Leishmania donovani pteridine reductase 1: biochemical properties and structure-modeling studies

Pteridine reductase 1 (PTR1, EC 1.5.1.33) is a NADPH dependent short-chain reductase (SDR) responsible for the salvage of pterins in the protozoan parasite Leishmania. This enzyme acts as a metabolic bypass for drugs targeting dihydrofolate reductase, therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Based on homology model drawn on recombinant pteridine reductase isolated from a clinical isolate of L. donovani, we carried out molecular modeling and docking studies with two compounds of dihydrofolate reductase specificity showing promising antileishmanial activity in vitro. Both the inhibitors appeared to fit well in the active pocket revealing the tight binding of the carboxylic acid ethyl ester group of pyridine moiety to pteridine reductase and identify the important interactions necessary to assist the structure based development of novel pteridine reductase inhibitors.     

Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes

Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution. (1)H(N), (15)N, and (13)C(alpha) resonance assignments have been made for the ternary complex with folate and oxidized NADP(+) cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6-dihydroNADPH. The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme). Analysis of (1)H(N) and (15)N chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme. Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide-ribose binding sites of the NADPH cofactor. The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X-ray crystallography; there is no evidence that the open conformation observed in some X-ray structures of E. coli DHFR are populated in solution.     

The crystal structure of the bifunctional deaminase/reductase RibD of the riboflavin biosynthetic pathway in Escherichia coli: implications for the reductive mechanism

We have determined the crystal structure of the bi-functional deaminase/reductase enzyme from Escherichia coli (EcRibD) that catalyzes two consecutive reactions during riboflavin biosynthesis. The polypeptide chain of EcRibD is folded into two domains where the 3D structure of the N-terminal domain (1-145) is similar to cytosine deaminase and the C-terminal domain (146-367) is similar to dihydrofolate reductase. We showed that EcRibD is dimeric and compared our structure to tetrameric RibG, an ortholog from Bacillus subtilis (BsRibG). We have also determined the structure of EcRibD in two binary complexes with the oxidized cofactor (NADP(+)) and with the substrate analogue ribose-5-phosphate (RP5) and superposed these two in order to mimic the ternary complex. Based on this superposition we propose that the invariant Asp200 initiates the reductive reaction by abstracting a proton from the bound substrate and that the pro-R proton from C4 of the cofactor is transferred to C1 of the substrate. A highly flexible loop is found in the reductase active site (159-173) that appears to control cofactor and substrate binding to the reductase active site and was therefore compared to the corresponding Met20 loop of E. coli dihydrofolate reductase (EcDHFR). Lys152, identified by comparing substrate analogue (RP5) coordination in the reductase active site of EcRibD with the homologous reductase from Methanocaldococcus jannaschii (MjaRED), is invariant among bacterial RibD enzymes and could contribute to the various pathways taken during riboflavin biosynthesis in bacteria and yeast.     

Quinazoline folate analogs inhibit the catalytic activity of thymidylate synthase but allow binding of 5-fluorodeoxyuridylate

We have investigated some unusual aspects of the inhibition of mammalian thymidylate synthase (TS) by the folate antimetabolite, 10-propargyl-5,8-dideaza-folic acid (CB 3717). From our results, we conclude that binding of CB 3717 metabolites to one subunit of L1210 TS modified the conformation of the second active site of this enzyme so that it retained the ability to bind 5-fluro-2'-deoxyuridine-5'-monophosphate (FdUMP) but not its catalytic activity. Exposure of intact mouse L1210 cells to CB 3717 resulted in inactivation of cellular TS activity, yet desalted cytosol preparations from these cells retained the ability to bind FdUMP. The same effect was found with several analogs of CB 3717. Complexes of FdUMP formed in vitro with TS from cells exposed to CB 3717 were covalent and co-migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with complexes of FdUMP, folate cofactor, and TS from cells not exposed to CB 3717. In the presence of dUMP, a tightly bound complex rapidly formed between isolated pure TS and the pentaglutamate of CB 3717 but not the monoglutamate form of this compound. Binding experiments using CB 3717 pentaglutamate-inhibited TS suggested a stoichiometry of 1 mol of FdUMP bound per mol of dimeric TS.     

Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate

The protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP+ and the inhibitor determined at 2.2 A resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the beta6-alpha6 loop and alpha6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.     

Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites

Pteridine reductase (PTR1) is a short-chain reductase (SDR) responsible for the salvage of pterins in parasitic trypanosomatids. PTR1 catalyzes the NADPH-dependent two-step reduction of oxidized pterins to the active tetrahydro-forms and reduces susceptibility to antifolates by alleviating dihydrofolate reductase (DHFR) inhibition. Crystal structures of PTR1 complexed with cofactor and 7,8-dihydrobiopterin (DHB) or methotrexate (MTX) delineate the enzyme mechanism, broad spectrum of activity and inhibition by substrate or an antifolate. PTR1 applies two distinct reductive mechanisms to substrates bound in one orientation. The first reduction uses the generic SDR mechanism, whereas the second shares similarities with the mechanism proposed for DHFR. Both DHB and MTX form extensive hydrogen bonding networks with NADP(H) but differ in the orientation of the pteridine.     

Circular-dichroism studies of ligand binding to dihydrofolate reductase from Lactobacillus casei MTX/R.

Circular-dichroism spectra (200--450 nm) were recorded for Lactobacillus casei MTX/R dihydrofolate reductase and its complexes with substrates, inhibitors and coenzymes. These spectra are compared with those reported by others for dihydrofolate reductase from other sources. The binding of NADP+ or NADPH is associated with the perturbation of one or more aromatic amino acid residues, and there is marked enhancement of the negative c.d. band at 340 nm arising from the dihydronicotinamide chromophore of NADPH. The substrates folate and dihydrofolate give rise to substantial extrinsic c.d. bands on binding, which show a number of specific differences between enzymes from different sources. The binary complexes between the enzyme and the inhibitors methotrexate or trimethoprim also show strong c.d. bands, and these are qualitatively very similar for all dihydrofolate reductases studied so far. The ternary complexes between enzyme, NADPH and trimethoprim or methotrexate are very different from the sum of the spectra of the binary complexes. Trimethoprim leads to the disappearance of the 340 nm c.d. band of bound NADPH, whereas in the methotrexate--NADPH--enzyme ternary complex a "couplet" c.d. spectrum is observed at long wavelengths. Analysis of this latter feature suggests that it arises from a direct interaction between the dihydronicotinamide and pteridine rings in the ternary complex.     

Cofactor triggers the conformational change in thymidylate synthase: implications for an ordered binding mechanism.

We have solved crystal structures of two complexes with Escherichia coli thymidylate synthase (TS) bound either to the cofactor analog N10-propargyl-5,8-dideazafolate (CB3717) or to a tighter binding polygutamyl derivative of CB3717. These structures suggest that cofactor binding alone is sufficient to induce the conformational change in TS; dUMP binding is not required. Because polyglutamyl folates are the primary cofactor form in vivo, and because they can bind more tightly than dUMP to TS, these structures may represent a key intermediate along the TS reaction pathway. These structures further suggest that the dUMP binding site is accessible in the TS-cofactor analog binary complexes. Conformational flexibility of the binary complex may permit dUMP to enter the active site of TS while the cofactor is bound. Alternatively, dUMP may enter the active site from the opposite side that the cofactor appears to enter; that is, through a portal flanked by arginines that also coordinate the phosphate group in the active site. Entry of dUMP through this portal may allow dUMP to bind to a TS-cofactor binary complex in which the complex has completed its conformational transition to the catalytically competent structure.     

Overexpression in Escherichia coli and purification of pteridine reductase (PTR1) from a clinical isolate of Leishmania donovani

Pteridine reductase 1 (PTR1) is part of a novel metabolic pathway in Leishmania associated with folate metabolism. Its main function is to salvage pterins but a second one is to reduce folates. The novelty and possible uniqueness of the pathway in which PTR1 is involved opens the possibility of developing specific inhibitors, which in combination with dihydrofolate reductase inhibitors could be highly effective against Leishmania. In order to increase our understanding of this putative important chemotherapeutic target, we present here the cloning, overexpression and purification of this enzyme from a clinical isolate of Leishmania donovani causing kala azar in India. This recombinant enzyme will set the basis for inhibition studies as well as for structure-function relationships.     

Circular dichroism studies of dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428: ternary complexes.

