ThiH is the radical S-adenosyl-L-methionine 2-iminoacetate synthase (tyrosine lyase) of the thiamine thiazole branch in Escherichia coli and other facultative and obligate anaerobes. Using a single [4Fe-4S] cluster that binds and reductively cleaves SAM to a 5'-deoxyadenosyl radical, ThiH cleaves the Calpha-Cbeta bond of L-tyrosine to give 2-iminoacetate (dehydroglycine) and 4-methylphenol (p-cresol). ThiH forms a tight heterodimeric complex with ThiG, the thiazole synthase, which condenses the dehydroglycine with 1-deoxy-D-xylulose 5-phosphate and the thiocarboxylated sulfur carrier ThiS (made by ThiF and the IscS/ThiI sulfur relay) to form the thiazole phosphate moiety of thiamine. ThiH supplies the C2-N3 unit of the thiazole; it does not itself carry out the thiazole cyclization. thiH mutants require the thiazole for thiamine synthesis. In aerobes such as Bacillus subtilis the oxygen-dependent glycine oxidase ThiO replaces ThiH.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: Root catalytic-activity term from the radical SAM signature; ThiH has a specific, experimentally established reaction. Reason: Per the RADICAL_SAM treatment rule, replaced by GO:0036355 2-iminoacetate synthase activity, which the gene carries by IDA, IBA and IEA. Proposed replacements: 2-iminoacetate synthase activity Supporting Evidence: PMID:19923213 ThiH is a tyrosine lyase that cleaves the C alpha-C beta bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. |
| GO:0005506 iron ion binding | IEA GO_REF:0000002 | MODIFY | Summary: ThiH binds iron only as part of its [4Fe-4S] cluster. Reason: Lateral correction to GO:0051539, applying the RADICAL_SAM treatment rule for metal ion binding (GO:0046872) to its child iron ion binding. There is no separate mononuclear iron site; the iron and sulfide analyses of ThiGH are accounted for by the cluster. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:ECOLI/thiH/thiH-uniprot.txt Binds 1 [4Fe-4S] cluster per subunit. The cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:0005515 protein binding | IPI PMID:12650933 Thiamine biosynthesis in Escherichia coli: isolation and ini... | REMOVE | Summary: ThiH co-purifies with ThiG as a stable complex. Reason: The interaction is real, but generic protein binding is uninformative. The ThiG-ThiH association is captured by the accepted complex term GO:1902508 (2-iminoacetate synthase complex), and ThiH's own activity by GO:0036355. Removal does not mean the interaction is false. Supporting Evidence: PMID:12650933 When isolated under anaerobic conditions, ThiG and ThiH-His co-purify as a large multimeric non-covalent complex. |
| GO:0006974 DNA damage response | IEP PMID:11967071 Over 1000 genes are involved in the DNA damage response of E... | MARK AS OVER ANNOTATED | Summary: Expression change of thiH in a genome-wide microarray after mitomycin C treatment, in which nearly 30% of genes changed. Reason: A transcript-level change in a global stress profile does not show that ThiH does any work in the DNA damage response; its only known activity is tyrosine cleavage for thiamine synthesis. Consistent with other E. coli reviews of rows from this paper (pstA, pstS, glgX). Supporting Evidence: PMID:11967071 Unexpectedly, a large number of genes (nearly 30% of all genes) displayed significant changes in their expression level. |
| GO:0009228 thiamine biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: ThiH performs the dehydroglycine-forming step of thiazole synthesis for thiamine. Reason: ThiH catalyses a step of the pathway, so the participation test is met. The node is seeded by E. coli ThiH itself, which reflects its experimental grounding. Supporting Evidence: PMID:17403671 the resultant substrate radical cleaves to yield dehydroglycine, which is required by ThiG for the thiazole cyclization reaction |
