MetaCyc Enzyme: thymidylate synthase

Gene: thyA Accession Number: G-12127 (MetaCyc)

Species: Mycobacterium tuberculosis H37Rv

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

Thymidylate synthase is a key enzyme in cellular DNA synthesis. It converts dUMP to dTMP in the pathway for de novo biosynthesis of the DNA building block dTTP.

In 2002 it was discovered that the genomes of some organisms such as Mycobacterium tuberculosis contain two genes, thyA and thyX, encoding thymidylate synthase. The reason for this is poorly understood but may be related to phase of the growth cycle. Mycobacterium tuberculosis also lacks gene tdk encoding the salvage enzyme thymidylate kinase, making de novo dTMP synthesis essential. ThyA is the classical thymidylate synthase (EC (this enzyme), whereas ThyX is an alternate, novel flavin-dependent thymidylate synthase (EC (see flavin-dependent thymidylate synthase). Most organisms posses only ThyA. Some organisms lack ThyA and have only ThyX (in [Park10, Hunter08, Myllykallio02, Lesley02, Graziani06, Leduc07] and reviewed in [Murzin02, Leduc04, Koehn10]).

ThyA and ThyX show no sequence homology with each other and show substantial differences in crystal structure and reaction mechanism. All thymidylate synthases catalyze a reductive methylation involving the transfer of the methylene group of 5,10-methylenetetrahydropteroyl mono-L-glutamate to the C5-position of dUMP and a two electron reduction of the methylene group to a methyl group, producing the thymine moiety of dTMP. However, the reductive mechanism of the classical thymidylate synthase ThyA is distinctly different from ThyX. The ThyA reductive mechanism uses folate as both a 1-carbon donor and a source of reducing equivalents, producing dUMP and 7,8-dihydrofolate monoglutamate as products, and does not involve a flavin coenzyme or a third substrate. In contrast, the ThyX mechanism uses a flavin coenzyme as a source of reducing equivalents, which are derived from a reducing substrate. This NAD(P)H oxidase uses FAD to mediate hydride transfer in a methylation reaction that results in tetrahydropteroyl mono-L-glutamate as a product, rather than 7,8-dihydrofolate monoglutamate produced in the ThyA reaction (reviewed in [Koehn10, Koehn09]).

Recombinant, His-tagged ThyA and ThyX enzymes from Mycobacterium tuberculosis H37Rv have been expressed in Escherichia. coli and characterized both kinetically and for their ligand binding preferences. Unlike some ThyA proteins that can bind their cognate mRNA coding sequences and inhibit their own translation, neither ThyA nor ThyX bound to their own mRNA, suggesting no regulation by autologous translational feedback [Hunter08].

The native apparent molecular mass of ThyA from Mycobacterium tuberculosis H37Rv was determined by size exclusion chromatography. The data suggested that the native enzyme is a homodimer [Hunter08].

Map Position: [3,073,680 <- 3,074,471]

Molecular Weight of Polypeptide: 29.853 kD (from nucleotide sequence)

Molecular Weight of Multimer: 57.0 kD (experimental) [Hunter08]

Unification Links: Entrez-gene:887728 , Pride:P67044 , Protein Model Portal:P9WFR8 , SMR:P67044 , String:83332.Rv2764c , UniProt:P67044

Relationship Links: InterPro:IN-FAMILY:IPR000398 , InterPro:IN-FAMILY:IPR020940 , InterPro:IN-FAMILY:IPR023451 , Panther:IN-FAMILY:PTHR11549:SF2 , PDB:Structure:3QJ7 , PDB:Structure:4FOA , PDB:Structure:4FOG , PDB:Structure:4FOX , PDB:Structure:4FQS , Pfam:IN-FAMILY:PF00303 , Prints:IN-FAMILY:PR00108 , Prosite:IN-FAMILY:PS00091

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Created 02-Aug-2010 by Fulcher CA , SRI International

Enzymatic reaction of: dTMP synthase (thymidylate synthase)

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 Enzyme Commission system.

The reaction is favored in the direction shown.

In Pathways: superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis (E. coli) , superpathway of pyrimidine deoxyribonucleoside salvage , pyrimidine deoxyribonucleotides de novo biosynthesis II , pyrimidine deoxyribonucleosides salvage

A tritium release assay was used and the dTMP product was confirmed by HPLC. F-dUMP and 1843U89 were strong inhibitors of this enzyme [Hunter08].

Inhibitors (Unknown Mechanism): F-dUMP [Hunter08] , 1843U89 [Hunter08]

Kinetic Parameters:

Km (μM)
a 5,10-methylene-tetrahydrofolate


Graziani06: Graziani S, Bernauer J, Skouloubris S, Graille M, Zhou CZ, Marchand C, Decottignies P, van Tilbeurgh H, Myllykallio H, Liebl U (2006). "Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX." J Biol Chem 281(33);24048-57. PMID: 16707489

Hunter08: Hunter JH, Gujjar R, Pang CK, Rathod PK (2008). "Kinetics and ligand-binding preferences of Mycobacterium tuberculosis thymidylate synthases, ThyA and ThyX." PLoS One 3(5);e2237. PMID: 18493582

Koehn09: Koehn EM, Fleischmann T, Conrad JA, Palfey BA, Lesley SA, Mathews II, Kohen A (2009). "An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene." Nature 458(7240);919-23. PMID: 19370033

Koehn10: Koehn EM, Kohen A (2010). "Flavin-dependent thymidylate synthase: a novel pathway towards thymine." Arch Biochem Biophys 493(1);96-102. PMID: 19643076

Leduc04: Leduc D, Graziani S, Meslet-Cladiere L, Sodolescu A, Liebl U, Myllykallio H (2004). "Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic Bacteria and Archaea." Biochem Soc Trans 32(Pt 2);231-5. PMID: 15046578

Leduc07: Leduc D, Escartin F, Nijhout HF, Reed MC, Liebl U, Skouloubris S, Myllykallio H (2007). "Flavin-dependent thymidylate synthase ThyX activity: implications for the folate cycle in bacteria." J Bacteriol 189(23);8537-45. PMID: 17890305

Lesley02: Lesley SA, Kuhn P, Godzik A, Deacon AM, Mathews I, Kreusch A, Spraggon G, Klock HE, McMullan D, Shin T, Vincent J, Robb A, Brinen LS, Miller MD, McPhillips TM, Miller MA, Scheibe D, Canaves JM, Guda C, Jaroszewski L, Selby TL, Elsliger MA, Wooley J, Taylor SS, Hodgson KO, Wilson IA, Schultz PG, Stevens RC (2002). "Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline." Proc Natl Acad Sci U S A 99(18);11664-9. PMID: 12193646

Murzin02: Murzin AG (2002). "Biochemistry. DNA building block reinvented." Science 297(5578);61-2. PMID: 12029066

Myllykallio02: Myllykallio H, Lipowski G, Leduc D, Filee J, Forterre P, Liebl U (2002). "An alternative flavin-dependent mechanism for thymidylate synthesis." Science 297(5578);105-7. PMID: 12029065

Park10: Park M, Cho S, Lee H, Sibley CH, Rhie H (2010). "Alternative thymidylate synthase, ThyX, involved in Corynebacterium glutamicum ATCC 13032 survival during stationary growth phase." FEMS Microbiol Lett 307(2);128-34. PMID: 20636973

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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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