Circular dichroism has been used to monitor the binding of pyridine nucleotide cofactors to enzyme-folate analog complexes of dihydrofolate reductase from Escherichia coli B (MB 1428). The enzyme binds one molar equivalent of many folate analogs and two molar equivalents of several pyridine nucleotide cofactors. The apo-enzyme has very low optical activity. The binding of folate analogs including folate, dihydrofolate, methotrexate, trimethoprim and pyrimethamine induce large Cotton effects. Pyridine nucleotides when bound to the enzyme-folate analog complexes also induce new optically active bands; all the effects being due to the first molar equivalent of cofactor bound. NADPH and NADP+ induce very similar bands when bound to the enzyme-methotrexate complex suggesting that the geometry of the complexes formed are very similar. The oxidized and reduced cofactor likewise have similar effects on the enzyme-folate complex. However, NADPH and NADP+ addition to both the enzyme-trimethoprim and enzyme-pyrimethamine complexes have significantly different effects on the circular dichroism spectra, suggesting that the inhibitors which are less homologous to the natural dihydrofolate substrate allow more conformational freedom in the enzyme-inhibitor-cofactor complex. In most cases the prior binding of the folate analog greatly increases the binding of the first molar equivalent of cofactor so that at concentrations of approx. 5-20 muM the binding appears stoichiometric. Pyrimethamine is an exception in that it apparently has no effect on the binding of NADPH to the enzyme.     

Molybdopterin guanine dinucleotide is the organic moiety of the molybdenum cofactor in respiratory nitrate reductase from Pseudomonas stutzeri

Abstract Respiratory nitrate reductase from the denitrifying bacterium Pseudomonas stutzeri is an iron-sulfur enzyme containing the molybdenum cofactor. Hydrolysis of native nitrate reductase with aqueous sulfuric acid revealed 0.92 mol of 5'-GMP per mol of enzyme. The pterin present in the molybdenum cofactor was liberated from the protein and reacted with iodoacetamide. The resulting di(carboxamidomethyl) (cam) derivative was purified on a C₁₈-cartridge and analyzed for its structural elements. Treatment of the cam derivative with nucleotide pyrophosphatase and subsequent HPLC analysis revealed the formation of di(cam)molybdopterin and 5'-GMP at a 1:1 molar ratio and with a yield of 79% with respect to the molybdenum content of the enzyme. Treatment of the cam derivative with nucleotide pyrophosphatase and alkaline phosphatase led to the liberation of 0.51 mol dephosphodi(cam)molybdopterin and of 0.59 mol guanosine per mol of enzyme, which is equal to a molar ratio of 1:2.2. The results indicate, that the organic moiety of the molybdenum cofactor of nitrate reductase from P. stutzeri is molybdopterin guanine dinucleotide of which one mol is contained per mol of nitrate reductase.     

Conformational change of the methionine 20 loop of Escherichia coli dihydrofolate reductase modulates pKa of the bound dihydrofolate

We evaluate the pK(a) of dihydrofolate (H(2)F) at the N(5) position in three ternary complexes with Escherichia coli dihydrofolate reductase (ecDHFR), namely ecDHFR(NADP(+):H(2)F) in the closed form (1), and the Michaelis complexes ecDHFR(NADPH:H(2)F) in the closed (2) and occluded (3) forms, by performing free energy perturbation with molecular dynamics simulations (FEP/MD). Our simulations suggest that in the Michaelis complex the pK(a) is modulated by the Met20 loop fluctuations, providing the largest pK(a) shift in substates with a "tightly closed" loop conformation; in the "partially closed/open" substates, the pK(a) is similar to that in the occluded complex. Conducive to the protonation, tightly closing the Met20 loop enhances the interactions of the cofactor and the substrate with the Met20 side chain and aligns the nicotinamide ring of the cofactor coplanar with the pterin ring of the substrate. Overall, the present study favors the hypothesis that N(5) is protonated directly from solution and provides further insights into the mechanism of the substrate protonation.     

Structure–activity relationship for enantiomers of potent inhibitors of B. anthracis dihydrofolate reductase

Background: Bacterial resistance to antibiotic therapies is increasing and new treatment options are badly needed. There is an overlap between these resistant bacteria and organisms classified as likely bioterror weapons. For example, Bacillus anthracis is innately resistant to the anti-folate trimethoprim due to sequence changes found in the dihydrofolate reductase enzyme. Development of new inhibitors provides an opportunity to enhance the current arsenal of anti-folate antibiotics while also expanding the coverage of the anti-folate class. Methods: We have characterized inhibitors of B. anthracis dihydrofolate reductase by measuring the K_i and MIC values and calculating the energetics of binding. This series contains a core diaminopyrimidine ring, a central dimethoxybenzyl ring, and a dihydrophthalazine moiety. We have altered the chemical groups extended from a chiral center on the dihydropyridazine ring of the phthalazine moiety. The interactions for the most potent compounds were visualized by X-ray structure determination. Results: We find that the potency of individual enantiomers is divergent with clear preference for the S-enantiomer, while maintaining a high conservation of contacts within the binding site. The preference for enantiomers seems to be predicated largely by differential interactions with protein residues Leu29, Gln30 and Arg53. Conclusions: These studies have clarified the activity of modifications and of individual enantiomers, and highlighted the role of the less-active R-enantiomer in effectively diluting the more active S-enantiomer in racemic solutions. This directly contributes to the development of new antimicrobials, combating trimethoprim resistance, and treatment options for potential bioterrorism agents.     

