Pathway Tools
Intro Tutorial
discounted registration ends Sept 5, 2015 (Sat)
Pathway Tools
Intro Tutorial
discounted registration ends Sept 5, 2015 (Sat)
Pathway Tools
Intro Tutorial
discounted registration ends Sept 5, 2015 (Sat)
Pathway Tools
Intro Tutorial
discounted registration ends Sept 5, 2015 (Sat)
Pathway Tools
Intro Tutorial
discounted registration ends Sept 5, 2015 (Sat)

Escherichia coli K-12 substr. MG1655 Enzyme: thymidylate synthase

Gene: thyA Accession Numbers: EG11002 (EcoCyc), b2827, ECK2823

Regulation Summary Diagram: ?

Regulation summary diagram for thyA

Subunit composition of thymidylate synthase = [ThyA]2
         thymidylate synthase = ThyA

Thymidylate synthase plays a key role in DNA synthesis. The conversion of dUMP to dTMP is the main pathway of de novo dTMP synthesis in the cell [Haertle79, Belfort83]. The enzyme undergoes conformational changes during the initial binding of dUMP, with even larger changes during the binding of the cofactor. The catalytic mechanism has been studied [Agrawal04].

The enzyme has been crystallized [Montfort90]. Crystal structures of mutant forms of the enzyme have also been determined [SoteloMundo06, Roberts06, Newby06].

Detailed studies of the overall mechanism of thymidylate synthase [Kanaan07], and of the proton abstraction and hydride transfer steps using purified wild-type and mutant enzymes [Hong07], have provided new insight into the reaction mechanism. The nature of the dimeric state has also been studied using wild-type and mutant enzymes [Saxl07].

In Escherichia coli K-12 the thyA gene encoding this enzyme contains a natural hotspot for T to A transversion mutations at base 131 of the coding sequence. This hotspot occurs within a 17 base pair quasi-palindrome and a template-switch mechanism of mutagenesis was confirmed and studied in detail [Dutra06].

Immunofluorescence microscopy studies showed E. coli thymidylate synthase to be located throughout the cell, which does not support the existence of a dNTP-synthesizing complex exclusively located near the replication factory [denBlaauwen06].

Inhibitors of the E. coli and human enzymes have been synthesized and studied with the aim of developing antitumor agents. These antifolate inhibitors of thymidylate synthase were novel 2-amino-4-oxo-5-arylthio-substituted-pyrrolo[2,3-d]pyrimidines [Gangjee05].

thyA expression may be directly or indirectly repressed by OmpR [Brombacher03].

Gene Citations: [Gan95]

Locations: cytosol

Map Position: [2,962,383 <- 2,963,177] (63.85 centisomes, 230°)
Length: 795 bp / 264 aa

Molecular Weight of Polypeptide: 30.48 kD (from nucleotide sequence)

pI: 5.99

Unification Links: ASAP:ABE-0009268 , CGSC:104 , DIP:DIP-48261N , EchoBASE:EB0995 , EcoGene:EG11002 , EcoliWiki:b2827 , Entrez-gene:949035 , Mint:MINT-1265510 , ModBase:P0A884 , OU-Microarray:b2827 , PortEco:thyA , Pride:P0A884 , Protein Model Portal:P0A884 , RefSeq:NP_417304 , RegulonDB:EG11002 , SMR:P0A884 , String:511145.b2827 , UniProt:P0A884

Relationship Links: InterPro:IN-FAMILY:IPR000398 , InterPro:IN-FAMILY:IPR020940 , InterPro:IN-FAMILY:IPR023451 , PDB:Structure:1AIQ , PDB:Structure:1AJM , PDB:Structure:1AN5 , PDB:Structure:1AOB , PDB:Structure:1AXW , PDB:Structure:1BDU , PDB:Structure:1BID , PDB:Structure:1BJG , PDB:Structure:1BQ1 , PDB:Structure:1BQ2 , PDB:Structure:1DDU , PDB:Structure:1DNA , PDB:Structure:1EV5 , PDB:Structure:1EV8 , PDB:Structure:1EVF , PDB:Structure:1EVG , PDB:Structure:1F4B , PDB:Structure:1F4C , PDB:Structure:1F4D , PDB:Structure:1F4E , PDB:Structure:1F4F , PDB:Structure:1F4G , PDB:Structure:1FFL , PDB:Structure:1FWM , PDB:Structure:1JG0 , PDB:Structure:1JTQ , PDB:Structure:1JTU , PDB:Structure:1JUT , PDB:Structure:1KCE , PDB:Structure:1KZI , PDB:Structure:1KZJ , PDB:Structure:1NCE , PDB:Structure:1QQQ , PDB:Structure:1SYN , PDB:Structure:1TDU , PDB:Structure:1TJS , PDB:Structure:1TLC , PDB:Structure:1TLS , PDB:Structure:1TRG , PDB:Structure:1TSD , PDB:Structure:1TSN , PDB:Structure:1TYS , PDB:Structure:1ZPR , PDB:Structure:2A9W , PDB:Structure:2BBQ , PDB:Structure:2FTN , PDB:Structure:2FTO , PDB:Structure:2FTQ , PDB:Structure:2g8x , PDB:Structure:2KCE , PDB:Structure:2TSC , PDB:Structure:2vet , PDB:Structure:2vf0 , PDB:Structure:3B5B , PDB:Structure:3B9H , PDB:Structure:3BFI , PDB:Structure:3BGX , PDB:Structure:3BHL , PDB:Structure:3BHR , PDB:Structure:3TMS , PDB:Structure:4F2V , PDB:Structure:4GEV , PDB:Structure:4ISK , PDB:Structure:4KNZ , Pfam:IN-FAMILY:PF00303 , Prints:IN-FAMILY:PR00108 , Prosite:IN-FAMILY:PS00091

