason: Uninformative generic term from a high-throughput Y2H screen, with no functional follow-up linking YafJ to biotin synthesis. Removal does not mean the reported interaction is false. Supporting Evidence: PMID:24561554 Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys |
| GO:0009102 biotin biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA for biotin biosynthetic process from the biotin synthase node PTN000540961. Reason: BioB catalyses the final step of the pathway, converting dethiobiotin to biotin, so it does part of the work of the process and passes the participation test. The node placement is sound and E. coli BioB is itself one of the experimental descendants. Supporting Evidence: PMID:17014080 catalyzes the final step of the biotin biosynthetic pathway file:ECOLI/bioB/bioB-uniprot.txt Cofactor biosynthesis; biotin biosynthesis; biotin from 7,8-diaminononanoate |
| GO:0009102 biotin biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Biotin biosynthetic process from combined automated methods (UniRule, InterPro, UniPathway). Reason: Consistent with the experimental evidence that BioB catalyses the terminal step of biotin synthesis. Supporting Evidence: PMID:22326745 Biotin synthase is an S-adenosyl-l-methionine (SAM or AdoMet) radical enzyme that catalyzes reduction of the AdoMet sulfonium to produce 5'-deoxyadenosyl radicals, high-energy carbon radicals that can directly abstract hydrogen atoms from dethiobiotin. |
| GO:0009102 biotin biosynthetic process | IMP PMID:4921685 Lambda phage transduction of the bio A locus of Escherichia ... | ACCEPT | Summary: Biotin biosynthetic process from a classical genetic study of the E. coli bio locus (Rolfe 1970, lambda transduction of the bio genes). Reason: The cached record has no abstract, so the specific bioB mutant assay cannot be checked here. The claim is nonetheless clearly correct: BioB catalyses the last step of biotin synthesis (PMID:8142361, PMID:17014080). Deferring to the curator, who had the full text. Supporting Evidence: PMID:8142361 The bioB gene product we have isolated is active in the conversion of dethiobiotin to biotin in vitro |
| GO:0042803 protein homodimerization activity | IDA PMID:8142361 Biotin synthase: purification, characterization as a [2Fe-2S... | KEEP AS NON CORE | Summary: Sanyal et al. 1994 found that purified BioB runs on native gels as an 82 kDa species, about twice the 38.7 kDa monomer. Reason: BioB is a homodimer: shown by native gel and gel filtration (PMID:8142361, PMID:11444982) and by the crystal structure (PMID:14704425, PDB 1R30, two chains). The dimer is the stable form of the enzyme, but each subunit carries its own clusters and active site, so dimerization is a structural feature rather than the core function. Supporting Evidence: PMID:8142361 One species of this protein runs on native gels with an electrophoretic mobility typical of a protein with m = 82 kDa, suggesting the protein is a dimer PMID:11444982 Biotin synthase (BioB) is aerobically purified as a dimer of 38.4 kDa monomers |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000120 | MODIFY | Summary: Iron-sulfur cluster binding from combined automated methods. Reason: Following the RADICAL_SAM project rule: correct but general. BioB binds a [4Fe-4S] radical SAM cluster and a [2Fe-2S] auxiliary cluster, both shown spectroscopically and structurally for the E. coli enzyme (PMID:14967041, PMID:14704425). The rule names GO:0051539; because BioB also has the [2Fe-2S] cluster, both supported descendants are listed, and the gene already carries both. Proposed replacements: 4 iron, 4 sulfur cluster binding 2 iron, 2 sulfur cluster binding Supporting Evidence: PMID:14704425 The structure places the substrates between the Fe4S4 cluster, essential for radical generation, and the Fe2S2 cluster, postulated to be the source of sulfur |
| GO:0051537 2 iron, 2 sulfur cluster binding | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA for 2 iron, 2 sulfur cluster binding from the biotin synthase node PTN000540961, seeded by E. coli BioB. Reason: The auxiliary [2Fe-2S] cluster, with its unusual Arg ligand, is a conserved feature of the biotin synthase family. E. coli BioB is the experimental donor; a single well-characterised donor is sound support for this node. Supporting Evidence: PMID:17014080 a [2Fe-2S](2+) cluster liganded by Cys-97, Cys-128, Cys-188, and Arg-260 |
