MiaB is the cytosolic radical S-adenosyl-L-methionine (SAM) methylthiotransferase of Escherichia coli that converts N6-dimethylallyladenosine (i6A) at position 37 of tRNAs reading codons that begin with uridine into 2-methylthio-N6-dimethylallyladenosine (ms2i6A). The reaction inserts a sulfur atom at C2 of the adenine ring and then methylates it, consuming two SAM: one is reductively cleaved to a 5'-deoxyadenosyl radical that abstracts the C2 hydrogen, and the other donates the methyl group. MiaB is a monomer with an N-terminal MTTase domain, a central radical SAM domain and a C-terminal TRAM domain, and binds two [4Fe-4S] clusters: the radical SAM cluster (Cys157, Cys161, Cys164) and an auxiliary cluster in the MTTase domain that activates an exogenous sulfur cosubstrate and is not consumed during turnover. MiaB acts after the prenyltransferase MiaA; in E. coli ms2i6A is the final product, whereas in Salmonella MiaE further hydroxylates it to ms2io6A. The ms2 group stabilises codon-anticodon pairing; miaB mutants lack the modification and show an antisuppressor phenotype but no growth defect under standard conditions. Fe-S cluster loading of MiaB in vivo depends on the carrier NfuA and the monothiol glutaredoxin GrxD.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro radical SAM signature mapping (IPR006638, IPR007197) to the root catalytic activity term. Reason: True but uninformative. Per the RADICAL_SAM treatment rule, catalytic activity is refined to the specific reaction MiaB is shown to catalyse, i6A37 to ms2i6A37 methylthiolation (EC 2.8.4.3, RHEA:37067), GO:0035597, which the gene already carries by IDA, IMP, IBA and IEA. Proposed replacements: tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity Supporting Evidence: PMID:15339930 we have reconstituted in vitro the conversion of i(6)A to ms(2)i(6)A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. file:ECOLI/miaB/miaB-uniprot.txt Catalyzes the methylthiolation of N6-(dimethylallyl)adenosine (i(6)A) |
| GO:0005515 protein binding | IPI PMID:23543739 Physical and functional interactions of a monothiol glutared... | REMOVE | Summary: MiaB physically interacts with the monothiol glutaredoxin GrxD (P0AC69) and the Fe-S carrier NfuA (P63020); NfuA transfers its cluster to MiaB in vitro. Reason: Generic protein binding is uninformative. The interactions are real and biologically meaningful, but they describe Fe-S cluster delivery to MiaB as a client apoprotein: the activity in the interaction belongs to the carrier (NfuA cluster transfer), while MiaB is the acceptor. No more specific MiaB molecular function follows from the interaction data, so the row is removed rather than modified. Removal does not mean the interactions are false. Supporting Evidence: PMID:23543739 We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. PMID:23543739 We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, whereas GrxD is unable to do so. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | MODIFY | Summary: UniProt/HAMAP subcellular location: cytoplasm. Reason: Correct but general. E. coli has no internal compartments that would hold MiaB other than the cytosol, and the protein was detected in the cytosolic fraction (PMID:18304323); the gene already carries cytosol (GO:0005829) by IDA and IBA. Proposed replacements: cytosol Supporting Evidence: PMID:18304323 we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100 file:ECOLI/miaB/miaB-uniprot.txt Cytoplasm {ECO:0000255|HAMAP-Rule:MF_01864} |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000228824 (MiaB/CDK5RAP1 clade) is_active_in cytosol. Reason: MiaB is a soluble monomeric tRNA-modifying enzyme acting on cytosolic tRNA; the IBA agrees with the direct proteomic detection in the E. coli cytosol. The target appearing in WITH/FROM reflects its own experimental cytosol annotation. Supporting Evidence: PMID:18304323 we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100 |