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0006231 - dTMP biosynthetic process Inferred by computational analysis [GOA06, GOA01a]
GO:0006235 - dTTP biosynthetic process Inferred by computational analysis [UniProtGOA12]
GO:0006417 - regulation of translation Inferred by computational analysis [UniProtGOA11a]
GO:0009165 - nucleotide biosynthetic process Inferred by computational analysis [UniProtGOA11a]
GO:0032259 - methylation Inferred by computational analysis [UniProtGOA11a]
Molecular Function: GO:0004799 - thymidylate synthase activity Inferred from experiment Inferred by computational analysis [GOA06, GOA01, GOA01a, Haertle79]
GO:0003723 - RNA binding Inferred by computational analysis [UniProtGOA11a]
GO:0008168 - methyltransferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08]
GO:0005737 - cytoplasm Inferred by computational analysis [UniProtGOA11, UniProtGOA11a, GOA06]

MultiFun Terms: metabolism central intermediary metabolism formyl-THF biosynthesis
metabolism central intermediary metabolism nucleotide and nucleoside conversions

Essentiality data for thyA knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 1]
M9 medium with 0.4% glucose No 37 Aerobic 7.2 0.27 No [Patrick07, Comment 2]
M9 medium with 1% glycerol No 37 Aerobic 7.2 0.35 No [Joyce06]
MOPS medium with 0.4% glucose No 37 Aerobic 7.2 0.22 No [Baba06, Comment 1]
No [Feist07, Comment 3]

Last-Curated ? 24-Mar-2008 by Fulcher C , SRI International
Reviewed 02-Mar-2010 by Sarker M

Enzymatic reaction of: thymidylate synthase

Synonyms: 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase

EC Number:

a 5,10-methylene-tetrahydrofolate + dUMP <=> dTMP + a 7,8-dihydrofolate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is irreversible in the direction shown. [Kanaan09]

Alternative Substrates for dUMP: arabinouracil 5'-monophosphate [Haertle79 ] , 2-fluoro-2-deoxy-uridine 5-monophosphate [Haertle79 ]

In Pathways: superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis (E. coli) , superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis , pyrimidine deoxyribonucleotides de novo biosynthesis I , pyrimidine deoxyribonucleotides de novo biosynthesis II , N10-formyl-tetrahydrofolate biosynthesis

Thymidylate synthase catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dTMP) using the cofactor N5,N10-methylenetetrahydrofolate as a donor of both methylene and hydride, and producing 7,8-dihydrofolate. Hydride transfer from the 6S position of tetrahydrofolate is the rate limiting step of the overall reaction [Haertle79, SoteloMundo06, Newby06, Kanaan07, Hong07, Kanaan09].

Cofactors or Prosthetic Groups: Mg2+ [Haertle79]

Activators (Unknown Mechanism): 2-mercaptoethanol [Haertle79]

Inhibitors (Competitive): stereoisomer (-)CH2H4folate [Comment 4]

pH(opt): 7 [BRENDA14, Galova92], 7.5 [BRENDA14, Horinishi72]

Sequence Features

Protein sequence of thymidylate synthase with features indicated

Feature Class Location Citations Comment
Formylation-Modification 1
UniProt: N-formylmethionine; Non-Experimental Qualifier: probable;
Active-Site 146

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Unit:

Transcription-unit diagram


10/20/97 Gene b2827 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11002; confirmed by SwissProt match.


Agrawal04: Agrawal N, Hong B, Mihai C, Kohen A (2004). "Vibrationally enhanced hydrogen tunneling in the Escherichia coli thymidylate synthase catalyzed reaction." Biochemistry 43(7);1998-2006. PMID: 14967040

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Belfort83: Belfort M, Maley G, Pedersen-Lane J, Maley F (1983). "Primary structure of the Escherichia coli thyA gene and its thymidylate synthase product." Proc Natl Acad Sci U S A 1983;80(16);4914-8. PMID: 6308660

BRENDA14: BRENDA team (2014). "Imported from BRENDA version existing on Aug 2014."