| GO:0051537 2 iron, 2 sulfur cluster binding | IDA PMID:14967041 Characterization of the cofactor composition of Escherichia ... | ACCEPT | Summary: Cosper et al. 2004 used UV-visible, resonance Raman and Mossbauer spectroscopy to show a [2Fe-2S] cluster site with partial non-cysteinyl ligation in E. coli BioB, present in vivo. Reason: Core cofactor binding. The [2Fe-2S] cluster is the immediate sulfur donor: it is destroyed during a single turnover while the [4Fe-4S] cluster survives (PMID:11444982, PMID:12873140), and it lies next to dethiobiotin in the crystal structure (PMID:14704425). Without it there is no sulfur insertion, so it is part of the core catalytic function, not a peripheral feature. Supporting Evidence: PMID:14967041 One site accommodates a [2Fe-2S](2+) cluster with partial noncysteinyl ligation PMID:11444982 during catalysis the [4Fe-4S](2+) cluster is preserved while the [2Fe-2S](2+) cluster is destroyed PMID:12873140 the (2Fe-2S)(2+) is the sulfur source for biotin |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:14967041 Characterization of the cofactor composition of Escherichia ... | ACCEPT | Summary: Cosper et al. 2004 showed a second site in E. coli BioB that holds a [4Fe-4S] cluster binding SAM at a unique iron. Reason: Core cofactor binding. The radical SAM [4Fe-4S] cluster (Cys53, Cys57, Cys60) reductively cleaves SAM to the 5'-deoxyadenosyl radical; mutating any of its cysteines abolishes activity (PMID:17014080). The structure shows it in contact with bound SAM (PMID:14704425). Supporting Evidence: PMID:14967041 The other site accommodates a [4Fe-4S](2+,+) cluster that binds S-adenosylmethionine (SAM) at a unique Fe site of the [4Fe-4S](2+) cluster PMID:17014080 a [4Fe-4S](2+) cluster liganded by Cys-53, Cys-57, and Cys-60 and the S-adenosylmethionine (AdoMet or SAM) cosubstrate |
| GO:1904047 S-adenosyl-L-methionine binding | IDA PMID:14704425 Crystal structure of biotin synthase, an S-adenosylmethionin... | NEW | Summary: The E. coli BioB crystal structure was solved with SAM bound, coordinated to the unique iron of the radical SAM cluster; spectroscopy shows the same SAM-cluster interaction in solution. Reason: GOA has no SAM-binding row for BioB, although the RADICAL_SAM project treats GO:1904047 as a core cosubstrate-binding function of every active radical SAM enzyme. This is a molecular function, not a process, so the participation test is met directly: BioB itself binds SAM and cleaves it (two equivalents per biotin). Direct evidence: the co-crystal structure with SAM (PMID:14704425) and Mossbauer evidence for SAM bound at the unique cluster iron (PMID:14967041). 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: PMID:14704425 The crystal structure of biotin synthase from Escherichia coli in complex with S-adenosyl-L-methionine and dethiobiotin has been determined to 3.4 angstrom resolution. PMID:14967041 binds S-adenosylmethionine (SAM) at a unique Fe site of the [4Fe-4S](2+) cluster |
Loading supporting content…
Download this section (compressed HTML)Q: Which E. coli iron-sulfur carriers (IscU with HscA/HscB, NfuA, ErpA, or SUF components) rebuild the BioB [2Fe-2S] cluster after each turnover in vivo, and is a damaged [2Fe-2S] remnant repaired in place or replaced by a newly delivered cluster?
Q: How does catalysis mark BioB for proteolysis in vivo, and which protease is responsible?
Experiment: Measure biotin output per BioB molecule in vivo (dethiobiotin-fed bioB strains expressing tagged BioB at defined levels) in iscU, hscA, nfuA, erpA and suf deletion backgrounds and their combinations, and follow 34S from labelled cluster donors into biotin by mass spectrometry.
Hypothesis: The BioB [2Fe-2S] cluster is rebuilt in vivo by IscU-HscA, with a separate pathway able to substitute.
Type: genetics and isotope labelling
Experiment: Pulse-chase BioB stability during active biotin synthesis in lon, clpP, hslV and ftsH mutant backgrounds, with and without dethiobiotin.
Hypothesis: Catalysis-linked degradation of BioB is carried out by a specific ATP-dependent protease.
Type: biochemical analysis
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)