Kinetic and thermodynamic characterization of dUTP hydrolysis by Plasmodium falciparum dUTPase

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and plays an important role in nucleotide metabolism and DNA replication controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. Isothermal titration calorimetry has been applied to the determination of the kinetic and thermodynamic parameters of the trimeric Plasmodium falciparum dUTPase, a potential drug target against malaria. The role of divalent ions in binding, and inhibition by different uridine derivatives has been assessed. When dUTP hydrolysis in the presence of EDTA was evaluated, a 105-fold decrease and a 12-fold increase of the k(cat) and K(m) values, respectively, were observed when compared with the dUTP.Mg(2+) complex. Calculation of the activation energy, E(a), and the thermodynamic activation parameters showed that the energetic barrier was ~4-fold higher when Mg(2+) was depleted. Other divalent ions such as Co(2+) or Mn(2+) can substitute the physiological cofactor, however the k(cat) was significantly reduced compared to dUTP.Mg(2+). Binding and inhibition by dU, dUMP, dUDP, and alpha,beta-imido-dUTP were analysed by ITC and compared with data obtained by spectrophotometric methods and binding equilibrium studies. Product inhibition (K(ip) dUMP: 99.34 muM) was insignificant yet K(i) values for dUDP and alpha,beta-imido-dUTP were in the low micromolar range. The effect of ionic strength on protein stability was also monitored. DSC analysis evidenced a slight increase in the unfolding temperature, T(m), with increasing salt concentrations. Moreover, the thermal unfolding pathway in the presence of salt fits adequately to an irreversible two-state model (N(3)-->3D).     

Kinetic properties and specificity of trimeric Plasmodium falciparum and human dUTPases

Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. Hydrolysis of other nucleotides similar in structure to dUTP would be physiologically negative and therefore high substrate specificity is essential. Binding and hydrolysis of nucleotides different to dUTP by the dUTPases from Plasmodium falciparum (PfdUTPase) and human (hdUTPase) was evaluated by applying isothermal titration calorimetry (ITC). The ribo and deoxyribonucleoside triphosphates dGTP, dATP, dCTP, dTTP, UTP, FdUTP and IdUTP have been analysed. dUTP and FdUTP were the most specific substrates for both enzymes. The specificity constants (k_cat/K_m) for the remaining ones, except for the IdUTP, were very similar for both enzymes, although PfdUTPase showed a slightly higher specificity for dCTP and UTP and the human enzyme for dTTP and dCTP. PfdUTPase was very efficient in using FdUTP as substrate indicating that small size substituents in the 5′ position are well tolerated. In addition product inhibition was assessed by binding studies with the nucleoside monophosphate derivatives and thermodynamic parameters were established. When FdUTP hydrolysis was monitored, Plasmodium dUTPase was more sensitive to end-product inhibition by FdUMP than the human enzyme. Taken together these results highlight further significant differences between the human and Plasmodium enzymes that may be exploitable in selective inhibitor design.     

The crystal structure of Trypanosoma cruzi dUTPase reveals a novel dUTP/dUDP binding fold

dUTPase is an essential enzyme involved with nucleotide metabolism and replication. We report here the X-ray structure of Trypanosoma cruzi dUTPase in its native conformation and as a complex with dUDP. These reveal a novel protein fold that displays no structural similarities to previously described dUTPases. The molecular unit is a dimer with two active sites. Nucleotide binding promotes extensive structural rearrangements, secondary structure remodeling, and rigid body displacements of 20 A or more, which effectively bury the substrate within the enzyme core for the purpose of hydrolysis. The molecular complex is a trapped enzyme-substrate arrangement which clearly demonstrates structure-induced specificity and catalytic potential. This enzyme is a novel dUTPase and therefore a potential drug target in the treatment of Chagas' disease.     

dUTPase as a platform for antimalarial drug design: structural basis for the selectivity of a class of nucleoside inhibitors

Pyrimidine metabolism is a major route for therapeutic intervention against malaria. Here we report inhibition and structural studies on the deoxyuridine nucleotidohydrolase from the malaria parasite Plasmodium falciparum (PfdUTPase). We have identified a series of triphenylmethane derivatives of deoxyuridine with antimalarial activity in vitro which inhibit specifically the Plasmodium dUTPase versus the human enzyme. A 2.4 Angstrom crystal structure of PfdUTPase in complex with one of these inhibitors reveals an atypical trimeric enzyme in which the triphenylmethane derivative can be seen to select for PfdUTPase by way of interactions between the trityl group and the side chains of residues Phe46 and Ile117. Immunofluorescence microscopy studies of parasitized red blood cells reveal that enzyme concentrations are highest during the trophozoite/schizont stages, suggesting that PfdUTPase has a major role in DNA replication. Taken together the data show that PfdUTPase may be considered as an antimalarial drug target.     

