Applied Microbiology and Biotechnology

, Volume 74, Issue 2, pp 282–289

Thymidyl biosynthesis enzymes as antibiotic targets

  • Anatoly Chernyshev
  • Todd Fleischmann
  • Amnon Kohen


The two long-known “classical” enzymes of uridyl-5-methylation, thymidylate synthase and ribothymidyl synthase, have been joined by two alternative methylation enzymes, flavin-dependent thymidylate synthase and folate-dependent ribothymidyl synthase. These two newly discovered enzymes have much in common: both contain flavin cofactors, utilize methylenetetrahydrofolate as a source of methyl group, and perform thymidylate synthesis via chemical pathways distinct from those of their classic counterparts. Several severe human pathogens (e.g., typhus, anthrax, tuberculosis, and more) depend on these “alternative” enzymes for reproduction. These and other distinctive properties make the alternative enzymes and their corresponding genes appealing targets for new antibiotics.


Thymine Biosynthesis Flavin Thymidylate Synthase 


  1. Agrawal N, Lesley SA, Kuhn P, Kohen A (2004) Mechanistic studies of a flavin-dependent thymidylate synthase. Biochemistry 43:10295–10301CrossRefGoogle Scholar
  2. Atreya CE, Anderson KS (2004) Kinetic characterization of bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis: a paradigm shift for ts activity and channeling behavior. J Biol Chem 279:18314–18322CrossRefGoogle Scholar
  3. Atreya CE, Johnson EF, Irwin JJ, Dow A, Massimine KM, Coppens I, Stempliuk V, Beverley S, Joiner KA, Shoichet BK, Anderson KS (2003) A molecular docking strategy identifies Eosin B as a non-active site inhibitor of protozoal bifunctional thymidylate synthase-dihydrofolate reductase. J Biol Chem 278:14092–14100CrossRefGoogle Scholar
  4. Barrett JE, Maltby DA, Santi DV, Schultz PG (1998) Trapping of the C5 methylene intermediate in thymidylate synthase. J Am Chem Soc 120:449–450CrossRefGoogle Scholar
  5. Carreras CW, Santi DV (1995) The catalytic mechanism and structure of thymidylate synthase. Annu Rev Biochem 64:721–762CrossRefGoogle Scholar
  6. Chu E, Voeller D, Koeller DM, Drake JC, Takimoto CH, Maley GF, Maley F, Allegra CJ (1993) Identification of an RNA binding site for human thymidylate synthase. Proc Natl Acad Sci USA 90:517–521CrossRefGoogle Scholar
  7. Danenberg PV, Malli H, Swenson S (1999) Thymidylate synthase inhibitors. Semin Oncol 26:621–631Google Scholar
  8. Delk AS, Rabinowitz JC (1975) Biosynthesis of ribosylthymine in the transfer RNA of Streptococcus faecalis: a folate-dependent methylation not involving S-adenosylmethionine. Proc Natl Acad Sci USA 72:528–530CrossRefGoogle Scholar
  9. Delk AS, Romeo JM, Nagle DP Jr, Rabinowitz JC (1976) Biosynthesis of ribothymidine in the transfer RNA of Streptococcus faecalis and Bacillus subtilis. A methylation of RNA involving 5,10-methylenetetrahydrofolate. J Biol Chem 251:7649–7656Google Scholar
  10. Delk AS, Nagle DP Jr, Rabinowitz JC, Straub KM (1979) The methylenetetrahydrofolate-mediated biosynthesis of ribothymidine in the transfer-RNA of Streptococcus faecalis: incorporation of hydrogen from solvent into the methyl moiety. Biochem Biophys Res Commun 86:244–251CrossRefGoogle Scholar
  11. Delk AS, Nagle DP Jr, Rabinowitz JC (1980) Methylenetetrahydrofolate-dependent biosynthesis of ribothymidine in transfer RNA of Streptococcus faecalis. Evidence for reduction of the 1-carbon unit by FADH2. J Biol Chem 255:4387–4390Google Scholar
