Thymidylate synthase (TYMS, EC 2.1.1.45) catalyses the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to 2'-deoxythymidine-5'-monophosphate (dTMP), using (6R)-5,10-methylenetetrahydrofolate as both the one-carbon donor and the reductant, which is oxidised in the reaction to 7,8-dihydrofolate (DHF; regenerated by dihydrofolate reductase). This is the sole de novo route to thymidylate and is essential for DNA replication and repair. The enzyme is a cytosolic homodimer with an active-site cysteine nucleophile; a de novo thymidylate synthesis multienzyme complex (with SHMT1/SHMT2 and DHFR/DHFRL1) also operates in the nucleus at replication forks during S phase and in mitochondria, supplying dTMP for nuclear and mitochondrial DNA. TYMS is a central chemotherapy target, inhibited by the 5-fluorouracil metabolite FdUMP and by antifolates such as raltitrexed and pemetrexed. Beyond catalysis, TYMS moonlights as an RNA-binding translational repressor, binding its own mRNA to autoregulate its translation.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0006231 dTMP biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic (IBA) propagation of the deeply conserved role of thymidylate synthase in de novo dTMP synthesis. This is the core biological process of TYMS and is directly supported by experimental annotations and by the enzyme's reaction. Reason: Correct core BP for TYMS; consistent with the catalysed reaction (dUMP -> dTMP) and the UniProt PATHWAY assignment. Well supported at the right level of specificity by orthologs across the thymidylate synthase family. Supporting Evidence: file:human/TYMS/TYMS-uniprot.txt PATHWAY: Pyrimidine metabolism; dTTP biosynthesis. |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA propagation of a mitochondrial location. A de novo dTMP synthesis pathway (SHMT2, TYMS, DHFRL1) operates in mammalian mitochondria and TYMS is experimentally localized there, so a mitochondrial annotation is supported, though it is a secondary compartment relative to the cytosol. Reason: TYMS was experimentally localized to mitochondria (matrix and inner membrane) as part of a mitochondrial de novo dTMP pathway, so the IBA location is defensible. It is a non-core compartment relative to the predominant cytosolic/nuclear activity. Supporting Evidence: PMID:21876188 Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane |
| GO:0004799 thymidylate synthase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA propagation of thymidylate synthase catalytic activity, the defining molecular function of TYMS and its orthologs. Directly corroborated by multiple experimental (IDA) annotations and by the UniProt catalytic-activity record (EC 2.1.1.45, RHEA:12104). Reason: This is the core molecular function of TYMS and is unambiguous across the family. Correct term at the correct level of specificity. Supporting Evidence: file:human/TYMS/TYMS-uniprot.txt Catalyzes the reductive methylation of 2'-deoxyuridine 5'- |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: IBA propagation of a cytosolic location. Thymidylate synthase is classically a cytosolic enzyme, consistent with Reactome and with the general subcellular distribution of nucleotide biosynthetic enzymes. Reason: Cytosol is the principal site of TYMS activity and is well established. This is the core cellular component annotation. Supporting Evidence: Reactome:R-HSA-73605 Cytosolic thymidylate synthase catalyzes the reaction of dUMP and N5,N10-methylene tetrahydrofolate to form TMP and dihydrofolate |
| GO:0004799 thymidylate synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic (multiple-method) assignment of thymidylate synthase activity based on InterPro/ARBA signatures, RHEA:12104 and EC 2.1.1.45. Redundant with, and fully consistent with, the experimental and IBA annotations of the same term. Reason: Correct MF; the InterPro/RHEA/EC mapping is accurate for this well-characterized enzyme. Supporting Evidence: file:human/TYMS/TYMS-uniprot.txt EC=2.1.1.45 |