Methylene substitution at the alpha-beta bridging position within the phosphate chain of dUDP profoundly perturbs ligand accommodation into the dUTPase active site

dUTP pyrophosphatase, a preventive DNA repair enzyme, contributes to maintain the appropriate cellular dUTP/dTTP ratio by catalyzing dUTP hydrolysis. dUTPase is essential for viability in bacteria and eukaryotes alike. Identification of species-specific antagonists of bacterial dUTPases is expected to contribute to the development of novel antimicrobial agents. As a first general step, design of dUTPase inhibitors should be based on modifications of the substrate dUTP phosphate chain, as modifications in either base or sugar moieties strongly impair ligand binding. Based on structural differences between bacterial and human dUTPases, derivatization of dUTP-analogous compounds will be required as a second step to invoke species-specific character. Studies performed with dUTP analogues also offer insights into substrate binding characteristics of this important and structurally peculiar enzyme. In this study, alpha,beta-methylene-dUDP was synthesized and its complex with dUTPase was characterized. Enzymatic phosphorylation of this substrate analogue by pyruvate kinase was not possible in contrast to the successful enzymatic phosphorylation of alpha,beta-imino-dUDP. One explanation for this finding is that the different bond angles and the presence of the methylene group may preclude formation of a catalytically competent complex with the kinase. Crystal structure of E. coli dUTPase:alpha,beta-methylene-dUDP and E. coli dUTPase:dUDP:Mn complexes were determined and analyzed in comparison with previous data. Results show that the "trans" alpha-phosphate conformation of alpha,beta-methylene-dUDP differs from the catalytically competent "gauche" alpha-phosphate conformation of the imino analogue and the oxo substrate, manifested in the shifted position of the alpha-phosphorus by more than 3 A. The three-dimensional structures determined in this work show that the binding of the methylene analogue with the alpha-phosphorus in the "gauche" conformation would result in steric clash of the methylene group with the protein atoms. In addition, the metal ion cofactor was not bound in the crystal of the complex with the methylene analogue while it was clearly visible as coordinated to dUDP, arguing that the altered phosphate chain conformation also perturbs metal ion complexation. Isothermal calorimetry titrations indicate that the binding affinity of alpha,beta-methylene-dUDP toward dUTPase is drastically decreased when compared with that of dUDP. In conclusion, the present data suggest that while alpha,beta-methylene-dUDP seems to be practically nonhydrolyzable, it is not a strong binding inhibitor of dUTPase probably due to the altered binding mode of the phosphate chain. Results indicate that in some cases methylene analogues may not faithfully reflect the competent substrate ligand properties, especially if the methylene hydrogens are in steric conflict with the protein.     

Cell cycle- and differentiation stage-dependent variation of dUTPase activity in higher plant cells

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a key enzyme in pyrimidine nucleotide metabolism, specifically hydrolyzes deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate and inorganic pyrophosphate. This enzyme activity has been studied in cellular extracts from Allium cepa root meristem cells with two specific aims: (i) to determine how the properties of the plant enzyme compare with those of dUTPase purified from other sources, and (ii) to analyze the relationship between dUTPase activity and cell proliferation and cell differentiation. Plant dUTPase is highly specific for dUTP, with an apparent Km of 6 microM, is highly sensitive to EDTA and it is probably a metalloenzyme. Our results demonstrate the presence of high levels of dUTPase in both resting and proliferating root meristem cells. The enzyme activity appears to be tightly regulated during the cell cycle. dUTPase activity increases at the G1/S boundary, remains high throughout S phase, and shows almost undetectable levels during G1 and G2. We have also found that dUTPase activity in differentiated cells, located in the mature portion of the root, is barely detectable. Altogether our results indicate that dUTPase activity is modulated by the proliferation rate and that this activity progressively decreases as cells initiate their differentiation program.     

Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from Campylobacter jejuni

The enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) catalyses the hydrolysis of dUTP to dUMP and PPi thus controlling the incorporation of uracil into DNA genomes. In Campylobacter jejuni dUTPase exhibits structural properties of dimeric proteins characteristic of protozoa of the Kinetoplastidae family. In the present study we perform a kinetic analysis of Campylobacter dUTPase using the continuous spectrophotometric method and show that the enzyme is highly specific for deoxyuridine nucleotides. The Michaelis-Menten constant for dUTP was 0.66 microM while the k(cat) was 12.3 s(- 1). dUDP was also efficiently hydrolysed although the specificity constant, k(cat)/K(m), was five fold lower than for dUTP. The reaction product and the non hydrolysable analogue alpha,beta imido dUDP are potent inhibitors of the enzyme while several analogues of dUMP with substituents at the 3'- and 5'-positions active against trimeric dUTPases, show poor inhibitory activity. Apparent structural and kinetic differences with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against campylobacteriosis.     