Mutant PTR1 proteins from Leishmania tarentolae: comparative kinetic properties and active-site labeling.

PTR1, the gene promoting MTX resistance following gene amplification or DNA transfection in Leishmania tarentolae and selected mutants, has been cloned and heavily overexpressed (>100 mg/liter) in Escherichia coli strain BL21 (DE3). Protein has been purified, essentially to homogeneity, in two steps, via ammonium sulfate precipitation and chromatography on DEAE-Trisacryl. The active proteins are tetramers and display optimal pteridine reductase activity at pH 6.0 using biopterin as substrate and NADPH as the reduced dinucleotide cofactor. 2,4-Diaminopteridine substrate analogues are strong competitive inhibitors (K(i) approximately 38 --> 3 nM) against the pterin substrate and both NADP(+) and folate are inhibitors although somewhat weaker. Dihydropteridines are poor substrates compared to the fully oxidized pteridine. Kinetic analysis affords the usual Michaelis constants and in addition shows that inhibition by NADP(+) allows the formation of ternary nonproductive complexes with folate. The kinetic results are consistent with a sequential ordered bi-bi kinetic mechanism in which first NADPH and then pteridine bind to the free enzyme. Sequence comparisons suggest that PTR1 belongs to the short-chain dehydrogenase/reductase (SDR) family containing an amino-terminal glycine-rich dinucleotide binding site plus a catalytic Y(Xaa)(3)K motif. In accord with this observation, the mutants K16A, Y37D, and R39A and the double mutants K17A:R39A and Y37D:R39A all show a two- to threefold lower binding affinity for NADPH and exhibit low or zero activity. Two Y(Xaa)(3)K regions are present in wild-type PTR1 at 152 and 194. Only Y194F gives protein with zero activity. This observation coupled with affinity labeling of PTR1 by oNADP(+) (2', 3'-dialdehyde derivative of NADP(+)) followed by NaBH(4) reduction, V8 protease digestion, and mass spectral analysis suggests that the motif participating in catalysis is that at 194. The mutation K198Q eliminates inactivation by oNADP(+) supporting the hypothesis that K198 is associated with nucleotide orientation, as has been demonstrated for similar lysine residues in other members of the SDR family.     

Importance of substrate and cofactor polarization in the active site of dihydrofolate reductase

By using a combined quantum-mechanical and molecular-mechanical potential in molecular dynamics simulations, we have investigated the effects of the enzyme electric field of dihydrofolate reductase on the electronic polarization of its 5-protonated dihydrofolate substrate at various stages of the catalyzed hydride transfer reaction. Energy decomposition of the total electrostatic interaction energy between the ligands and the enzyme shows that the polarization effect is 4% of the total electrostatic interaction energy, and, significantly, it accounts for 9kcal/mol of transition state stabilization relative to the reactant state. Therefore it is essential to take account of substrate polarization for quantitative interpretation of enzymatic function and for calculation of binding free energies of inhibitors to a protein. Atomic polarizations are calculated as the differences in the average atomic charges on the atoms in gas phase and in molecular simulations of the enzyme; this analysis shows that the glutamate tail and the pterin ring are the highly polarized regions of the substrate. Electron density difference plots of the reactant and product complexes at instantaneous configurations in the enzyme active center confirm the inferences made on the basis of partial atomic charges.     

Inactivation of the Leishmania tarentolae pterin transporter (BT1) and reductase (PTR1) genes leads to viable parasites with changes in folate metabolism and hypersensitivity to the antifolate methotrexate

The protozoan parasite Leishmania is a folate and pterin auxotroph. The main biopterin transporter (BT1) and pterin reductase (PTR1) have already been characterized in Leishmania. In this study, we have succeeded in generating a BT1 and PTR1 null mutant in the same Leishmania tarentolae strain. These cells are viable with growth properties indistinguishable from wildtype cells. However, in response to the inactivation of BT1 and PTR1, at least one of the folate transporter genes was deleted, and the level of the folylpolyglutamate synthetase activity was increased, leading to increased polyglutamylation of both folate and methotrexate (MTX). Secondary events following gene inactivation should be considered when analyzing a phenotype in Leishmania. The BT1/PTR1 null mutant is hypersensitive to MTX, but in a step-by-step fashion, we could induce resistance to MTX in these cells. Several resistance mechanisms were found to co-exist including a reduced folate and MTX accumulation, demonstrating that cells with no measurable biopterin uptake but also greatly reduced folate uptake are viable, despite their auxotrophy for each of these substrates. The resistant cells have also amplified the gene coding for the MTX target dihydrofolate reductase. Finally, we found a marked reduction in MTX polyglutamylation in resistant cells. These studies further highlight the formidable ability of Leishmania cells to bypass the blockage of key metabolic pathways.     