  12. Gattis SG, Palfey BA (2005) Direct observation of the participation of flavin in product formation by thyX-encoded thymidylate synthase. J Am Chem Soc 127:832–833CrossRefGoogle Scholar
  13. Graziani S et al. (2004) Functional analysis of FAD-dependent thymidylate synthase ThyX from paramecium bursaria chlorella virus-1. J Biol Chem 279:54340–54347CrossRefGoogle Scholar
  14. 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):24048–24057CrossRefGoogle Scholar
  15. Griffin J, Roshick C, Iliffe-Lee E, McClarty G (2005) Catalytic mechanism of Chlamydia trachomatis flavin-dependent thymidylate synthase. J Biol Chem 280:5456–5467CrossRefGoogle Scholar
  16. Ivanetich KM, Santi DV (1990) Bifunctional thymidylate synthase-dihydrofolate reductase in protozoa. Faseb J 4:1591–1597Google Scholar
  17. Johnson EF, Hinz W, Atreya CE, Maley F, Anderson KS (2002) Mechanistic characterization of Toxoplasma gondii thymidylate synthase (TS-DHFR)-dihydrofolate reductase. Evidence for a TS intermediate and TS half-sites reactivity. J Biol Chem 277:43126–43136CrossRefGoogle Scholar
  18. Kanai F, Sawa T, Hamada M, Naganawa H, Takeuchi T, Umezawa H (1983) Vanoxonin, a new inhibitor of thymidylate synthetase. J Antibiot (Tokyo) 36:656–660Google Scholar
  19. Kanai F, Isshiki K, Umezawa Y, Morishima H, Naganawa H, Takita T, Takeuchi T, Umezawa H (1985a) Vanoxonin, a new inhibitor of thymidylate synthetase. II. Structure determination and total synthesis. J Antibiot (Tokyo) 38:31–38Google Scholar
  20. Kanai F, Kaneko T, Morishima H, Isshiki K, Takita T, Takeuchi T, Umezawa H (1985b) Vanoxonin, a new inhibitor of thymidylate synthetase. III. Inhibition of thymidylate synthetase by vanoxonin-vanadium complex. J Antibiot (Tokyo) 38:39–50Google Scholar
  21. Kanai A, Sato A, Imoto J, Tomita M (2006) Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein. Biochem J 393:373–379CrossRefGoogle Scholar
  22. Kealey JT, Santi DV (1995) Stereochemistry of tRNA(m5U54)-methyltransferase catalysis: 19F NMR spectroscopy of an enzyme-FUraRNA covalent complex. Biochemistry 34:2441–2446CrossRefGoogle Scholar
  23. Kealey JT, Gu X, Santi DV (1994) Enzymatic mechanism of tRNA (m5U54)methyltransferase. Biochimie 76:1133–1142CrossRefGoogle Scholar
  24. 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, Mon 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 Å resolution. Protein Struc Funct Genet 49:142–145CrossRefGoogle Scholar
  25. Leduc D, Graziani S, Lipowski G, Marchand C, Le Maréchal P, Liebl U, Myllykallio H (2004a) Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers. Proc Natl Acad Sci USA 101:7252–7257CrossRefGoogle Scholar
  26. Leduc D, Graziani S, Meslet-Cladiere L, Sodolescu A, Liebl U, Myllykallio H (2004b) Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic Bacteria and Archaea. Biochem Soc Trans 32:231–235CrossRefGoogle Scholar
  27. Lee TT, Agarwalla S, Stroud RM (2004) Crystal structure of RumA, an iron–sulfur cluster containing E. coli ribosomal RNA 5-methyluridine methyltransferase. Structure 12:397–407CrossRefGoogle Scholar
  28. Lee TT, Agarwalla S, Stroud RM (2005) A unique RNA Fold in the RumA-RNA-cofactor ternary complex contributes to substrate selectivity and enzymatic function. Cell 120:599–611CrossRefGoogle Scholar
  29. 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 M-A, 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 USA 99:11664–11669CrossRefGoogle Scholar