Multidimensional NMR identifies the conformational shift essential for catalytic competence in the 60-kDa Drosophila melanogaster dUTPase trimer

The catalytic mechanism of dUTP pyrophosphatase (dUTPase), responsible for the prevention of uracil incorporation into DNA, involves ordering of the flexible C terminus of the enzyme. This conformational shift is investigated by multidimensional NMR on the Drosophila enzyme. Flexible segments of the homotrimer give rise to sharp resonances in the (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra, which are clearly distinguishable from the background resonances of the well folded protein globule. Binding of the product dUMP or the analogues dUDP and alpha,beta-imino-dUTP to the enzyme induces a conformational change reflected in the disappearance of eight sharp resonances. This phenomenon is interpreted as nucleotide binding-induced ordering of some residues upon the folded protein globule. Three-dimensional (15)N-edited (1)H-(15)N HSQC total correlation spectroscopy (TOCSY) and (1)H-(15)N HSQC nuclear Overhauser effect spectroscopy measurements allowed clear assignment of these eight specific resonance peaks. The residues identified correspond to the conserved C-terminal sequence motif, indicating that (i) this conformational shift is amenable to NMR studies in solution even in the large trimeric molecule and (ii) formation of the closed enzyme conformer in the case of the Drosophila enzyme does not require the complete triphosphate chain of the substrate. NMR titration of the enzyme with the nucleotide ligands as well as kinetic data indicated significant deviation from the model of independent active sites within the homotrimer. The results suggest allosterism in the eukaryotic dUTPase.     

β-Branched acyclic nucleoside analogues as inhibitors of Plasmodium falciparum dUTPase

We report a series of β-branched acyclic tritylated deoxyuridine analogues as inhibitors of Plasmodium falciparum deoxyuridine-5'-triphosphate nucleotidohydrolase (PfdUTPase), an enzyme involved in nucleotide metabolism that acts as first line of defence against uracil incorporation into DNA. Compounds were assayed against both PfdUTPase and intact parasites showing a correlation between enzyme inhibition and cellular assays. β-Branched acyclic uridine analogues described here showed equal or slightly better potency and selectivity compared with previously reported analogues. The best inhibitor gave a K(i) of 0.5 μM against PfdUTPase with selectivity greater than 200-fold compared to the corresponding human enzyme and sub-micromolar growth inhibition of P. falciparum (EC(50) 0.6 μM). A crystal structure of the complex of PfdUTPase with one of the inhibitors shows that this acyclic derivative binds to the active site in a similar manner to that previously reported for a tritylated cyclic deoxyuridine derivative.     

Mechanism of Unfolding of Goat Lung Cystatin During Urea and Guanidine Hydrochloride Induced Denaturation

Cystatins essentially regulate lysosomal cysteine protease besides affecting several physiological processes. In the present study, denaturation of a high molecular weight cystatin (Mr 66.4 kDa) purified from goat lung (GLC-I) has been studied by monitoring its inhibitory activity, intrinsic fluorescence, circular dichroism (CD), and binding of ANS. It was found that increasing concentration of GdnHCl significantly enhances the inactivation and unfolding of the purified inhibitor (GLC-I) with complete loss of inhibitory activity at 4 M GdnHCl. Denaturation of GLC-I in the presence of GdnHCl is accompanied by red shift (15 nm) of the emission maximum as shown by intrinsic fluorescence. The inhibitory activity of GLC-I was increased by 1.5 fold at 2 M urea; however, it decreased with further increased of the urea concentration. Intrinsic fluorescence studies of GLC-I in the presence of 0–3 M urea shows blue shift of 5 nm, suggesting stabilization of the inhibitor followed by 5 nm red shift at higher concentration. ANS binding studies in the presence of urea indicate significant changes in the tertiary structure of the inhibitor. Thus, our result shows denaturation profile of GLC-I following simple two state transitions in the presence of GdnHCl while it proceeds through an intermediate state in the presence of urea.     

