The Catalytic Mechanism and Structure of Thymidylate Synthase

Carreras, Christopher W.; Santi, Daniel V.

doi:10.1146/annurev.bi.64.070195.003445

Cited by 695 publications

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“…The amino acid sequence of TS is highly conserved across species, particularly among those residues that form the substrate and cofactor binding pockets (12). These residues also interact closely with inhibitors such as 5-fluorouridylate, 1 Abbreviations: TS, thymidylate synthase; dUMP, 2′-deoxyuridine monophosphate; CH2H4folate, N 5 ,N 10 -methylene tetrahydrofolate; dTMP, 2′-deoxythymidine monophosphate; 5-FU, 5-fluorouracil; CB3717, 10-propargyl-5,8-dideazafolate; EcTS, Escherichia coli thymidylate synthase; LcTS, Lactobacillus casei thymidylate synthase; HTS, Homo sapiens (human) thymidylate synthase; 1, phenolphthalein; 2, diphenol-2,3-naphthalein; 3, diphenol-1,8-naphthalein; 4, 3′,3′′-dichlorophenol-1,8-naphthalein; 5, diphenol-5-nitro-1,8-naphthalein; 6, 3′,3′′-dichlorophenolphthalein; 7, 3′,3′′-dichlorophenol-4-chloro-1,8-naphthalein; 8, 3′-chlorophenol-4-nitro-1,8-naphthalein; DMSO, dimethyl sulfoxide; MR, molecular replacement; F o, observed structure factor; Fc, calculated structure factor; rcalc, phases derived from the atomic coordinates.…”

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Structure-Based Design of Inhibitors Specific for Bacterial Thymidylate Synthase^,

et al. 1999

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Thymidylate synthase is an attractive target for antiproliferative drug design because of its key role in the synthesis of DNA. As such, the enzyme has been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In practice, TS is highly conserved across species, and it has proven to be difficult to develop inhibitors that are selective for microbial TS enzymes over the human enzyme. Using the structure of TS from Lactobacillus casei in complex with the nonsubstrate analogue phenolphthalein, inhibitors were designed to take advantage of features of the bacterial enzyme that differ from those of the human enzyme. Upon synthesis and testing, these inhibitors were found to be up to 40-fold selective for the bacterial enzyme over the human enzyme. The crystal structures of two of these inhibitors in complex with TS suggested the design of further compounds. Subsequent synthesis and testing showed that these second-round compounds inhibit the bacterial enzyme at sub-micromolar concentrations, while the human enzyme was not inhibited at detectable levels (selectivities of 100-1000-fold or greater). Although these inhibitors share chemical similarities, X-ray crystal structures reveal that the analogues bind to the enzyme in substantially different orientations. Site-directed mutagenesis experiments suggest that the individual inhibitors may adopt multiple configurations in their complexes with TS.Thymidylate synthase (TS) 1 is an attractive target for the design of drugs used against proliferative diseases because of its central role in the production of DNA. TS catalyzes the methylation of 2′-deoxyuridine 5′-monophosphate (dUMP) by N 5 ,N 10 -methylene tetrahydrofolate (CH 2 H 4 folate). This reaction is the terminal step in the only de novo synthetic pathway to thymidylate, which is essential for DNA production. Inhibition of TS stops the production of DNA, disrupting the progression through the cell cycle and eventually leading to "thymineless" cell death (1).Much effort in drug design against TS has focused on inhibitors that resemble the substrate, dUMP, or the cofactor, CH 2 H 4 folate. A mechanism-based inhibitor of TS (2), 5-fluorouridylate, which is administered as the premetabolite, 5-fluorouracil (5-FU), is used in chemotherapy. The TS inhibitor, 10-propargyl-5,8-dideazafolate (CB3717), is a mimic of the cofactor, CH 2 H 4 folate (3). Although CB3717 is a potent inhibitor of TS [K i of 40 nM (4)], it shows liver and kidney toxicity in a small number of patients (5). Recent structure-based drug design efforts against TS (6-11, 41, 42) have resulted in a series of potent compounds such as AG337 and Tomudex that bind in the folate binding site of the enzyme. These new compounds show promise as cancer chemotherapeutics.The amino acid sequence of TS is highly conserved across species, particularly among those residues that form the substrate and cofactor binding pockets (12). These residues also interact closely with inhi...

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Structure-Based Design of Inhibitors Specific for Bacterial Thymidylate Synthase^,

et al. 1999

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“…Asp 229 Oδ 1 is 2.8 Å 2 has moved significantly back toward the wall of the active site cavity relative to its position in the TS N229D-dCMP complex. The orientation of the dCMP is essentially identical to the orientation of dUMP in wild-type TS-dUMP, as seen when the TS-dUMP and H199A/N229D-dCMP complexes are overlapped ( Figure 5).…”

Section: Ts H199a/n229d-dcmp Structurementioning

confidence: 99%

“…Comparison of sequences of TS from ∼30 different organisms reveals that it is one of the most highly conserved enzymes (1,2). A large number of three-dimensional structures of free and ligand-bound TSs and TS mutants have been determined and provide a structural understanding of substrate recognition, stereochemical aspects of the reaction pathway (3), and roles of specific residues in the reaction chemistry (4).…”