Brombacher03: Brombacher E, Dorel C, Zehnder AJ, Landini P (2003). "The curli biosynthesis regulator CsgD co-ordinates the expression of both positive and negative determinants for biofilm formation in Escherichia coli." Microbiology 149(Pt 10);2847-57. PMID: 14523117

denBlaauwen06: den Blaauwen T, Aarsman ME, Wheeler LJ, Nanninga N (2006). "Pre-replication assembly of E. coli replisome components." Mol Microbiol 62(3);695-708. PMID: 16999830

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

Dutra06: Dutra BE, Lovett ST (2006). "Cis and trans-acting effects on a mutational hotspot involving a replication template switch." J Mol Biol 356(2);300-11. PMID: 16376936

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Galova92: Galova M, Koptidesova D, Rusznakova D, Racay P, Kollarova M (1992). "Characteristics of NADPH-dependent thymidylate synthetase purified from Streptomyces aureofaciens." Arch Biochem Biophys 296(1);81-7. PMID: 1605647

Gan95: Gan K, Sankaran K, Williams MG, Aldea M, Rudd KE, Kushner SR, Wu HC (1995). "The umpA gene of Escherichia coli encodes phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (lgt) and regulates thymidylate synthase levels through translational coupling." J Bacteriol 1995;177(7);1879-82. PMID: 7896715

Gangjee05: Gangjee A, Jain HD, Kisliuk RL (2005). "Novel 2-amino-4-oxo-5-arylthio-substituted-pyrrolo[2,3-d]pyrimidines as nonclassical antifolate inhibitors of thymidylate synthase." Bioorg Med Chem Lett 15(9);2225-30. PMID: 15837298

GOA01: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Haertle79: Haertle T, Wohlrab F, Guschlbauer W (1979). "Thymidylate synthetase from Escherichia coli K12. Purification, and dependence of kinetic properties on sugar conformation and size of the 2' substituent." Eur J Biochem 1979;102(1);223-30. PMID: 42538

Hong07: Hong B, Maley F, Kohen A (2007). "Role of Y94 in proton and hydride transfers catalyzed by thymidylate synthase." Biochemistry 46(49);14188-97. PMID: 17999469

Horinishi72: Horinishi H, Greenberg DM (1972). "Purification and properties of thymidylate synthase from calf thymus." Biochim Biophys Acta 258(3);741-52. PMID: 5017698

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Kanaan07: Kanaan N, Marti S, Moliner V, Kohen A (2007). "A quantum mechanics/molecular mechanics study of the catalytic mechanism of the thymidylate synthase." Biochemistry 46(12);3704-13. PMID: 17328531

Kanaan09: Kanaan N, Martí S, Moliner V, Kohen A (2009). "QM/MM Study of Thymidylate Synthase: Enzymatic Motions and the Temperature Dependence of the Rate Limiting Step (dagger)." J Phys Chem A. PMID: 19182971

Montfort90: Montfort WR, Perry KM, Fauman EB, Finer-Moore JS, Maley GF, Hardy L, Maley F, Stroud RM (1990). "Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate." Biochemistry 1990;29(30);6964-77. PMID: 2223754

Newby06: Newby Z, Lee TT, Morse RJ, Liu Y, Liu L, Venkatraman P, Santi DV, Finer-Moore JS, Stroud RM (2006). "The role of protein dynamics in thymidylate synthase catalysis: variants of conserved 2'-deoxyuridine 5'-monophosphate (dUMP)-binding Tyr-261." Biochemistry 45(24);7415-28. PMID: 16768437

Patrick07: Patrick WM, Quandt EM, Swartzlander DB, Matsumura I (2007). "Multicopy suppression underpins metabolic evolvability." Mol Biol Evol 24(12);2716-22. PMID: 17884825

Roberts06: Roberts SA, Hyatt DC, Honts JE, Changchien L, Maley GF, Maley F, Montfort WR (2006). "Structure of the Y94F mutant of Escherichia coli thymidylate synthase." Acta Crystallogr Sect F Struct Biol Cryst Commun 62(Pt 9);840-3. PMID: 16946460

Saxl07: Saxl RL, Maley GF, Hauer CR, Maccoll R, Changchien L, Maley F (2007). "Significance of mutations on the structural perturbation of thymidylate synthase: implications for their involvement in subunit exchange." Protein Sci 16(7);1439-48. PMID: 17586776

SoteloMundo06: Sotelo-Mundo RR, Changchien L, Maley F, Montfort WR (2006). "Crystal structures of thymidylate synthase mutant R166Q: structural basis for the nearly complete loss of catalytic activity." J Biochem Mol Toxicol 20(2);88-92. PMID: 16615077

UniProt10: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt15: UniProt Consortium (2015). "UniProt version 2015-01 released on 2015-01-16 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on the manual assignment of UniProtKB Subcellular Location terms in UniProtKB/Swiss-Prot entries."

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 19.0 on Wed Sep 2, 2015, biocyc13.