Effect of urea denaturation on tryptophan fluorescence and nucleotide binding on tubulin studied by fluorescence and NMR spectroscopic methods

Tubulin, the major protein of microtubules, has been shown to be an example of protein undergoing multistep unfolding. Local unfolding and stepwise loss of a number of characteristic functions were demonstrated. In order to understand urea induced effects on tryptophan fluorescence and nucleotide binding on tubulin, both fluorescence and NMR techniques were used. Tubulin was denatured by different urea concentrations. The present experiments were carried out at concentrations of tubulin (to approximately 10 microM) at which most of the protein will be in the dimeric state. Quenching studies in the presence of KI suggest that all the tryptophans are fairly solvent exposed. Similar studies using acrylamide as quencher, suggest unfolding of tubulin at these protein concentrations to be an apparent two state process between the native and the completely unfolded states unlike at low concentrations where a partially folded intermediate was observed. No observable effects of the nucleotide or the metal ion on tryptophan fluorescence were observed. An attempt was made using NMR to monitor the changes in the nucleotide interaction with tubulin as the protein is unfolded by urea denaturation. No significant effects were observed in the binding of the nucleotide to tubulin by urea denaturation.     

Presence of dUTPase in the Various Human Endogenous Retrovirus K (HERV-K) Families

Various retroviruses have been shown to encode dUTPase. The overall phylogeny of dUTPase is unclear, though. The human genome contains a significant amount of human endogenous retroviruses (HERV) representing fossilized sequences of ancient exogenous retroviruses. A few HERV families have been reported to harbor dUTPase domains. We surveyed the various HERV families for the presence of dUTPase and found that ancestors of all HERV-K families but one encoded dUTPase. With two exceptions phylogenetic analysis shows a monophyletic origin of dUTPase for the different HERV-K dUTPases. Sequences of consensus dUTPase domains suggest that the various exogenous ancestors of HERV-K once encoded active enzymes. Our analysis provides informations on dUTPase phylogeny and further shows that endogenous retroviruses provide important informations regarding retrovirus evolution.     

Mechanistic studies of dUTPases

The essential enzyme dUTPase is responsible for preventive DNA repair via exclusion of uracil. Lack or inhibition of the enzyme induces thymine-less cell death in cells performing active DNA synthesis, serving therefore as an important chemotherapeutic target. In the present work, employing differential circular dichroism spectroscopy, we show that D. mel. dUTPase, a recently described eukaryotic model, has a similar affinity of binding towards alpha,beta-imino-dUTP as compared to the prokaryotic E. coli enzyme. However, in contrast to the prokaryotic dUTPase, the nucleotide exerts significant protection against tryptic digestion at a specific tryptic site 20 A far from the active site in the fly enzyme. This result indicates that binding of the nucleotide in the active site induces an allosteric conformational change within the central threefold channel of the homotrimer exclusively in the eukaryotic enzyme. Nucleotide binding induced allosterism in the D. mel. dUTPase, but not in the E. coli enzyme, might be associated with the altered hydropathy of subunit interfaces in these two proteins.     

The turnover of deoxyuridine triphosphate during the HeLa cell cycle

The synthesis and breakdown of deoxyuridine triphosphate (dUTP) was studied to determine whether a dUTP pool is present at any stage of the HeLa cell cycle. Although cell extracts were found to be capable of phosphorylating dUMP to dUTP, only minimal quantities of intracellular dUMP, dUDP or dUTP could be detected. When thymidylate synthetase was blocked with FUdR the dUMP pool increased but no substantial increase in dUDP or dUTP was seen. A powerful and specific dUTP nucleotidohydrolase (dUTPase, EC3.6.1.23) which hydrolyses dUTP to dUMP and PPi was detected. The activity of this enzyme as well as that of the dUTP synthesizing enzymes was low in G1, rose through S and G2 and reached a maximum just prior to cell division. Pulsing experiments with [5-³H]UdR and [¹⁴C]TdR suggest that the size of the dUTP pool is 1% of the dTTP pool.     

Effect of an Asp80Ala substitution on the binding of dUTP and dUMP to Trypanosoma cruzi dUTPase

