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 Å 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.

Kinetic properties of the dimeric enzyme dUTPase from Leishmania major were studied using a continuous spectrophotometric method. dUTP was the natural substrate and dUMP and PPi the products of the hydrolysis. The trypanosomatid enzyme exhibited a low K m value for dUTP (2.11 μM), a k cat of 49 s −1 , strict Michaelis–Menten kinetics and is a potent catalyst of dUDP hydrolysis, whereas in other dUTPases described, this compound acts as a competitive inhibitor. Discrimination is achieved for the base and sugar moiety showing specificity constants for different dNTPs similar to those of bacterial, viral, and human enzymes. In the alkaline range, the K m for dUTP increases with the dissociation of ionizable groups showing pK a values of 8.8, identified as the uracil moiety of dUTP and 10, whereas in the acidic range, K m is regulated by an enzyme residue exhibiting a pK a of 7.1. Activity is strongly inhibited by the nucleoside triphosphate analog α‐β‐imido‐dUTP, indicating that the enzyme can bind triphosphate analogs. The existence of specific inhibition and the apparent structural and kinetic differences (reflected in different binding strength of dNTPs) with other eukaryotic dUTPases suggest that the present enzyme might be exploited as a target for new drugs against leishmaniasis.

We have previously reported the presence, in the parasitic protozoan Leishmania major, of an enzyme involved in controlling intracellular dUTP levels. The gene encoding this enzyme has now been overexpressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical and enzymic analyses of the Leishmania enzyme show that it is a novel nucleotidohydrolase highly specific for deoxyuridine 5'-triphosphate. The enzyme has proved to be a dimer by gel filtration and is able to hydrolyse both dUTP and dUDP quite efficiently, acting as a dUTP nucleotidohydrolase (dUTPase)-dUDP nucleotidohydrolase but has a limited capacity to act upon other nucleoside di- or triphosphates. The reaction products are dUMP and PP(i) when dUTP is the substrate and dUMP and P(i) in the case of dUDP. The enzyme is sensitive to inhibition by the reaction product dUMP but not by PP(i). dUTPase activity is highly dependent on Mg(2+) concentrations and markedly sensitive to the phosphatase inhibitor, NaF. In summary, Leishmania dUTPase appears to be markedly different to other proteins characterized previously that accomplish the same function.

Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity. We have isolated cDNAs from the Trypanosomatidae Leishmania major and Trypanosoma cruzi capable of complementing the deficiency of exonuclease III and dUTPase in the Escherichia coli mutant BW286. This double mutant is non-viable at 37 degreesC due to an accumulation of non-repaired sites following excision of uracil from DNA. The genes were expressed as beta-galactosidase-AP endonuclease fusion proteins and as such are active in repair of AP sites in E. coli. The Trypanosoma and Leishmania sequences have unique N-termini containing sequences that correspond to probable nuclear transport signals, while the C-terminal domains exhibit pronounced similarity to exonuclease III. The L.major gene was overexpressed as a histidine-tagged protein and recombinant enzyme exhibited endonuclease activity on AP DNA in vitro. Furthermore, expression of the enzymes in AP endonuclease-deficient E.coli mutants conferred significant resistance to killing by methylmethane sulphonate and peroxides. This study constitutes one of the first descriptions of DNA repair enzymes in these pathogenic organisms where oxidative stress is an important mechanism of both drug-mediated and intracellular killing.

Crystals of Trypanosoma cruzi dUTPase have been grown. Two different morphologies are observed, depending on the molecular weight of the PEG used as precipitating agent in the mother liquor, both having a hexagonal unit cell with similar dimensions. Complete X-ray diffraction data have been collected to low resolution for one of the forms. The space group is P6322, with unit-cell dimensions a = 134.15, c = 147.05 A. Peaks in the self-rotation function and the solvent content are consistent with two molecules of dUTPase per asymmetric unit.

Isothermal titration microcalorimetry and equilibrium dialysis have been used to characterize the binding of 2′‐deoxycytidine 5′‐monophosphate (dCMP) to the Asn229Asp mutant of Lactobacillus casei recombinant thymidylate synthase at pH 7.4 over a temperature range of 15°C to 35°C. Equilibrium dialysis analysis shows that dCMP binds to two sites in the dimer of both wild‐type and mutant thymidylate synthase. A concomitant net uptake of protons with binding of dCMP to both enzymes, was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The change in protonation for binding of dCMP to wild‐type enzyme is lower than that obtained for binding of this nucleotide to TS N229D, which suggests that the pK value of Asp‐229 is increased upon dCMP binding to the mutant enzyme. At 25°C, although the binding of dCMP to wild‐type and N229D TS is favoured by both enthalpy and entropy changes, the enthalpy change is more negative for the mutant protein. Thus, the substitution of Asn 229 for Asp results in a higher affinity of TS for dCMP due to a more favourable enthalpic contribution. The Gibbs energy change of binding of dCMP to the mutant enzyme is weakly temperature‐dependent, because of the enthalpy‐entropy compensation arising from a negative heat capacity change of binding equal to −0.83±0.02 kJ K−1 per mol of dCMP bound.