THI4 (YGR144W) encodes thiamine thiazole synthase (ADP-thiazole synthase, EC 2.4.2.60), the single enzyme that forms the thiazole moiety of thiamine (vitamin B1) in Saccharomyces cerevisiae and other eukaryotes. Thi4 condenses NAD+ and glycine into the adenylated thiazole intermediate ADP-5-ethyl-4-methylthiazole-2-carboxylate (ADT), releasing nicotinamide; the thiazole sulfur is donated by its own active-site Cys205, which is converted to dehydroalanine in an Fe(II)-dependent sulfide transfer. Each Thi4 polypeptide therefore performs a single turnover (a suicide enzyme). Thi4 forms a homo-octamer (tetramer of dimers), binds one iron per subunit, is one of the most abundant THI-regulon proteins under thiamine limitation, and is strongly repressed by thiamine. It is cytosolic (also detected in the nucleus); thi4 mutants are thiamine auxotrophs rescued by hydroxyethylthiazole. Additional roles in stress tolerance and mitochondrial DNA damage tolerance have been reported.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005506 iron ion binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA from THI4 family node (Methanococcus Thi4 Q58018 and yeast Thi4 donors). Iron is a required catalytic cofactor of the sulfide transfer; this is a cofactor-binding aspect of the thiazole synthase activity, not an independent function. Supporting Evidence: PMID:22031445 These observations suggested that the THI4p catalyzed sulfur incorporation reactions are iron-dependent. |
| GO:0005506 iron ion binding | IEA GO_REF:0000104 | KEEP AS NON CORE | Summary: UniRule (HAMAP MF_03158) iron ion binding. Correct cofactor binding, non-core. Supporting Evidence: PMID:22031445 These observations suggested that the THI4p catalyzed sulfur incorporation reactions are iron-dependent. |
| GO:0005634 nucleus | EXP PMID:16850348 Pdc2 coordinates expression of the THI regulon in the yeast ... | KEEP AS NON CORE | Summary: Mojzita & Hohmann 2006 report nuclear as well as cytoplasmic Thi4 (UniProt cites this for the nucleus). Abstract-only cache; deferring to curator. Catalytic function is cytosolic, so nuclear presence is non-core (possibly related to reported DNA-damage-tolerance roles). Supporting Evidence: file:yeast/THI4/THI4-uniprot.txt ECO:0000269|PubMed:16850348}. Nucleus {ECO:0000269|PubMed:16850348}. |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: UniProt subcellular-location mapping (SL-0191) derived from the same nuclear observation. Non-core. Supporting Evidence: file:yeast/THI4/THI4-uniprot.txt ECO:0000269|PubMed:16850348}. Nucleus {ECO:0000269|PubMed:16850348}. |
| GO:0005737 cytoplasm | EXP PMID:15544818 Neurospora crassa CyPBP37: a cytosolic stress protein that i... | ACCEPT | Summary: Faou & Tropschug 2004 cellular fractionation shows Thi4 is cytosolic. Supporting Evidence: PMID:15544818 Cellular fractionation studies revealed that Thi4p is a cytosolic protein in S.cerevisiae, like its ortholog CyPBP37 in N.crassa. |
| GO:0005737 cytoplasm | EXP PMID:16850348 Pdc2 coordinates expression of the THI regulon in the yeast ... | ACCEPT | Summary: Mojzita & Hohmann 2006 localisation of Thi4 to the cytoplasm (cited by UniProt). Accepted. Supporting Evidence: file:yeast/THI4/THI4-uniprot.txt -!- SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:15544818, |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA (ARBA, UniProt SubCell SL-0086). Correct; cytosol is the more specific term also annotated. Supporting Evidence: PMID:15544818 Cellular fractionation studies revealed that Thi4p is a cytosolic protein in S.cerevisiae, like its ortholog CyPBP37 in N.crassa. |
| GO:0005829 cytosol | IDA PMID:15544818 Neurospora crassa CyPBP37: a cytosolic stress protein that i... | ACCEPT | Summary: Fractionation places Thi4 in the soluble cytosolic fraction; thiamine thiazole synthesis occurs in the cytosol. Supporting Evidence: PMID:15544818 Cellular fractionation studies revealed that Thi4p is a cytosolic protein in S.cerevisiae, like its ortholog CyPBP37 in N.crassa. |
