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



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

Regulation Summary Diagram: ?

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

Summary:
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)
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:4KNZ , Pfam:IN-FAMILY:PF00303 , Prints:IN-FAMILY:PR00108 , Prosite:IN-FAMILY:PS00091

Gene-Reaction Schematic: ?

GO Terms:

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

Credits:
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: 2.1.1.45

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

Summary:
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

Feature Class Location Citations Comment
Formylation-Modification 1
[UniProt10a]
UniProt: N-formylmethionine; Non-Experimental Qualifier: probable;
Active-Site 146
[UniProt10]


Gene Local Context (not to scale): ?

Transcription Unit:

Notes:

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


References

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." http://www.brenda-enzymes.org.

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

GOA01a: GOA, MGI (2001). "Gene Ontology annotation based on Enzyme Commission mapping." Genomics 74;121-128.

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 2010-11 released on 2010-11-02 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-07 released on 2010-06-15 00:00:00." Database.

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

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

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


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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 18.5 on Thu Nov 27, 2014, BIOCYC13A.