Dihydrofolate reductase-thymidylate synthase

Toyoda T, Brobey RKB, Sano G, Horii T, Tomioka N, Itai Akiko. Lead discovery of inhibitors of the dihydrofolate reductase domain of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase. Biochem Biophys Res Commun 1997 235 515-19. 

The methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA. The one-carbon fragment of methy-lene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division. Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anticancer drug. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme inhibitors of these enzymes can be used as antibacterial drugs, eg, trimethoprim, and anti-malarial drugs, eg, pyrimethamine. 

Dihydrofolate reductase-thymidylate synthase bifunctional enzyme Apicomplexa and Kinetoplastida Pyrimethamine... 

Dihydrofolate Reductase-Thymidylate Synthase Bifunctional Enzyme... 

Bzik, D. J., Li, W. B., Horii, T., and Inselburg, J. (1987). Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene. Proc. Natl. Acad. Sci. USA 84, 8360-8364. 

Mudeppa, D. G., Pang, C. K., Tsuboi, T., Endo, Y., Buckner, F. S., Varani, G., and Rathod, P. K. (2007). Cell-free production of functional Plasmodium falciparum dihydrofolate reductase-thymidylate synthase. Mol. Biochem. Parasitol. 151,216-219. 

Yuthavong, Y., Yuvaniyama, J., Chitnumsub, P., Vanichtanankul, J., Chusacultanachai, S., Tarnchompoo, B., Vilaivan, T., and Kamchonwongpaisan, S. (2005). Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase Structural basis for antifolate resistance and development of effective inhibitors. Parasitology 130,249-259. 

Consistent with the metabolic data, there is no dihydrofolate reductase/thymidylate synthase activity (75). Thymidine is salvaged by a phosphotransferase. Uracil PRTase, uridine phosphorylase, cytidine deaminase and uridine and cytidine phosphotransferases were found. The major pyrimidine salvaged is uracil via its PRTase. The lack of incorporation of salvaged uracil into DNA and the lack of thymidylate synthase indicates that this parasite cannot synthesize TMP from UMP. It is dependent on salvage for its thymidine requirements. This parasite possesses a hydroxyurea-resistant ribonucleotide reductase and can synthesize deoxycytidine nucleotides from cytidine nucleotides. 

Res. Commun., 235, 515 (1997). Lead Discovery of Inhibitors of the Dihydrofolate Reductase Domain of Plasmodium falciparum Dihydrofolate Reductase-Thymidylate Synthase. 

Foote SJ, Galatis D, Cowman AF. Amino adds in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium foldparum involved in cycloguanil resistance differ feom those involved in pyrimethamine resistance. Proc Nad Acad Sd USA 1990 87(8) 3014-3017. 

Maiate dehydrogenase-citrate syndiase Dihydrofolate reductase-thymidylate synthase... 

Chinese hamster fibroblasts co-sedimentation on sucrose gradients kinetic coupling nascent and template DNA, ribonucleotide reductase, thymidylate synthase, dihydrofolate reductase, thymidine kinase, NDP kinase, DNA polymerase 83, 273, 353... 

UMP, the product of both the biosynthetic and salvage pathways, serves as the precursor for all other piyrimidine nudeotides. It is converted to UDP and UTP by nudeotide kinases, UTP is aminated to CTP by CTP s)mthetase (CTPS), deoxyribonudeotides are synthesized fix>m ribonudeoside diphosphates by ribonudeotide reductase (RIO, and thymidylate nudeotides ate produced via reductive methylation of dUMP utilizing N, N °-meth)denetetrahydtofolate as the methyl donor, a reaaion catalyzed by a bifuncdonal dihydrofolate teductase-thymidylate synthase (DHFR-TS) protein. 

Studies using free energy calculations for the design and analysis of potential drug candidates are reviewed in section five. The chapters in this section cover drug discovery programs targeting fructose 1,6-bisphosphatase (diabetes), COX-2 (inflammation), SRC SH2 domain (osteoporosis and cancer), HIV reverse transcriptase (AIDS), HIV-1 protease (AIDS), thymidylate synthase (cancer), dihydrofolate reductase (cancer) and adenosine deaminase (immunosuppression, myocardial ischemia). 

Fig. 14.1 Cellular pathway of methotrexate. ABCBl, ABCCl-4, ABC transporters ADA, adenosine deaminase ADP, adenosine diphosphate AICAR, aminoimidazole carboxamide ribonucleotide AMP, adenosine monophosphate ATIC, AICAR transformylase ATP, adenosine triphosphate SjlO-CH -THF, 5,10-methylene tetrahydrofolate 5-CHj-THF, 5-methyl tetrahydro-folate DHFR, dihydrofolate reductase dTMP, deoxythymidine monophosphate dUMP, deoxy-uridine monophosphate FAICAR, 10-formyl AICAR FH, dihydrofolate FPGS, folylpolyglutamyl synthase GGH, y-glutamyl hydrolase IMP, inosine monophosphate MTHFR, methylene tetrahydrofolate reductase MTR, methyl tetrahydrofolate reductase MTX-PG, methotrexate polyglutamate RFCl, reduced folate carrier 1 TYMS, thymidylate synthase. Italicized genes have been targets of pharmacogenetic analyses in studies published so far. (Reproduced from ref. 73 by permission of John Wiley and Sons Inc.)...