| GO:0005829 cytosol | IDA PMID:18304323 Protein abundance profiling of the Escherichia coli cytosol. | ACCEPT | Summary: Detected in the cytosolic fraction of E. coli MC4100 by LC-MS/MS abundance profiling. Reason: Direct, though high-throughput, evidence that MiaB is a cytosolic protein, consistent with its role on tRNA and its lack of membrane or export signals. Supporting Evidence: PMID:18304323 we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100 |
| GO:0006400 tRNA modification | IEA GO_REF:0000120 | MODIFY | Summary: UniRule/InterPro mapping (UR000086054) to tRNA modification. Reason: True but general. MiaB performs one specific tRNA modification, 2-methylthiolation of i6A37, so the process is refined to tRNA methylthiolation (GO:0035600), which the gene already carries (IBA). Same treatment as the human orthologs CDK5RAP1 and CDKAL1. Proposed replacements: tRNA methylthiolation Supporting Evidence: PMID:10572129 The tRNA from the E. coli miaB strains showed an accumulation of i 6 A, whereas ms 2 i 6 A was absent (data not shown). PMID:15339930 we have reconstituted in vitro the conversion of i(6)A to ms(2)i(6)A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. |
| GO:0016740 transferase activity | IEA GO_REF:0000120 | MODIFY | Summary: UniRule/InterPro mapping to the root transferase activity term. Reason: True but general; refined to the specific activity GO:0035597, which the gene already carries. Same treatment as CDK5RAP1 and CDKAL1. Proposed replacements: tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity Supporting Evidence: PMID:15339930 we have reconstituted in vitro the conversion of i(6)A to ms(2)i(6)A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. |
| GO:0030488 tRNA methylation | IMP PMID:10572129 Identification of the miaB gene, involved in methylthiolatio... | MODIFY | Summary: miaB insertion mutants of S. typhimurium and E. coli accumulate i6A in tRNA and lack ms2i6A/ms2io6A; complementation restores the modification. Reason: The genetic evidence is sound and MiaB does transfer a methyl group from a second SAM onto the newly inserted sulfur (PMID:15339930), so the term is not false. But the process MiaB performs is methylthiolation (sulfur insertion plus S-methylation) at C2 of i6A37, for which GO has the specific term tRNA methylthiolation (GO:0035600). tRNA methylation is not an ancestor of GO:0035600, so this is a lateral correction to the precise process the evidence supports. Note the paper itself argued (from weak AdoMet-motif similarity) that MiaB acts only in thiolation and not methylation; that was later overturned by in vitro reconstitution. Proposed replacements: tRNA methylthiolation Supporting Evidence: PMID:10572129 The tRNA from the E. coli miaB strains showed an accumulation of i 6 A, whereas ms 2 i 6 A was absent (data not shown). PMID:10572129 Taken together, the data from both S. typhimurium and E. coli firmly identifies f474 as the miaB gene. PMID:15339930 This study demonstrates that MiaB protein is a bifunctional system, involved in both thiolation and methylation of i(6)A. |
| GO:0035596 methylthiotransferase activity | IEA GO_REF:0000120 | MODIFY | Summary: ARBA/InterPro mapping to methylthiotransferase activity. Reason: Correct but general; the specific methylthiotransferase activity is GO:0035597 (i6A37 tRNA methylthiotransferase), a descendant the gene already carries. Same treatment as CDK5RAP1. Proposed replacements: tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity Supporting Evidence: PMID:15339930 we have reconstituted in vitro the conversion of i(6)A to ms(2)i(6)A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. file:ECOLI/miaB/miaB-uniprot.txt Belongs to the methylthiotransferase family. MiaB subfamily. |