| GO:0005634 nucleus | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Electronic transfer from the UniProt Subcellular Location vocabulary (nucleus). TYMS localizes to the nucleus during S and G2/M as part of the de novo thymidylate complex at replication forks; this IEA is corroborated by experimental annotations. Reason: Nuclear localization is experimentally documented and cell-cycle dependent; it is a genuine but secondary compartment relative to the bulk cytosolic enzyme. Supporting Evidence: file:human/TYMS/TYMS-uniprot.txt Localized to the nucleus during S |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: Electronic transfer from the UniProt Subcellular Location vocabulary (cytoplasm). This is the general parent of the more specific and better-supported cytosol annotation. Reason: Correct but general; TYMS is a cytoplasmic/cytosolic enzyme. The more specific cytosol term (GO:0005829) is preferred for the core annotation, but this broader term is not wrong. Supporting Evidence: PMID:21876188 Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Electronic transfer from the UniProt Subcellular Location vocabulary (mitochondrion), corroborated by experimental localization of TYMS to mitochondria as part of a mitochondrial de novo dTMP pathway. Reason: Experimentally supported non-core compartment; consistent with the IDA mitochondrion annotation from PMID:21876188. Supporting Evidence: PMID:21876188 de novo dTMP synthesis occurs within mitochondria |
| GO:0005743 mitochondrial inner membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Electronic transfer from the UniProt Subcellular Location vocabulary (mitochondrion inner membrane), corroborated by experimental localization of TYMS to the mitochondrial inner membrane. Reason: Supported by the experimental IDA annotation (PMID:21876188) placing TYMS at the mitochondrial matrix/inner membrane; a non-core compartment. Supporting Evidence: PMID:21876188 Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane |
| GO:0005759 mitochondrial matrix | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Electronic transfer from the UniProt Subcellular Location vocabulary (mitochondrion matrix), corroborated by experimental localization of TYMS to the mitochondrial matrix. Reason: Supported by the experimental IDA annotation (PMID:21876188); a non-core compartment. Supporting Evidence: PMID:21876188 Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane |
| GO:0006231 dTMP biosynthetic process | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO electronic mapping of the thymidylate synthase domain to de novo dTMP biosynthesis. Redundant with, and consistent with, the experimental and IBA annotations of this core process. Reason: Correct core BP; the InterPro-to-GO mapping is accurate for TYMS. Supporting Evidence: file:human/TYMS/TYMS-uniprot.txt PATHWAY: Pyrimidine metabolism; dTTP biosynthesis. |
| GO:0016741 transferase activity, transferring one-carbon groups | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: InterPro2GO electronic mapping to a broad one-carbon transferase parent. This is a general ancestor of the specific and experimentally supported thymidylate synthase activity (GO:0004799) already annotated to TYMS. Reason: Not wrong (TYMS transfers a one-carbon methylene/methyl group), but it is a high-level parent term that is uninformative given the specific GO:0004799 annotation. The specific term should carry the molecular-function annotation. Supporting Evidence: file:human/TYMS/TYMS-uniprot.txt methylenetetrahydrofolate (CH2H4folate) as a 1- |
| GO:0006235 dTTP biosynthetic process | IEA GO_REF:0000041 | ACCEPT | Summary: UniPathway-derived electronic mapping to dTTP biosynthesis. TYMS provides dTMP, the committed de novo precursor that is subsequently phosphorylated to dTTP; UniProt assigns the pathway 'Pyrimidine metabolism; dTTP biosynthesis'. Reason: Correct pathway-level BP; consistent with the UniProt PATHWAY statement. TYMS is the de novo entry point to the dTTP pathway. Supporting Evidence: file:human/TYMS/TYMS-uniprot.txt PATHWAY: Pyrimidine metabolism; dTTP biosynthesis. |
| GO:0005634 nucleus | EXP PMID:22235121 Serine hydroxymethyltransferase anchors de novo thymidylate ... | KEEP AS NON CORE | Summary: Experimental localization of the de novo thymidylate synthesis complex (including TYMS) to the nuclear lamina and to sites of DNA replication initiation during S phase. Reason: Experimentally supported nuclear localization; a genuine but cell-cycle-restricted, non-core compartment relative to the bulk cytosolic enzyme. Supporting Evidence: PMID:22235121 The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen |
| GO:0006260 DNA replication | IDA PMID:22235121 Serine hydroxymethyltransferase anchors de novo thymidylate ... | KEEP AS NON CORE | Summary: TYMS, as part of the de novo thymidylate synthesis complex localized at replication forks, supplies dTMP for DNA replication; the complex is enriched at DNA replication initiation sites and associates with PCNA and the replication machinery. Reason: TYMS supports DNA replication by providing the dTMP building block rather than acting directly in the replication reaction; this is a downstream, contextual process, so it is retained as non-core. Supporting Evidence: PMID:22235121 de novo thymidylate biosynthesis occurs at replication forks |