| GO:0005829 cytosol | IDA PMID:16850348 Pdc2 coordinates expression of the THI regulon in the yeast ... | ACCEPT | Summary: Cytosolic Thi4 in Mojzita & Hohmann 2006 (abstract-only cache; deferring to curator). Supporting Evidence: file:yeast/THI4/THI4-uniprot.txt -!- SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:15544818, |
| GO:0005829 cytosol | IEA GO_REF:0000104 | ACCEPT | Summary: UniRule MF_03158 cytosol; correct. Supporting Evidence: PMID:15544818 Cellular fractionation studies revealed that Thi4p is a cytosolic protein in S.cerevisiae, like its ortholog CyPBP37 in N.crassa. |
| GO:0005829 cytosol | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways PWY3O-17 cytosolic step; consistent with experimental data. Supporting Evidence: PMID:15544818 Cellular fractionation studies revealed that Thi4p is a cytosolic protein in S.cerevisiae, like its ortholog CyPBP37 in N.crassa. |
| GO:0008198 ferrous iron binding | IDA PMID:22031445 Saccharomyces cerevisiae THI4p is a suicide thiamine thiazol... | KEEP AS NON CORE | Summary: Chatterjee et al. 2011 showed the sulfide-transfer reaction is iron dependent with Fe(II) as the active oxidation state, and UniProt records one Fe per subunit. Correct but a cofactor-binding facet of the core activity. Supporting Evidence: PMID:22031445 The oxygen sensitivity of the reaction suggests that Fe+2 is the catalytically active iron oxidation state. PMID:22031445 These observations suggested that the THI4p catalyzed sulfur incorporation reactions are iron-dependent. |
| GO:0009228 thiamine biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA (ARBA, InterPro IPR027495, UniRule). Correct. Supporting Evidence: PMID:22031445 In contrast, only one gene product is involved in thiazole biosynthesis in eukaryotes (THI4p in Saccharomyces cerevisiae). |
| GO:0009228 thiamine biosynthetic process | IMP PMID:7941734 Regulation of THI4 (MOL1), a thiamine-biosynthetic gene of S... | ACCEPT | Summary: Praekelt et al. 1994: thi4 disruption causes thiamine auxotrophy rescued by hydroxyethylthiazole. Supporting Evidence: PMID:7941734 A thi4::URA3 disruption strain is auxotrophic for thiamine, but can grow in the presence of hydroxyethyl thiazole, indicating that the gene product is involved in the biosynthetic pathway leading to the formation of the thiazole precursor of thiamine. |
| GO:0009228 thiamine biosynthetic process | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways PWY3O-17 membership; correct. Supporting Evidence: PMID:22031445 In contrast, only one gene product is involved in thiazole biosynthesis in eukaryotes (THI4p in Saccharomyces cerevisiae). |
| GO:0016763 pentosyltransferase activity | IEA GO_REF:0000104 | MODIFY | Summary: UniRule pentosyltransferase activity is the direct parent of GO:0160205 (the ADP-ribosyl moiety of NAD+ is transferred to glycine-derived intermediate). Correct but uninformative; the specific child term is already annotated. Proposed replacements: cysteine-dependent adenosine diphosphate thiazole synthase activity Supporting Evidence: PMID:22031445 Here we report the preparation of fully active recombinant wild-type THI4p, the identification of an iron-dependent sulphide transfer reaction from a conserved cysteine residue of the protein to a reaction intermediate and the demonstration that THI4p is a suicide enzyme undergoing only a single turnover. |
| GO:0052837 thiazole biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from the THI4 family node (Methanococcus, S. pombe, S. cerevisiae donors). Thiazole formation is the defining process of the family. Supporting Evidence: PMID:22031445 In contrast, only one gene product is involved in thiazole biosynthesis in eukaryotes (THI4p in Saccharomyces cerevisiae). |