| GO:0035597 tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000228824 (MiaB subfamily; WITH/FROM includes human CDK5RAP1, mouse Cdk5rap1, T. maritima MiaB and E. coli MiaB itself). Reason: Core molecular function. MiaB converts i6A37 to ms2i6A37 in tRNAs reading UNN codons, using two SAM (one cleaved to 5'-deoxyadenosine, one as methyl donor); the activity was reconstituted in vitro with purified MiaB (PMID:15339930) and is lost in miaB mutants (PMID:10572129). The IBA places the function at the MiaB subfamily node, inside which E. coli MiaB is a founding experimental member. Supporting Evidence: PMID:15339930 we have reconstituted in vitro the conversion of i(6)A to ms(2)i(6)A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. file:ECOLI/miaB/miaB-uniprot.txt Belongs to the methylthiotransferase family. MiaB subfamily. PMID:15339930 In this process, one molecule of AdoMet is converted to 5'-deoxyadenosine, probably through reductive cleavage and intermediate formation ofa5'-deoxyadenosyl radical as observed in other "Radical-AdoMet" enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. |
| GO:0035597 tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity | IDA PMID:11882645 Enzymatic modification of tRNAs: MiaB is an iron-sulfur prot... | ACCEPT | Summary: Purified E. coli MiaB is a [4Fe-4S] protein whose radical SAM cluster cysteines are required for activity. Reason: Core molecular function. MiaB converts i6A37 to ms2i6A37 in tRNAs reading UNN codons, using two SAM (one cleaved to 5'-deoxyadenosine, one as methyl donor); the activity was reconstituted in vitro with purified MiaB (PMID:15339930) and is lost in miaB mutants (PMID:10572129). The IBA places the function at the MiaB subfamily node, inside which E. coli MiaB is a founding experimental member. Supporting Evidence: PMID:11882645 Preliminary site-directed mutagenesis studies suggest that Cys(157), Cys(161), and Cys(164) are involved in iron chelation and that the cluster is essential for activity. PMID:15339930 In this process, one molecule of AdoMet is converted to 5'-deoxyadenosine, probably through reductive cleavage and intermediate formation ofa5'-deoxyadenosyl radical as observed in other "Radical-AdoMet" enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. |
| GO:0035597 tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: UniRule/Rhea mapping (RHEA:37067, EC 2.8.4.3). Reason: Core molecular function. MiaB converts i6A37 to ms2i6A37 in tRNAs reading UNN codons, using two SAM (one cleaved to 5'-deoxyadenosine, one as methyl donor); the activity was reconstituted in vitro with purified MiaB (PMID:15339930) and is lost in miaB mutants (PMID:10572129). The IBA places the function at the MiaB subfamily node, inside which E. coli MiaB is a founding experimental member. Supporting Evidence: file:ECOLI/miaB/miaB-uniprot.txt Catalyzes the methylthiolation of N6-(dimethylallyl)adenosine (i(6)A) PMID:15339930 In this process, one molecule of AdoMet is converted to 5'-deoxyadenosine, probably through reductive cleavage and intermediate formation ofa5'-deoxyadenosyl radical as observed in other "Radical-AdoMet" enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. |
| GO:0035597 tRNA-2-methylthio-N(6)-dimethylallyladenosine(37) synthase activity | IMP PMID:10572129 Identification of the miaB gene, involved in methylthiolatio... | ACCEPT | Summary: miaB mutant tRNA accumulates i6A and lacks ms2i6A; the E. coli miaB+ clone complements. Reason: Core molecular function. MiaB converts i6A37 to ms2i6A37 in tRNAs reading UNN codons, using two SAM (one cleaved to 5'-deoxyadenosine, one as methyl donor); the activity was reconstituted in vitro with purified MiaB (PMID:15339930) and is lost in miaB mutants (PMID:10572129). The IBA places the function at the MiaB subfamily node, inside which E. coli MiaB is a founding experimental member. Supporting Evidence: PMID:10572129 The tRNA from the E. coli miaB strains showed an accumulation of i 6 A, whereas ms 2 i 6 A was absent (data not shown). PMID:10572129 Taken together, the data from both S. typhimurium and E. coli firmly identifies f474 as the miaB gene. PMID:15339930 In this process, one molecule of AdoMet is converted to 5'-deoxyadenosine, probably through reductive cleavage and intermediate formation ofa5'-deoxyadenosyl radical as observed in other "Radical-AdoMet" enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. |