The DNA component deoxythymidine triphosphate (dTTP) is synthesized from UDP in several steps. The base thymine, which only occurs in DNA (see p. 80), is formed by meth-ylation of dUMP at the nucleoside monophosphate level. Thymidylate synthase and its helper enzyme dihydrofolate reductase are important target enzymes for cytostatic drugs (see p. 402). 

Dihydrofolate reductase acts as an auxiliary enzyme for thymidylate synthase. It is involved in the regeneration of the coenzyme N, N -methylene-THF, initially reducing DHF to THF with NADPH as the reductant (see p. 418). The folic acid analogue methotrexate, a frequently used cytostatic agent, is an extremely effective competitive inhibitor of dihydrofolate reductase. It leads to the depletion of N, N -methylene-THF in the cells and thus to cessation of DNA synthesis. 

RGURE 22-44 Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate reductase. Serine hydroxymethyltransferase is required for regeneration of the /V5,/V10-methylene form of tetrahy-... 

Conversion of dUMP to dTMP is catalyzed by thy-midylate synthase. A one-carbon unit at the hydroxymethyl (—CH2OH) oxidation level (see Fig. 18-17) is transferred from Af5,Af10-methylenetetrahydrofolate to dUMP, then reduced to a methyl group (Fig. 22-44). The reduction occurs at the expense of oxidation of tetrahydrofolate to dihydrofolate, which is unusual in tetrahydrofolate-requiring reactions. (The mechanism of this reaction is shown in Fig. 22-50.) The dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase—a regeneration that is essential for the many processes that require tetrahydrofolate. In plants and at least one protist, thymidylate synthase and dihy-drofolate reductase reside on a single bifunctional protein. 

Other useful targets for pharmaceutical agents are thymidylate synthase and dihydrofolate reductase, enzymes that provide the only cellular pathway for thymine synthesis (Fig. 22-49). One inhibitor that acts on thymidylate synthase, fluorouracil, is an important chemotherapeutic agent. Fluorouracil itself is not the enzyme inhibitor. In the cell, salvage pathways convert it to the deoxynucleoside monophosphate FdUMP, which binds to and inactivates the enzyme. Inhibition by FdUMP (Fig. 22-50) is a classic example of mechanism-based enzyme inactivation. Another prominent chemotherapeutic agent, methotrexate, is an inhibitor of dihydrofolate reductase. This folate analog acts as a competitive inhibitor the enzyme binds methotrexate with about 100 times higher affinity than dihydrofolate. Aminopterin is a related compound that acts similarly. 

During thymine formation the coenzyme is oxidized to dihydrofolate, which must be reduced by dihydrofolate reductase to complete the catalytic cycle. A possible mechanistic sequence for thymidylate synthase, an enzyme of known three-dimensional structure,354/418-4213 is given in Fig. 15-21. In the first step (a) a thiolate anion, from the side chain of Cys 198 of the 316-residue Lactobacillus enzyme, adds to the 5 position of the substrate 2 -deoxyuridine monophosphate... 

In protozoa thymidylate synthase and dihydrofolate reductase exist as a single bifunctional protein. 

Thymidylate synthase requires methylene tetrahydro-folate as a reductant and the reduction of dihydrofolate is also an important part of the process. In protozoa dihydrofolate reductase and thymidylate synthase occur as a singlechain bifunctional enzyme.f As has been pointed out in the main text, such folic acid analogs as methotrexate are among the most useful anticancer drugs. By inhibiting dihydrofolate reductase they deprive thymidylate synthase of an essential substrate. 

A four-base overlap between dihydrofolate reductase and thymidylate synthase has been found201 in the DNA of phage T4. A transposable DNA insertion sequence (see Section D,5) in E. coli encodes two genes, one of which is contained within the other and which is transcribed from the opposite strand of DNA.202 The double-stranded RNA of a reovirus produces two peptides from the same sequence using two different AUG initiation codons in different reading frames 203... 

Young DW (1994) Studies on thymidylate synthase and dihydrofolate reductase - two enzymes involved in the synthesis of thymidine. Chem Soc Rev 23, 119-128. 

Tetrahydrofolate cofactors participate in one-carbon transfer reactions. As described above in the section on vitamin B12, one of these essential reactions produces the dTMP needed for DNA synthesis. In this reaction, the enzyme thymidylate synthase catalyzes the transfer of the one-carbon unit of N 5,N 10-methylenetetrahydrofolate to deoxyuridine monophosphate (dUMP) to form dTMP (Figure 33-2, reaction 2). Unlike all of the other enzymatic reactions that utilize folate cofactors, in this reaction the cofactor is oxidized to dihydrofolate, and for each mole of dTMP produced, one mole of tetrahydrofolate is consumed. In rapidly proliferating tissues, considerable amounts of tetrahydrofolate can be consumed in this reaction, and continued DNA synthesis requires continued regeneration of tetrahydrofolate by reduction of dihydrofolate, catalyzed by the enzyme dihydrofolate reductase. The tetrahydrofolate thus produced can then reform the cofactor N 5,N 10-methylenetetrahydrofolate by the action of serine transhydroxy- methylase and thus allow for the continued synthesis of dTMP. The combined catalytic activities of dTMP synthase, dihydrofolate reductase, and serine transhydroxymethylase are often referred to as the dTMP synthesis cycle. Enzymes in the dTMP cycle are the targets of two anticancer drugs methotrexate inhibits dihydrofolate reductase, and a metabolite of 5-fluorouracil inhibits thymidylate synthase (see Chapter 55 Cancer Chemotherapy). 

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