Thymidylate biosynthesis

Methylenetetrahydrofohc acid (5,10-CH2-H4 folate) (5) is a coen2yme in thymidylate biosynthesis the natural (6R)-stereoisomer is prepared by en2ymatic reduction of H2 folate (2), foUowed by condensation with formaldehyde (54). 

Thymidylate biosynthesis. (R = p-aminobenzoyl-L-glutamate.) Red symbols indicate precursors of the methyl group of thymidylate. The dTMP is formed from dUMP. 5,10-Methylenetetrahydrofolate donates the methyl group in this reaction. This methyl group donor is... 

Figure 2. Inhibition of dihydrofolate reductase and de novo thymidylate biosynthesis in (A) nonresistant H35 and (B) resistant H35Ro, hepatoma cells, after treatment with MTX and MTX(poly-L-lysine). (Reproduced, with permission, from Ref. 23. Copyright 1981, American Society for Pharmacology and Experimental Therapeutics.)...

High thymidylate synthase level was accompanied (Table 1) by high specific activities of other enzymes involved in thymidylate biosynthesis, dihydrofolate reductase (14 2 nmol/min/mg protein N = 21) and dUTPase (0.68 0.16 nmol/ min/mg protein N = 14), again comparable with the corresponding activities (67 nmol/min/mg protein for dihydrofolate reductase and 0.6-1.5 nmol/min/mg protein for dUTPase) found in regenerating rat Uver crude extracts. 

Winska P, Golos B, Ciesla J, Zielinski Z, Frqczyk T, Walajtys-Rode E, Rode W (2005) Developmental arrest in C. elegans dauer larvae leaves high expression of enzymes involved in thymidylate biosynthesis, similar to that found in TrichineUa muscle larvae. Parasitology 131 247-254... 

Clinical implications of methionine synthase inhibition The inhibition of methionine synthase due to vitamin B12 deficiency induces megaloblastic anemia that is clinically indistinguishable from that caused by folate deficiency. The hematological effect in both cases results in levels of 5,10-methyleneTHF that are inadequate to sustain thymidylate biosynthesis. Clinically, it is essential to ascertain whether the anemia is the result of folate deficiency or vitamin Bi2 deficiency by differential diagnostic techniques. Vitamin B12 is essential for the synthesis of myelin in nerve tissue, a function probably related to... 

The dihydrofolate reductase enzyme (DHFR) is involved in one-carbon metabolism and is required for the survival of prokaryotic and eukaryotic cells. The enzyme catalyzes the reduction of dihydrofolate to tetrahydrofolate, which is required for the biosynthesis of serine, methionine, purines, and thymidylate. The mouse dihydrofolate reductase (mDHFR) is a small (21 kD), monomeric enzyme that is highly homologous to the E. coli enzyme (29% identify) (Pelletier et al., 1998). The three-dimensional structure of DHFR indicates that it is comprised of three structural fragments F[l], F[2] andF[3] (Gegg etal., 1997). 

Reddy et al29 studied the molecule N6,N6-dimethyl-2,6-diaminobenz[cd]-indole, which is shown in Figure 6. The aim was to assess its suitability as an inhibitor of thymidylate synthase, an enzyme which is important in DNA biosynthesis. Three structures were investigated N1H, which is the amine form depicted, and N2H, the imine form, in anti- and syn-conformations, i.e. with the proton on Ni away from or towards the N2 proton, respectively. In the gas phase it was found that the order of stability is N1H > anti-N2H > syn-N2H, in a ratio of 73.1 20.3 6.6. Solvation then further favours the N1H form, and the ratios in solution are calculated to be 98.5 0.5 1.0. Unfortunately, the syn-N2H form is likely to be the conformation that binds most strongly to the enzyme, and so the calculations indicate that this molecule is unlikely to be a suitable inhibitor. 

The pyrimidine antagonists inhibit the biosynthesis of pyrimidine nucleotides or interfere with vital cellular functions, such as the synthesis or function of nucleic acids. The analogues of deoxycytidine and thymidine that are used are inhibitors of DNA synthesis while 5-fluorouracil (5-FU) an analogue of uracil, is an inhibitor of both RNA function and of the synthesis of thymidylate (see Fig. 2). PALA (N-phosphonoacetyl-L-aspartate), an inhibitor of as-... 