dUTPase (deoxyuridine 5'-triphosphate nucleotide hydrolase) is an enzyme responsible for maintaining low levels of intracellular dUTP and thus prevents uracil incorporation into DNA by DNA polymerases during replication and repair processes. The thermodynamics of binding for both dUTP and dUMP (deoxyuridine 5'-monophosphate) to the D80A mutant form of Trypanosoma cruzi dUTPase have been investigated by fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry. In the presence of magnesium, approximately a 30-fold decrease in the value of the k(cat) and a 15-fold increase in the K(m) for dUTP hydrolysis was calculated while a 5-fold decrease was observed in the affinity for dUMP. In the absence of magnesium, the affinity for dUTP binding was similar for both enzymes while that for dUMP was lowered 3-fold as a consequence of the mutation. Calorimetric titrations in several buffers with different ionization heats rendered similar proton exchanges during the binding of dUMP. Thus, apparently the side chain of Asp 80 does not seem to vary its protonation state during the binding process. The enthalpy change values for the D80A mutant hardly change with temperature and, in addition, were Mg(2+) independent. We conclude that the D80A mutation induces only a slight conformational change in the active site yet results in a significant alteration of nucleotide binding and modifies the ability of the enzyme to discriminate between dUTP and dUMP when magnesium is present.     

Structural characterization of protein kinase A as a function of nucleotide binding. Hydrogen-deuterium exchange studies using matrix-assisted laser desorption ionization-time of flight mass spectrometry detection

Transient state kinetic studies indicate that substrate phosphorylation in protein kinase A is partially rate-limited by conformational changes, some of which may be associated with nucleotide binding (Shaffer, J., and Adams, J. A. (1999) Biochemistry 38, 12072-12079). To assess whether specific structural changes are associated with the binding of nucleotides, hydrogen-deuterium exchange experiments were performed on the enzyme in the absence and presence of ADP. Four regions of the protein are protected from exchange in the presence of ADP. Two regions encompass the catalytic and glycine-rich loops and are integral parts of the active site. Conversely, protection of probes in the C terminus is consistent with nucleotide-induced domain closure. One protected probe encompasses a portion of helix C, a secondary structural element that does not make any direct contacts with the nucleotide but has been reported to undergo segmental motion upon the activation of some protein kinases. The combined data suggest that binding of the nucleotide has distal structural effects that may include stabilizing the closed state of the enzyme and altering the position of a critical helix outside the active site. The latter represents the first evidence that the nucleotide alone can induce changes in helix C in solution.     

Altered active site flexibility and a structural metal-binding site in eukaryotic dUTPase: kinetic characterization, folding, and crystallographic studies of the homotrimeric Drosophila enzyme

dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54 degrees C, 23 degrees C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg(2+) binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.     

Binding of mercury(II) to poly(dA-dT) studied by proton nuclear magnetic resonance

The binding of Hg(II) to poly(dA-dT) has been examined with proton NMR spectroscopy. Addition of HgCl2 between r (Hg2+/nucleotide) = 0 and 0.25 results in loss of the exchangeable imino N3H resonance of thymine, indicating preferential binding at this site. The nonexchangeable base resonances AH8, AH2, and TH6 shift their intensity downfield in a cooperative manner, indicating complexation which is slow on the NMR time scale and changes in the polymer conformation upon binding. At r = 0.25, the polymer is cross-linked, and an increase in temperature does not result in denaturation of the polymer, as evidenced by the thymine proton resonance chemical shifts. The chemical shifts of the AH2 and T(CH3)5 base resonances allow some general conclusions to be made about the stereochemistry of this complex.     

Differences in the processes of beta-lactoglobulin cold and heat denaturations.

The changes in beta-lactoglobulin upon cold and heat denaturation were studied by scanning calorimetry, CD, and NMR spectroscopy. It is shown that, in the presence of urea, these processes of beta-lactoglobulin denaturation below and above 308 K are accompanied by different structural and thermodynamic changes. Analysis of the NOE spectra of beta-lactoglobulin shows that changes in the spin diffusion of beta-lactoglobulin after disruption of the unique tertiary structure upon cold denaturation are much more substantial than those upon heat denaturation. In cold denatured beta-lactoglobulin, the network of residual interactions in hydrophobic and hydrophilic regions of the molecules is more extensive than after heat denaturation. This suggests that upon cold- and heat-induced unfolding, the molecule undergoes different structural rearrangements, passing through different denaturation intermediates. From this point of view, cold denaturation can be considered to be a two stage process with a stable intermediate. A similar equilibrium intermediate can be obtained at 35 degrees C in 6.0 M urea solution, where the molecule has no tertiary structure. Cooling or heating of the solution from this temperature leads to unfolding of the intermediate. However, these processes differ in cooperativity, showing noncommensurate sigmoidal-like changes in efficiency of spin diffusion, ellipticity at 222 nm, and partial heat capacity. The disruption with cooling is accompanied by cooperative changes in heat capacity, whereas with heating the heat capacity changes only gradually. Considering the sigmoidal shape of the heat capacity change an extended heat absorption peak, we propose that the intermediate state is stabilized by enthalpic interactions.