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MetaCyc Enzyme: flavin-dependent thymidylate synthase

Gene: thyX Accession Number: G-12103 (MetaCyc)

Synonyms: thy1, thymidylate synthase thyX, thymidylate synthase complementing protein, thymidylate synthase (FAD), complementing thymidylate synthase, flavin-dependent thymidylate synthase X, thymidylate synthase 1, thyX-encoded thymidylate synthase

Species: Thermotoga maritima

Subunit composition of flavin-dependent thymidylate synthase = [ThyX]4
         flavin-dependent thymidylate synthase subunit = ThyX

In 2002 it was discovered that some organisms do not posses the classical thymidylate synthase product of gene thyA (EC and also lacked gene tdk encoding the salvage enzyme thymidylate kinase. Instead, these organisms had an alternate, novel flavin-dependent thymidylate synthase (EC encoded by gene thyX. Homologs of the thyX gene have been identified in some eubacterial, archael, slime mold and viral genomes (as shown in [Myllykallio02]). The genomes of some of these organisms contain only thyX, while others contain both thyX and thyA, the reason for which is poorly understood but may be related to phase of the growth cycle. A ThyX motif RHRX7S has been identified in this protein family (Pfam Thy1, PF02511) (in [Park10, Myllykallio02, Lesley02, Graziani06, Leduc07] and reviewed in [Murzin02, Leduc04, Koehn10]).

ThyX showed no sequence homology with ThyA and it showed substantial differences from ThyA 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. The chemical and kinetic mechanisms of ThyX and progress in the design of specific inhibitors are reviewed in [Koehn10, Koehn09].

The hyperthermophile Thermotoga maritima has been shown to posses only ThyX [Leduc04]. Because ThyX alone is found in several human pathogens, it is of interest as a drug target [Myllykallio02].

The crystal structure of the Thermotoga maritima ThyX has been determined for wild-type [Kuhn02, Lesley02] and mutant [Koehn09] proteins. The wild-type enzyme was shown to be a homotetramer with four active sites (reviewed in [Koehn10]).

Map Position: [472,959 <- 473,621]

Molecular Weight of Polypeptide: 26.004 kD (from nucleotide sequence)

Unification Links: DIP:DIP-60076N, Entrez-gene:897468, Protein Model Portal:Q9WYT0, SMR:Q9WYT0, String:243274.TM0449, UniProt:Q9WYT0

Relationship Links: InterPro:IN-FAMILY:IPR003669, PDB:Structure:1KQ4, PDB:Structure:1O24, PDB:Structure:1O25, PDB:Structure:1O26, PDB:Structure:1O27, PDB:Structure:1O28, PDB:Structure:1O29, PDB:Structure:1O2A, PDB:Structure:1O2B, PDB:Structure:3G4A, PDB:Structure:3G4C, PDB:Structure:3N0B, PDB:Structure:3N0C, PDB:Structure:4GT9, PDB:Structure:4GTA, PDB:Structure:4GTB, PDB:Structure:4GTC, PDB:Structure:4GTD, PDB:Structure:4GTE, PDB:Structure:4GTF, PDB:Structure:4GTL, PDB:Structure:4KAR, PDB:Structure:4KAS, PDB:Structure:4KAT, Pfam:IN-FAMILY:PF02511, Prosite:IN-FAMILY:PS51331

Gene-Reaction Schematic

Gene-Reaction Schematic

Created 13-Jul-2010 by Fulcher CA, SRI International

Enzymatic reaction of: flavin-dependent thymidylate synthase

Inferred from experiment

EC Number:

dUMP + a 5,10-methylene-tetrahydrofolate + NADPH + H+ → dTMP + a tetrahydrofolate + NADP+

The direction shown, i.e. which substrates are on the left and right sides, is in accordance with the Enzyme Commission system.

The reaction is favored in the direction shown.

In Pathways: pyrimidine deoxyribonucleotides de novo biosynthesis III

Note that the Enzyme Commission reaction equation for EC does not include NAD(P)H as of this curation date. Here, NADPH is indicated as a cofactor for the Thermotoga maritima enzyme [Koehn10].

This flavoenzyme also showed an oxidase activity, catalyzing the reduction of oxygen to hydrogen peroxide using NADPH or other reducing agents. This oxidase activity was used to probe the kinetic mechanism of the synthase reaction [Wang09].

Cofactors or Prosthetic Groups: FAD [Agrawal04], NADPH [Agrawal04]


Agrawal04: Agrawal N, Lesley SA, Kuhn P, Kohen A (2004). "Mechanistic studies of a flavin-dependent thymidylate synthase." Biochemistry 43(32);10295-301. PMID: 15301527

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

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

Kuhn02: Kuhn P, Lesley SA, Mathews II, Canaves JM, Brinen LS, Dai X, Deacon AM, Elsliger MA, Eshaghi S, Floyd R, Godzik A, Grittini C, Grzechnik SK, Guda C, Hodgson KO, Jaroszewski L, Karlak C, Klock HE, Koesema E, Kovarik JM, Kreusch AT, McMullan D, McPhillips TM, Miller MA, Miller M, Morse A, Moy K, Ouyang J, Robb A, Rodrigues K, Selby TL, Spraggon G, Stevens RC, Taylor SS, van den Bedem H, Velasquez J, Vincent J, Wang X, West B, Wolf G, Wooley J, Wilson IA (2002). "Crystal structure of thy1, a thymidylate synthase complementing protein from Thermotoga maritima at 2.25 A resolution." Proteins 49(1);142-5. PMID: 12211025

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

Wang09: Wang Z, Chernyshev A, Koehn EM, Manuel TD, Lesley SA, Kohen A (2009). "Oxidase activity of a flavin-dependent thymidylate synthase." FEBS J 276(10);2801-10. PMID: 19459936

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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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