| GO:0005657 replication fork | IDA PMID:22235121 Serine hydroxymethyltransferase anchors de novo thymidylate ... | KEEP AS NON CORE | Summary: The de novo thymidylate synthesis complex (SHMT1, DHFR, TYMS) localizes to replication forks during DNA synthesis, placing TYMS activity at the fork. Reason: Experimentally supported localization of TYMS activity to replication forks, but this reflects the S-phase nuclear complex rather than the enzyme's core cytosolic site; retained as non-core. Supporting Evidence: PMID:22235121 de novo thymidylate biosynthesis occurs at replication forks |
| GO:0006207 'de novo' pyrimidine nucleobase biosynthetic process | IDA PMID:22235121 Serine hydroxymethyltransferase anchors de novo thymidylate ... | KEEP AS NON CORE | Summary: TYMS contributes to de novo synthesis of the pyrimidine nucleotide thymidylate. The strictly correct process is de novo dTMP/dTTP biosynthesis (already annotated); this term names the broader de novo pyrimidine nucleobase process. Reason: The essence is correct (de novo pyrimidine nucleotide synthesis), but the specific and directly supported processes are dTMP/dTTP biosynthesis (GO:0006231, GO:0006235). Retained as a non-core broader-process annotation rather than removed. Supporting Evidence: PMID:22235121 The de novo thymidylate biosynthetic pathway in mammalian cells translocates to |
| GO:0005515 protein binding | IPI PMID:22235121 Serine hydroxymethyltransferase anchors de novo thymidylate ... | MARK AS OVER ANNOTATED | Summary: IPI capturing physical interaction of TYMS with SHMT1 (P34896) and DHFR (P00374) within the de novo thymidylate synthesis complex. The specific interactions are biologically meaningful, but the GO term 'protein binding' itself is uninformative. Reason: Bare 'protein binding' does not convey a specific molecular function. The underlying SHMT1/DHFR complex interactions are real and are better captured by the complex/subunit description rather than this generic MF term. Supporting Evidence: PMID:22235121 this pathway forms a multienzyme complex that is associated with the nuclear lamina |
| GO:0004799 thymidylate synthase activity | IDA PMID:11278511 Structure of human thymidylate synthase suggests advantages ... | ACCEPT | Summary: Direct assay of human thymidylate synthase catalytic activity, with the 2.0 A crystal structure and kinetics of the dUMP reaction defining the enzyme's active-site mechanism. Reason: Core molecular function, experimentally demonstrated for the human enzyme. Supporting Evidence: PMID:11278511 analogs of its substrates, dUMP and CH(2)H(4)folate |
| GO:0004799 thymidylate synthase activity | IDA PMID:8845352 Crystal structure of human thymidylate synthase: a structura... | ACCEPT | Summary: Human thymidylate synthase catalytic activity supported by the 3.0 A crystal structure characterizing the active-site loop and substrate docking mechanism. Reason: Core molecular function; the structure resolves the human TS active site and confirms the catalytic enzyme. Supporting Evidence: PMID:8845352 The crystal structure of human thymidylate synthase, a target for anti-cancer drugs |
| GO:0006231 dTMP biosynthetic process | IDA PMID:11278511 Structure of human thymidylate synthase suggests advantages ... | ACCEPT | Summary: The catalytic characterization of human TS establishes its role in dTMP biosynthesis via the dUMP reaction. Reason: Core BP directly supported by the human enzyme's catalytic activity. Supporting Evidence: PMID:11278511 analogs of its substrates, dUMP and CH(2)H(4)folate |
| GO:0006231 dTMP biosynthetic process | IDA PMID:8845352 Crystal structure of human thymidylate synthase: a structura... | ACCEPT | Summary: The structural characterization of human TS as an anti-cancer drug target supports its role in dTMP biosynthesis. Reason: Core BP; consistent with the enzyme's dUMP-to-dTMP reaction. Supporting Evidence: PMID:8845352 The crystal structure of human thymidylate synthase, a target for anti-cancer drugs |
| GO:0004799 thymidylate synthase activity | TAS PMID:1924359 Autoregulation of human thymidylate synthase messenger RNA t... | ACCEPT | Summary: Traceable statement of thymidylate synthase catalytic activity (EC 2.1.1.45, 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase) in the context of the autoregulation study. Reason: Core molecular function, correctly stated. Supporting Evidence: PMID:1924359 essential for the de novo synthesis of |