| GO:0052837 thiazole biosynthetic process | IDA PMID:22031445 Saccharomyces cerevisiae THI4p is a suicide thiamine thiazol... | ACCEPT | Summary: Chatterjee et al. 2011 reconstituted ADT (thiazole) formation with purified Thi4. Supporting Evidence: PMID:22031445 The full reconstitution of ADT 5 formation, using purified wtTHI4p, in a reaction mixture lacking any exogenous sulfide donor, coupled with the observed loss of 34 Da from the protein and the structural characterization of a dehydroalanine residue at Cys205 provides compelling evidence that the thiazole sulfur is derived from Cys205 of wtTHI4p. |
| GO:0052837 thiazole biosynthetic process | IEA GO_REF:0000104 | ACCEPT | Summary: UniRule MF_03158; correct. Supporting Evidence: PMID:22031445 In contrast, only one gene product is involved in thiazole biosynthesis in eukaryotes (THI4p in Saccharomyces cerevisiae). |
| GO:0160205 cysteine-dependent adenosine diphosphate thiazole synthase activity | IDA PMID:22031445 Saccharomyces cerevisiae THI4p is a suicide thiamine thiazol... | ACCEPT | Summary: Chatterjee et al. 2011 demonstrated that purified Thi4 forms ADT from NAD+ and glycine using Cys205 as sulfur donor, in a single turnover. Exactly the GO:0160205 reaction; core activity. Supporting Evidence: PMID:22031445 Here we report the preparation of fully active recombinant wild-type THI4p, the identification of an iron-dependent sulphide transfer reaction from a conserved cysteine residue of the protein to a reaction intermediate and the demonstration that THI4p is a suicide enzyme undergoing only a single turnover. PMID:22031445 The full reconstitution of ADT 5 formation, using purified wtTHI4p, in a reaction mixture lacking any exogenous sulfide donor, coupled with the observed loss of 34 Da from the protein and the structural characterization of a dehydroalanine residue at Cys205 provides compelling evidence that the thiazole sulfur is derived from Cys205 of wtTHI4p. |
| GO:0160205 cysteine-dependent adenosine diphosphate thiazole synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA (ARBA, RHEA:55708, EC 2.4.2.60). Correct. Supporting Evidence: PMID:22031445 The full reconstitution of ADT 5 formation, using purified wtTHI4p, in a reaction mixture lacking any exogenous sulfide donor, coupled with the observed loss of 34 Da from the protein and the structural characterization of a dehydroalanine residue at Cys205 provides compelling evidence that the thiazole sulfur is derived from Cys205 of wtTHI4p. |
| GO:0160205 cysteine-dependent adenosine diphosphate thiazole synthase activity | RCA GO_REF:0000123 | ACCEPT | Summary: YeastPathways RCA for 'thiazole synthase + glycine + NAD+ -> ADT + nicotinamide'. Correct. Note that YeastPathways also attaches Thi4 to the subsequent ADT hydrolysis (ADT + H2O -> AMP + thiazole phosphate); no evidence that Thi4 catalyses that step was found and it is not represented by this GO row. Supporting Evidence: PMID:22031445 Here we report the preparation of fully active recombinant wild-type THI4p, the identification of an iron-dependent sulphide transfer reaction from a conserved cysteine residue of the protein to a reaction intermediate and the demonstration that THI4p is a suicide enzyme undergoing only a single turnover. |
Loading supporting content…
Download this section (compressed HTML)Q: Which enzyme converts Thi4's product ADT to the carboxythiazole phosphate used by Thi6 in yeast (YeastPathways assigns this hydrolysis to Thi4 without direct evidence)?
Q: Are the reported stress/mitochondrial DNA damage tolerance phenotypes of thi4 mutants independent of thiamine supply (e.g. via iron sequestration by the abundant spent enzyme)?
Experiment: Incubate purified ADT with yeast cytosolic extracts (and candidate Nudix hydrolases) to identify the ADT-to-thiazole-phosphate hydrolase; test thi4 Cys205Ser for rescue of non-thiamine phenotypes.
Thiazole synthase; abundant protein involved in the formation of the thiazole moiety of thiamine during thiamine biosynthesis; acts more as a co-substrate rather than an enzyme by providing the sulphur source for thiazole formation; undergoes a single turnover only; required for mitochondrial genome stability in response to DNA damaging agents
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)