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A Novel dCMP Methylase by Engineering Thymidylate Synthase

et al. 1997

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X-ray crystal structures of binary complexes of dUMP or dCMP with the Lactobacillus casei TS mutant N229D, a dCMP methylase, revealed that there is a steric clash between the 4-NH 2 of dCMP and His 199, a residue which normally H-bonds to the 4-O of dUMP but is not essential for activity. As a result, the cytosine moiety of dCMP is displaced from the active site and the catalytic thiol is moved from the C6 of the substrate about 0.5 Å further than in the wild-type TS-dUMP complex. We reasoned that combining the N229D mutation with mutations at residue 199 which did not impinge on the 4-NH 2 of dCMP should correct the displacements and further favor methylation of dCMP. We therefore prepared several TS N229D mutants and characterized their steady state kinetic parameters. TS H199A/N229D showed a 10 11 change in specificity for methylation of dCMP Versus dUMP. The structures of TS H199A/ N229D in complex with dCMP and dUMP confirmed that the position and orientation of bound dCMP closely approaches that of dUMP in wild-type TS, whereas dUMP was displaced from the optimal catalytic binding site.Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) by 5,10-methylene-5,6,7,8-tetrahydrofolate (CH 2 H 4 -folate) to form deoxythymidine-5′-monophosphate (dTMP) and 7,8-dihydrofolate (H 2 folate). Comparison of sequences of TS from ∼30 different organisms reveals that it is one of the most highly conserved enzymes (1, 2). A large number of three-dimensional structures of free and ligand-bound TSs and TS mutants have been determined and provide a structural understanding of substrate recognition, stereochemical aspects of the reaction pathway (3), and roles of specific residues in the reaction chemistry (4).Asn 229 is invariant in all TS's sequenced to date, and forms a cyclic hydrogen bond network with the 4-O and 3-NH of the substrate dUMP (Figure 2a) (5-7). Nevertheless, it can be replaced by numerous other residues, and the resultant mutants retain TS activity and also gain the capability to bind 2′-deoxycytidine 5′-monophosphate (dCMP) tightly (8,9). Moreover, alteration of Asn 229 to aspartate provides an enzyme which methylates dCMP more efficiently than dUMP, although the catalytic efficiency is still significantly lower than is the wild-type TS for its natural substrate dUMP (10, 11).

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“…The synthesis of the fourth triphosphate dTTP necessary to DNA synthesis is achieved from dUDP or dUTP, produced by RNRs, through dephosphorylation and phosphorylation processes and a key methylation step of dUMP catalyzed by the enzyme thymidylate synthase (TS) leading to dTMP. Three classes of TS have been identified and, in the main class, the enzymes called classical thymidylate synthases use the coenzyme N 5 ,N 10 -methylene-5,6,7,8-tetrahydrofolate (CH 2 H 4 folate) and dUMP to produce dihydrofolate (H 2 folate) and dTMP (Figures 2 and 4) [16,17]. covalent linkage between the thiol side chain of an active site cysteine residue and the C6 atom of the targeted base for C5-methylation [16,17].…”

Section: Bio Web Of Conferencesmentioning

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Key steps from the “RNA World” to the “DNA World”

Renard

Maurin

Chambert

et al. 2014

BIO Web of Conferences

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Abstract. In the « RNA World » hypothesis of the origin of life, RNAs are assumed to be the central macromolecules able to self-replicate, conserve information and catalyze the reactions necessary for a primitive metabolism and many enzymatic cofactors may be regarded as molecular fossils of the "RNA World". In the key steps involved in the transition from the RNA World to the DNA World, two main steps can be distinguished: (i) the synthesis of 2'-deoxyribonucleotides from ribonucleotides catalyzed nowadays by the enzyme ribonucleotide reductase and (ii) the synthesis of thymine, a base specific for DNA, from uracil which is a base specific for RNA, catalyzed today by the enzyme thymidylate synthase. In regard to the chemistry of sulfur used by both enzymes for achieving their respective catalysis, we were interested in the search for simple sulfur reactions able to catalyze such transformations and report here on first results in an approach from thionucleosides to the catalysis involved in the conversion of uracil to thymine. In the RNA World, the recruitment of cofactors was crucial to expand the catalytic repertoire of RNA and we also describe interesting preliminary results obtained in the prebiotic synthesis of pyridoxal (vitamin B6) that is the precursor of the key coenzyme pyridoxal phosphate (PLP) able to catalyze nowadays seven different enzymatic reactions.

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The Catalytic Mechanism and Structure of Thymidylate Synthase

Cited by 695 publications

References 1 publication

Structure-Based Design of Inhibitors Specific for Bacterial Thymidylate Synthase^,

Structure-Based Design of Inhibitors Specific for Bacterial Thymidylate Synthase^,

A Novel dCMP Methylase by Engineering Thymidylate Synthase

Key steps from the “RNA World” to the “DNA World”

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The Catalytic Mechanism and Structure of Thymidylate Synthase

Cited by 695 publications

References 1 publication

Structure-Based Design of Inhibitors Specific for Bacterial Thymidylate Synthase,

Structure-Based Design of Inhibitors Specific for Bacterial Thymidylate Synthase,

A Novel dCMP Methylase by Engineering Thymidylate Synthase

Key steps from the “RNA World” to the “DNA World”

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Resources

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Structure-Based Design of Inhibitors Specific for Bacterial Thymidylate Synthase^,

Structure-Based Design of Inhibitors Specific for Bacterial Thymidylate Synthase^,