| GO:0009228 thiamine biosynthetic process | IEA GO_REF:0000002 | ACCEPT | Summary: Same claim as the IBA row, from the ThiH family signature. Reason: Consistent with the IBA and IMP rows. Supporting Evidence: PMID:8432721 The thiE, thiF, thiG, and thiH gene products are required for synthesis of the thiazole. |
| GO:0009228 thiamine biosynthetic process | IMP PMID:8432721 Structural genes for thiamine biosynthetic enzymes (thiCEFGH... | ACCEPT | Summary: thiH mutants are thiazole auxotrophs. Reason: Genetic necessity evidence, consistent with the biochemically established catalytic step. Supporting Evidence: PMID:8432721 The thiE, thiF, thiG, and thiH gene products are required for synthesis of the thiazole. |
| GO:0009229 thiamine diphosphate biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: ThiH supplies the thiazole C2-N3 unit for thiamine diphosphate, the active cofactor. Reason: Correct; GO:0009229 is not an is_a or part_of relative of GO:0009228, so both are kept. This is the process used for ThiH in the thiamine_diphosphate_biosynthesis module. Supporting Evidence: file:ECOLI/thiH/thiH-uniprot.txt PATHWAY: Cofactor biosynthesis; thiamine diphosphate biosynthesis. |
| GO:0009229 thiamine diphosphate biosynthetic process | IDA PMID:12650933 Thiamine biosynthesis in Escherichia coli: isolation and ini... | ACCEPT | Summary: Isolation and characterisation of the ThiGH complex, which contains the iron-sulfur cluster of ThiH. Reason: Abstract-only cache; the abstract describes complex purification and cluster characterisation rather than an assay of ThDP formation, but the claim is correct and well supported by later reconstitution (PMID:17403671), so I defer to the curator. Supporting Evidence: PMID:12650933 In Escherichia coli, two of the proteins required for the biosynthesis of the thiazole moiety of thiamine (vitamin B(1)) are ThiG and ThiH |
| GO:0009229 thiamine diphosphate biosynthetic process | IEA GO_REF:0000041 | ACCEPT | Summary: UniPathway mapping for thiamine diphosphate biosynthesis. Reason: Consistent with the IBA and IDA rows. Supporting Evidence: PMID:17403671 Genetic evidence has identified thiH, thiG, thiS, and thiF as essential for thiazole biosynthesis in E. coli. |
| GO:0036355 2-iminoacetate synthase activity | IBA GO_REF:0000033 | ACCEPT | Summary: The core ThiH activity, inherited across the ThiH clade. Reason: Node placement is sound; the target appears among its own sources because of its own IDA annotation. Supporting Evidence: PMID:19923213 ThiH is a tyrosine lyase that cleaves the C alpha-C beta bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. |
| GO:0036355 2-iminoacetate synthase activity | IDA PMID:19923213 Catalytic activity of the anaerobic tyrosine lyase required ... | ACCEPT | Summary: Purified ThiGH shows catalytic, multiple-turnover tyrosine cleavage, forming 5'-deoxyadenosine and p-cresol. Reason: The tyrosine lyase reaction is a radical SAM reaction and belongs to the ThiH subunit, the only radical SAM protein in the ThiGH heterodimer; ThiG uses the dehydroglycine product. The assay used the complex because ThiH has been purified only with ThiG, so the activity is also captured at the level of GO:1902508. Supporting Evidence: PMID:19923213 Analysis of the time-dependent formation of the reaction products 5'-deoxyadenosine (DOA) and p-cresol has demonstrated catalytic behavior of the tyrosine lyase. |
| GO:0036355 2-iminoacetate synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: EC/Rhea mapping to the 2-iminoacetate synthase reaction. Reason: Consistent with the IDA and IBA rows. Supporting Evidence: file:ECOLI/thiH/thiH-uniprot.txt FUNCTION: Catalyzes the radical-mediated cleavage of tyrosine to 2- iminoacetate and 4-cresol. |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: Correct but general; the ThiH cluster is a [4Fe-4S] cluster. Reason: Per the RADICAL_SAM treatment rule, refined to GO:0051539, which the gene carries by IDA. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:17403671 Reconstitution of the [4Fe-4S] cluster in ThiH was essential for activity |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:17403671 Thiazole synthase from Escherichia coli: an investigation of... | ACCEPT | Summary: The ThiH [4Fe-4S] cluster was reconstituted and characterised spectroscopically in ThiGH, and is required for activity. Reason: Catalytic radical SAM cluster, core per the RADICAL_SAM treatment rule. Supporting Evidence: PMID:17403671 Reconstitution of the [4Fe-4S] cluster in ThiH was essential for activity |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000002 | ACCEPT | Summary: Same claim as the IDA row, from the ThiH family signature. Reason: Consistent with the IDA row. Supporting Evidence: file:ECOLI/thiH/thiH-uniprot.txt Binds 1 [4Fe-4S] cluster per subunit. The cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:1902508 2-iminoacetate synthase complex | IBA GO_REF:0000033 | ACCEPT | Summary: ThiH acts as part of the ThiGH heterodimer. Reason: Correct. ThiH is the catalytic subunit for the 2-iminoacetate synthase activity named by the complex term; ThiG (P30139) also carries this complex term in GOA and contributes the separate thiazole synthase activity. The node assumes ThiH partners with ThiG throughout the ThiH clade, which is reasonable because thiH and thiG are conserved together. Supporting Evidence: PMID:19923213 Thiazole synthase in Escherichia coli is an alphabeta heterodimer of ThiG and ThiH. |
| GO:1902508 2-iminoacetate synthase complex | IPI PMID:12650933 Thiamine biosynthesis in Escherichia coli: isolation and ini... | ACCEPT | Summary: ThiG and ThiH co-purify as a stable non-covalent complex. Reason: Direct evidence for the ThiGH complex. The complex can equally be described as the thiazole synthase (GO:1902507 thiazole synthase complex), since ThiG in it carries thiazole synthase activity, but the 2-iminoacetate synthase complex term is the one that matches the activity ThiH contributes. Supporting Evidence: PMID:12650933 When isolated under anaerobic conditions, ThiG and ThiH-His co-purify as a large multimeric non-covalent complex. |
| GO:1904047 S-adenosyl-L-methionine binding | IDA PMID:17403671 Thiazole synthase from Escherichia coli: an investigation of... | NEW | Summary: ThiH binds SAM at its [4Fe-4S] cluster and cleaves it to 5'-deoxyadenosine and methionine, with one SAM consumed per tyrosine cleaved. Reason: GOA has no SAM-binding row for ThiH, 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 that ThiH itself performs, so the participation test is met directly. Spectroscopy of ThiGH shows SAM binding to the cluster, and SAM is consumed stoichiometrically with tyrosine. 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:17403671 Spectroscopic studies with ThiGH strongly suggested that S-adenosylmethionine (AdoMet) bound to the [4Fe-4S] cluster, which became more susceptible to reduction to the +1 state. PMID:17403671 for each mole of thiazole-P formed, 1 eq of AdoMet and 1 eq of tyrosine were utilized, and 1 eq of 5'-deoxyadenosine was produced. |
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Download this section (compressed HTML)Q: Is ThiH folded and active without ThiG, and does ThiG simply sequester the unstable dehydroglycine product (which hydrolyses to glyoxylate and ammonium) or also tune ThiH catalysis?
Q: What is the physiological reductant of the ThiH radical SAM cluster in E. coli (flavodoxin FldA with flavodoxin reductase, or another donor)?
Q: Should the ThiGH heterodimer be annotated to the thiazole synthase complex (GO:1902507) as well as the 2-iminoacetate synthase complex (GO:1902508), given that it carries both activities?
Experiment: Express ThiH alone and with catalytically inactive ThiG variants; compare cluster content, SAM cleavage, p-cresol formation and glyoxylate release (uncoupled dehydroglycine hydrolysis) under anaerobic conditions.
Hypothesis: ThiG is required for ThiH folding or for coupling tyrosine cleavage to product channelling.
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