| GO:0000900 mRNA regulatory element binding translation repressor activity | IDA PMID:1924359 Autoregulation of human thymidylate synthase messenger RNA t... | KEEP AS NON CORE | Summary: TS protein binds its own mRNA and represses its translation in vitro; this autoregulatory RNA-binding repressor activity is a documented moonlighting function distinct from its catalytic role. Reason: Experimentally supported moonlighting activity (translational autoregulation), but not the core metabolic function of TYMS; retained as non-core. Supporting Evidence: PMID:1924359 translation of TS mRNA is controlled by its own protein end product, TS, in an autoregulatory manner |
| GO:0017148 negative regulation of translation | IDA PMID:1924359 Autoregulation of human thymidylate synthase messenger RNA t... | KEEP AS NON CORE | Summary: Addition of recombinant TS protein inhibits translation of TS mRNA in vitro, a specific negative regulation of its own translation. Reason: Experimentally supported moonlighting/regulatory role; not the core metabolic function, so retained as non-core. Supporting Evidence: PMID:1924359 recombinant TS protein to in vitro translation reactions inhibited translation of TS mRNA |
| GO:1990825 sequence-specific mRNA binding | IDA PMID:1924359 Autoregulation of human thymidylate synthase messenger RNA t... | KEEP AS NON CORE | Summary: Gel-retardation assays demonstrate a specific interaction between TS protein and its own mRNA but not unrelated mRNAs, supporting sequence-specific mRNA binding. Reason: Experimentally supported RNA-binding moonlighting activity underlying autoregulation; not the core metabolic function. Supporting Evidence: PMID:1924359 a specific interaction between TS protein and its corresponding mRNA but not with unrelated mRNAs |
| GO:0004799 thymidylate synthase activity | IDA PMID:15093541 Thymidylate synthase as an oncogene: a novel role for an ess... | ACCEPT | Summary: TYMS is treated as a catalytically active enzyme essential for DNA synthesis; catalytically dead point mutants lack the transforming activity, tying the phenotype to catalytic thymidylate synthase activity. Reason: Core molecular function; the study depends on and confirms catalytic TS activity. Supporting Evidence: PMID:15093541 ectopic expression of catalytically active |
| GO:0005542 folic acid binding | IC PMID:15093541 Thymidylate synthase as an oncogene: a novel role for an ess... | ACCEPT | Summary: Inferred from thymidylate synthase activity (GO:0004799); the enzyme binds the folate cosubstrate (6R)-5,10-methylenetetrahydrofolate as its one-carbon donor and reductant, consistent with folate binding. Reason: TYMS binds a folate cofactor during catalysis (UniProt records methylenetetrahydrofolate-binding residues), so folate binding is a well-justified molecular function inferred from its catalytic activity. Supporting Evidence: file:human/TYMS/TYMS-uniprot.txt methylenetetrahydrofolate (CH2H4folate) as a 1- |
| GO:0006231 dTMP biosynthetic process | IDA PMID:15093541 Thymidylate synthase as an oncogene: a novel role for an ess... | ACCEPT | Summary: TYMS is characterized as essential for DNA synthesis through de novo thymidylate production; its catalytic role in dTMP biosynthesis is central to the oncogene-like phenotype. Reason: Core BP; supported by the enzyme's essential role in de novo thymidylate synthesis. Supporting Evidence: PMID:15093541 is an E2F1-regulated enzyme that is essential for DNA |
| GO:0035999 tetrahydrofolate interconversion | IDA PMID:15093541 Thymidylate synthase as an oncogene: a novel role for an ess... | ACCEPT | Summary: The TYMS reaction oxidises 5,10-methylenetetrahydrofolate to 7,8-dihydrofolate, participating in the interconversion of folate cofactors that is coupled to dTMP synthesis. Reason: Mechanistically correct - TYMS consumes methylene-THF and produces DHF, a folate-interconversion step tightly linked to the thymidylate cycle. Supporting Evidence: file:human/TYMS/TYMS-uniprot.txt carbon donor and reductant and |
| GO:0071897 DNA biosynthetic process | IC PMID:15093541 Thymidylate synthase as an oncogene: a novel role for an ess... | KEEP AS NON CORE | Summary: Inferred (IC) from thymidylate synthase activity - by supplying dTMP/dTTP, TYMS enables DNA biosynthesis. This is a broad downstream process rather than a direct activity of the enzyme. Reason: TYMS enables DNA synthesis by providing the thymidine nucleotide precursor rather than participating directly in DNA polymerization; retained as a non-core downstream process. Supporting Evidence: PMID:15093541 is an E2F1-regulated enzyme that is essential for DNA |