Tetrahydrofolate is in turn converted to N, N °-methylenetetrahydrofolate, which is an essential cofactor for the synthesis of thymidylate, purines, methionine, and glycine. The major mechanism by which methotrexate brings about cell death appears to be inhibition of DNA synthesis through a blockage of the biosynthesis of thymidylate and purines. 

Thymidylate synthase (TS) is the enzyme that converts 2-deoxyuridine monophosphate into thymidine monophosphate. This is a key step in the biosynthesis of DNA. This enzymatic reaction of methylation involves the formation of a ternary complex between the substrate, the enzyme, and tetrahydrofolic acid (CH2FAH4). The catalytic cycle involves the dissociation of this complex and the elimination of FAH4. It is initiated by pulling out the proton H-5, thus generating an exocyclic methylene compound. As the release of a F" " ion is energetically forbidden, the fluorine atom that replaces the proton H-5 cannot be pulled out by the base. This leads to inhibition of the enzyme (Figure 7.2). 

FIGURE 22-43 Biosynthesis of thymidylate (dTMP). The pathways are shown beginning with the reaction catalyzed by ribonucleotide reductase. Figure 22-44 gives details of the thymidylate synthase reaction. 

A key step in DNA biosynthesis, that of conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP), is catalyzed by thymidylate synthetase which uses (25) as cofactor. This reaction involves both the transfer of a one carbon unit at the formaldehyde level and hydride transfer (from C-6 of (25)) to produce 7,8-dihydrofolate (27) and dTMP... 

Phosphorylation of dCDP to dCTP (step k, Fig. 25-14) completes the biosynthesis of the first of the pyrimidine precursors of DNA. The uridine nucleotides arise in two ways. Reduction of UDP yields dUDP (step), Fig. 25-14). However, the deoxycytidine nucleotides are more often hydrolytically deaminated (reactions / and / ) 274 Methylation of dUMP to form thymidylate, dTMP (step n, Fig. 25-14), is catalyzed by thymidylate synthase. The reaction involves transfer of a 1-carbon unit from methylene tetrahydrofolic acid with subsequent reduction using THF as the electron donor. A probable mechanism is shown in Fig. 15-21. See also Box 15-E. Some bacterial transfer RNAs contain 4-thiouridine (Fig. 5-33). The sulfur atom is introduced by a sulfurtransferase (the Thil gene product in E. coli). The same protein is essential for thiamin biosynthesis (Fig. 25-21)274a... 

Another group of inhibitors prevents nucleotide biosynthesis indirectly by depleting the level of intracellular tetrahydrofolate derivatives. Sulfonamides are structural analogs of p-aminobenzoic acid (fig. 23.19), and they competitively inhibit the bacterial biosynthesis of folic acid at a step in which p-aminobenzoic acid is incorporated into folic acid. Sulfonamides are widely used in medicine because they inhibit growth of many bacteria. When cultures of susceptible bacteria are treated with sulfonamides, they accumulate 4-carboxamide-5-aminoimidazole in the medium, because of a lack of 10-formyltetrahydrofolate for the penultimate step in the pathway to IMP (see fig. 23.10). Methotrexate, and a number of related compounds inhibit the reduction of dihydrofolate to tetrahydrofolate, a reaction catalyzed by dihydrofolate reductase. These inhibitors are structural analogs of folic acid (see fig. 23.19) and bind at the catalytic site of dihydrofolate reductase, an enzyme catalyzing one of the steps in the cycle of reactions involved in thymidylate synthesis (see fig. 23.16). These inhibitors therefore prevent synthesis of thymidylate in replicating... 

Figure 10.11 Biosynthesis of thymidylic acid from deoxyuridylic acid.

A large number of compounds in category 3 act at different sites in the pathways for purine and pyrimidine biosynthesis. These compounds are very toxic for rapidly growing tumors and bacteria, making them useful in cancer chemotherapy and treatment of bacterial infections. 6-Mercaptopurine is a potent inhibitor of purine biosynthesis, and 5-fluorouracil inhibits thymidylate synthesis. Some compounds, such as hydroxyurea and sulfonamides, inhibit the synthesis of both purine and pyrimidine nucleotides. These are only a few of the many compounds useful in treating cancer and infectious diseases (see Chapter 10). 

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