| GO:0035600 tRNA methylthiolation | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000228775 (MTTase family) involved_in tRNA methylthiolation. Reason: Core process: MiaB itself catalyses the methylthiolation step converting i6A37 to ms2i6A37 (participation test met: it is the enzyme). Same term as the CDK5RAP1 and CDKAL1 reviews. Supporting Evidence: PMID:15339930 we have reconstituted in vitro the conversion of i(6)A to ms(2)i(6)A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. PMID:10572129 The tRNA from the E. coli miaB strains showed an accumulation of i 6 A, whereas ms 2 i 6 A was absent (data not shown). |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro radical SAM mapping to iron-sulfur cluster binding. Reason: Per the RADICAL_SAM treatment rule, refined to 4 iron, 4 sulfur cluster binding (GO:0051539), which MiaB carries by IDA: it binds two [4Fe-4S] clusters. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:11882645 UV-visible and EPR spectroscopy of MiaB indicate the presence of a [4Fe-4S] cluster under reducing and anaerobic conditions file:ECOLI/miaB/miaB-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT node PTN000228775 (MTTase family). Reason: Core cofactor binding. MiaB binds two [4Fe-4S] clusters: the radical SAM cluster (CxxxCxxC, Cys157/161/164) that binds and cleaves SAM, and the MTTase N-terminal-domain cluster that activates the exogenous sulfur. Both clusters remain intact during catalysis (PMID:23542644). Supporting Evidence: PMID:11882645 UV-visible and EPR spectroscopy of MiaB indicate the presence of a [4Fe-4S] cluster under reducing and anaerobic conditions PMID:11882645 Preliminary site-directed mutagenesis studies suggest that Cys(157), Cys(161), and Cys(164) are involved in iron chelation and that the cluster is essential for activity. file:ECOLI/miaB/miaB-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:23542644 The biochemical and structural studies reported here demonstrate for the first time that the MTTases MiaB and RimO are true enzymes whose [4Fe-4S] clusters are not sacrificed as sulfur donors. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:11882645 Enzymatic modification of tRNAs: MiaB is an iron-sulfur prot... | ACCEPT | Summary: UV-visible and EPR spectroscopy of purified MiaB show a [4Fe-4S] cluster; Cys157/161/164 mutants lose iron chelation and activity. Reason: Core cofactor binding. MiaB binds two [4Fe-4S] clusters: the radical SAM cluster (CxxxCxxC, Cys157/161/164) that binds and cleaves SAM, and the MTTase N-terminal-domain cluster that activates the exogenous sulfur. Both clusters remain intact during catalysis (PMID:23542644). Supporting Evidence: PMID:11882645 UV-visible and EPR spectroscopy of MiaB indicate the presence of a [4Fe-4S] cluster under reducing and anaerobic conditions PMID:11882645 Preliminary site-directed mutagenesis studies suggest that Cys(157), Cys(161), and Cys(164) are involved in iron chelation and that the cluster is essential for activity. file:ECOLI/miaB/miaB-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:23542644 The biochemical and structural studies reported here demonstrate for the first time that the MTTases MiaB and RimO are true enzymes whose [4Fe-4S] clusters are not sacrificed as sulfur donors. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000120 | ACCEPT | Summary: UniRule/InterPro mapping (MF_01864). Reason: Core cofactor binding. MiaB binds two [4Fe-4S] clusters: the radical SAM cluster (CxxxCxxC, Cys157/161/164) that binds and cleaves SAM, and the MTTase N-terminal-domain cluster that activates the exogenous sulfur. Both clusters remain intact during catalysis (PMID:23542644). Supporting Evidence: PMID:11882645 UV-visible and EPR spectroscopy of MiaB indicate the presence of a [4Fe-4S] cluster under reducing and anaerobic conditions PMID:11882645 Preliminary site-directed mutagenesis studies suggest that Cys(157), Cys(161), and Cys(164) are involved in iron chelation and that the cluster is essential for activity. file:ECOLI/miaB/miaB-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:23542644 The biochemical and structural studies reported here demonstrate for the first time that the MTTases MiaB and RimO are true enzymes whose [4Fe-4S] clusters are not sacrificed as sulfur donors. |