  30. Liu J, Schmitz JC, Lin X, Tai N, Yan W, Farrell M, Bailly M, Chen T, Chu E (2002) Thymidylate synthase as a translational regulator of cellular gene expression. Biochim Biophys Acta 1587:174–182Google Scholar
  31. Mason A, Agrawal N, Washington MT, Lesley SA, Kohen A (2006) A lag-phase in the reduction of flavin dependent thymidylate synthase (FDTS) revealed a mechanistic missing link. Chem Commun 16:1781–1783CrossRefGoogle Scholar
  32. Mathews II, Deacon AM, Canaves JM, McMullan D, Lesley SA, Agarwalla S, Kuhn P (2003) Functional analysis of substrate and cofactor complex structures of a thymidylate synthase-complementing protein. Structure 11:677–690CrossRefGoogle Scholar
  33. Mattevi A (2006) To be or not to be an oxidase: challenging the oxygen reactivity of flavoenzymes. Trends Biochem Sci 31:276–283CrossRefGoogle Scholar
  34. Murzin AG (2002) Biochemistry. DNA building block reinvented. Science 297:61–62CrossRefGoogle Scholar
  35. Myllykallio H, Lipowski G, Leduc D, Filee J, Forterre P, Liebl U (2002) An alternative flavin-dependent mechanism of thymidylate synthesis. Science 297:105–107CrossRefGoogle Scholar
  36. Persson BC, Grustafsson C, Berg DE, Bjork GR (1992) The gene for a tRNA modifying enzyme, m5U54-methyltransferase, is essential for viability in Escherichia coli. Proc Natl Acad Sci USA 89:3995–3998CrossRefGoogle Scholar
  37. Pharkya P, Nikolaev EV, Maranas CD (2003) Review of the BRENDA Database. Metab Eng 5:71–73CrossRefGoogle Scholar
  38. Romeo JM, Delk AS, Rabinowitz JC (1974) The occurrence of a transmethylation reaction not involving S-adenosylmethionine in the formation of ribothymidine in Bacillus subtilis transfer-RNA. Biochem Biophys Res Commun 61:1256–1261CrossRefGoogle Scholar
  39. Sampathkumar P, Turley S, Ulmer JE, Rhie HG, Sibley CH, Hol WGJ (2005) Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0 resolution. J Mol Biol 352:1091–1104CrossRefGoogle Scholar
  40. Sampathkumar P, Turley S, Sibley CH, Hol WG (2006) NADP(+) expels both the co-factor and a substrate analog from the Mycobacterium tuberculosis ThyX active site: opportunities for anti-bacterial drug design. J Mol Biol 360:1–6Google Scholar
  41. Shoichet BK, Stroud RM, Santi DV, Kuntz ID, Perry KM (1993) Structure-based discovery of inhibitors of thymidylate synthase. Science 259:1445–1450CrossRefGoogle Scholar
  42. Stout TJ, Tondi D, Rinaldi M, Barlocco D, Pecorari P, Santi DV, Kuntz ID, Stroud RM, Shoichet BK, Costi MP (1999) Structure-based design of inhibitors specific for bacterial thymidylate synthase. Biochemistry 38:1607–1617CrossRefGoogle Scholar
  43. Tai N, Schmitz JC, Liu J, Lin X, Bailly M, Chen TM, Chu E (2004) Translational autoregulation of thymidylate synthase and dihydrofolate reductase. Front Biosci 9:2521–2526CrossRefGoogle Scholar
  44. Tondi D, Slomczynska U, Costi MP, Watterson DM, Ghelli S, Shoichet BK (1999) Structure-based discovery and in-parallel optimization of novel competitive inhibitors of thymidylate synthase. Chem Biol 6:319–331CrossRefGoogle Scholar
  45. Urbonavicius J, Skouloubris S, Myllykallio H, Grosjean H (2005) Identification of a novel gene encoding a flavin-dependent tRNA: m5U methyltransferase in bacteria-evolutionary implications. Nucleic Acids Res 33:3955–3964CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Anatoly Chernyshev
    • 1
  • Todd Fleischmann
    • 1
  • Amnon Kohen
    • 1
  1. 1.Department of ChemistryUniversity of IowaIowa CityUSA

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