| GO:0005829 cytosol | TAS Reactome:R-HSA-73605 | ACCEPT | Summary: Reactome traceable statement placing thymidylate synthase in the cytosol, where it catalyses the conversion of dUMP to dTMP as a homodimer. Reason: Cytosol is the principal, well-established site of TYMS activity; correct core cellular component. Supporting Evidence: Reactome:R-HSA-73605 Cytosolic thymidylate synthase catalyzes the reaction of dUMP and N5,N10-methylene tetrahydrofolate to form TMP and dihydrofolate |
| GO:0005829 cytosol | TAS Reactome:R-HSA-8962050 | ACCEPT | Summary: Reactome cytosol annotation associated with the TYMS gene expression event (E2F1-regulated expression). Consistent with the enzyme's established cytosolic localization. Reason: Correct core cellular component; redundant with the other cytosol annotations. Supporting Evidence: Reactome:R-HSA-73605 Cytosolic thymidylate synthase catalyzes the reaction of dUMP and N5,N10-methylene tetrahydrofolate to form TMP and dihydrofolate |
| GO:0005634 nucleus | IDA PMID:21876188 Identification of a de novo thymidylate biosynthesis pathway... | KEEP AS NON CORE | Summary: Experimental localization consistent with TYMS's nuclear presence as part of the de novo thymidylate pathway. The primary focus of PMID:21876188 is the mitochondrial pathway; the nuclear/S-phase localization is documented in the companion work PMID:22235121. Reason: Experimentally supported nuclear localization (cell-cycle restricted); a non-core compartment relative to the bulk cytosolic enzyme. Supporting Evidence: PMID:22235121 The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen |
| GO:0005737 cytoplasm | IDA PMID:21876188 Identification of a de novo thymidylate biosynthesis pathway... | ACCEPT | Summary: Experimental (IDA) cytoplasmic localization of TYMS. This is the general parent of the better-supported cytosol annotation. Reason: Correct but general; TYMS is a cytoplasmic/cytosolic enzyme. The specific cytosol term is preferred for the core annotation. Supporting Evidence: PMID:21876188 converted dUMP to dTMP in the presence of NADPH |
| GO:0005739 mitochondrion | IDA PMID:21876188 Identification of a de novo thymidylate biosynthesis pathway... | KEEP AS NON CORE | Summary: Direct experimental localization of TYMS to mitochondria, where it participates in a de novo dTMP synthesis pathway (with SHMT2 and DHFRL1) that protects mtDNA from uracil misincorporation. Reason: Experimentally supported mitochondrial localization; a genuine but non-core compartment for this predominantly cytosolic/nuclear enzyme. Supporting Evidence: PMID:21876188 de novo dTMP synthesis occurs within mitochondria |
| GO:0005743 mitochondrial inner membrane | IDA PMID:21876188 Identification of a de novo thymidylate biosynthesis pathway... | KEEP AS NON CORE | Summary: TYMS was localized to the mitochondrial matrix and inner membrane, confirming the mitochondrial de novo dTMP pathway. Reason: Directly experimentally supported; a non-core compartment. Supporting Evidence: PMID:21876188 Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane |
| GO:0005759 mitochondrial matrix | IDA PMID:21876188 Identification of a de novo thymidylate biosynthesis pathway... | KEEP AS NON CORE | Summary: TYMS was localized to the mitochondrial matrix and inner membrane, part of the mitochondrial de novo dTMP synthesis pathway. Reason: Directly experimentally supported; a non-core compartment. Supporting Evidence: PMID:21876188 Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane |
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Download this section (compressed HTML)Q: Is the moonlighting RNA-binding/translational-repressor activity of TYMS physiologically significant in vivo, or primarily an in vitro/regulatory phenomenon?
Q: What is the quantitative contribution of the mitochondrial de novo dTMP pathway (TYMS/SHMT2/DHFRL1) versus the cytosolic/nuclear pathway to total cellular dTMP?
Experiment: Compartment-specific rescue (cytosolic vs nuclear-targeted vs mitochondrial-targeted TYMS) in TYMS-null cells to dissect the relative importance of each localization for genome integrity.
Experiment: Structure-guided separation-of-function mutants that abolish mRNA binding while retaining catalysis, to test the biological role of translational autoregulation.
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