| GO:1904047 S-adenosyl-L-methionine binding | ISS PMID:15339930 MiaB protein is a bifunctional radical-S-adenosylmethionine ... | NEW | Summary: MiaB binds S-adenosyl-L-methionine at the unique iron of its radical SAM [4Fe-4S] cluster and cleaves it to 5'-deoxyadenosine; a second SAM supplies the methyl group of the methylthio product. Reason: Not in GOA. Added per the RADICAL_SAM treatment rule (SAM binding is a core cosubstrate-binding function of every radical SAM enzyme whose catalysis is established) and for consistency with the human MiaB-subfamily ortholog CDK5RAP1, where the same row was added by ISS from this very paper. This is a molecular function, so the participation test is met directly: MiaB itself binds and turns over SAM. Purified MiaB reconstituted in vitro converts one AdoMet to 5'-deoxyadenosine and uses a second as methyl donor (PMID:15339930). Coded ISS rather than IDA deliberately: the cached record of PMID:15339930 is abstract-only and names no source organism, UniProt P0AEI1 does not cite that paper at all (its experimental references are PMID:10572129 and PMID:11882645), and PubMed indexes it under both Escherichia coli Proteins and Thermotoga maritima, so the reconstituted protein cannot be shown from the accessible text to be the E. coli enzyme. What is established for E. coli MiaB specifically is the [4Fe-4S] cluster and its requirement for activity (PMID:11882645), together with UniProt's curated note of an exchangeable SAM on that cluster; SAM binding follows for this protein by similarity to the characterised enzyme. Upgrade to IDA if the full text confirms the organism. 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:15339930 we have reconstituted in vitro the conversion of i(6)A to ms(2)i(6)A within a tRNA substrate using the iron-sulfur MiaB protein, S-adenosylmethionine (AdoMet), and a reducing agent. PMID:15339930 In this process, one molecule of AdoMet is converted to 5'-deoxyadenosine, probably through reductive cleavage and intermediate formation ofa5'-deoxyadenosyl radical as observed in other "Radical-AdoMet" enzymes, and a second molecule of AdoMet is used as a methyl donor as shown by labeling experiments. file:ECOLI/miaB/miaB-uniprot.txt Binds 2 [4Fe-4S] clusters. One cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. PMID:23542644 One cluster binds S-adenosylmethionine and generates an Ado• radical via a well-established mechanism. PMID:11882645 The product of the miaB gene, MiaB, from Escherichia coli participates in the methylthiolation of the adenosine 37 residue |
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Download this section (compressed HTML)Q: What is the physiological sulfur donor for MiaB in E. coli: free sulfide/polysulfide, a persulfide carrier protein, or something else, and is it shared with RimO?
Q: Do the GrxD and NfuA interactions reflect first-time cluster loading of the auxiliary MTTase-domain cluster, repair after oxidative damage, or both?
Experiment: Reconstitute E. coli MiaB with 34S-labelled candidate sulfur donors (sulfide, polysulfide, persulfidated IscS/TusA-type carriers) and quantify ms2i6A formation and label incorporation by LC-MS to identify the physiological sulfur source.
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The physiological sulfur donor for MiaB-catalysed methylthiolation in vivo is unidentified; in vitro, an exogenous sulfur cosubstrate bound to the auxiliary cluster is used, but its cellular source is unknown.
BIOLOGY RESIDUAL_SUBGAP
What is known: MiaB is a true catalyst whose two [4Fe-4S] clusters are not sacrificed; sulfur is activated at an exchangeable site on the auxiliary cluster; two SAM are consumed per ms2i6A formed.
Significance: Determines how ms2i6A formation is coupled to cellular sulfur trafficking and Fe-S biogenesis.
What would resolve it: Isotope-labelled sulfur-donor reconstitution and in vivo labelling in sulfur-carrier mutants.
Provenance (the field's own admissions):
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