Dihydrofolate reductase from soybean seedlings: a fluorescence and circular dichroism study.

The fluorescence emission spectrum of soybean dihydrofolate reductase suggests that the emitting tryptophan residues are situated in a hydrophobic microenvironment. The dissociation constants determined from fluorescence and circular dichroism data reveal that the soybean enzyme has a lower affinity for substrates and substrate analogs than that determined for dihydrofolate reductases isolated from other sources. The binding of methotrexate to the soybean enzyme does not affect the binding of NADPH. Similarly, the binding of NADPH has no effect on subsequent methotrexate binding. Polarimetric study indicates that the enzyme has a low (ca. 5%) α-helical content. Addition of dihydrofolate to the soybean enzyme results in the generation of a positive ellipticity band at 298 nm with a molar ellipticity, [θ], of 186,000, whereas the binding of folate induces a negative ellipticity band at 280 nm with [θ] of ?181,000. The qualitative and quantitative differences in the circular dichroism of the enzyme-dihydrofolate and enzyme-folate complexes indicate that the mode of binding of these ligands may be different. The formation of an enzyme-NADPH complex is accompanied by a negative Cotton effect at 270 nm. These studies indicate that the binding of substrates or inhibitors causes significant conformational changes in the enzyme and also leads to the formation of a number of spectroscopically identifiable complexes.     

A sensitive high-performance liquid chromatographic-fluorometric assay for dihydrofolate reductase in adult rat brain, using 7,8-dihydrobiopterin as substrate

A liquid chromatographic-fluorometric assay has been developed to study the role of dihydrofolate reductase in adult rat brain since low levels of the enzyme preclude measurement by current spectrophotometric procedures. This method involves in vitro incubation of desalted, cell-free brain extracts with 7,8-dihydrobiopterin, NADPH, and an NADPH-regenerating system. The tetrahydrobiopterin formed is quantitatively converted to pterin using alkaline iodine oxidation, and the pterin formed is separated by liquid chromatography and detected fluorometrically. The method is linear from 100 fmol to greater than or equal to 1 nmol of product, and the sensitivity is at least 100 times greater than that of existing spectrophotometric assays. Enzyme activity of desalted brain extracts is linear with both time (to 100 min) and protein (from 50 to 620 micrograms). The enzyme shows an absolute requirement for NADPH, does not use NADH, and is completely inhibited by 10 nM methotrexate. The Km of the enzyme for NADPH was found to be 7.5 microM, while the Km for 7,8-dihydrobiopterin was 88 microM. Since brain dihydrobiopterin reductase has the same properties as dihydrofolate reductase, this fluorometric procedure can serve as a sensitive assay for dihydrofolate reductase.     

Crystal structure of Trypanosoma cruzi pteridine reductase 2 in complex with a substrate and an inhibitor

Reduced pteridines are required for a number of important cellular functions. Trypanosomatid parasites, unlike their mammalian hosts, are pteridine auxotrophs and salvage the precursor pteridines from the host and reduce them to the respective biologically active tetrahydro forms using parasite-encoded enzymes. These enzymes may offer selective drug targets. In Leishmania, pteridine reductase 1 (PTR1), the primary enzyme for reducing pterins, is also responsible for resistance to antifolate drugs. Typically, PTR1 is more active with fully oxidized biopterin and folate than with their reduced counterparts. We have identified an enzyme, TcPTR2 of Trypanosoma cruzi, which though very similar to PTR1 in its primary sequence, can reduce only dihydrobiopterin and dihydrofolate and not oxidized pteridines. The structures of an inhibitor (methotrexate) and a substrate (dihydrofolate) complex of this enzyme demonstrate that the orientation of the substrate and the inhibitor in the active site of TcPTR2 are different from each other. However, the orientation of each ligand is similar to that of the corresponding ligand in Leishmania major PTR1 complexes.     

Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase–dihydrofolate reductase

Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS–DHFR specific inhibitors.