Properties of Leishmania major dUTP nucleotidohydrolase, a distinct nucleotide-hydrolysing enzyme in kinetoplastids

Abstract

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.

The Crystal Structure of the Leishmania major Deoxyuridine Triphosphate Nucleotidohydrolase in Complex with Nucleotide Analogues, dUMP, and Deoxyuridine

“…These enzymes act as dimers and are able to hydrolyze not only dUTP but also dUDP (15), whereas dUDP has been shown to be an inhibitor of the trimeric enzyme in E. coli (16). They are, in addition, subject to product inhibition by dUMP (17). The structure of TcdUTPase revealed a dimeric all ␣-helical structure that is very distinct from the trimeric enzymes (18), with which they share no sequence similarity.…”

“…The metals were implicated in substrate binding and led to the proposal that these dimeric dUTPases utilize a mechanism of reaction similar to that used in the phosphoryl transfer reactions catalyzed by DNA polymerases (20,21). Indeed, the dimeric enzyme, like the trimeric one, requires divalent metal ions for activity (17).…”

Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool

“…Kinetic characterization of these novel forms of dUTPases showed that they accepted either dUDP or dUTP as a substrate, whereas the dUMP product was found to be their potent inhibitor, which clearly indicated the distinct mode of action of these enzymes [88,93]. The first dimer dUTPase structure (Trypanosoma cruzi dUTPase) revealed many structural and mechanistic aspects [94].…”

“…A group of dUTPases has been shown to form homodimers in solution and possess sequence conservation that is different from the monomeric or trimeric dUTPases [88]. These results, combined with phylogenetic analysis, allowed the definition of a new superfamily of d(C/U)TPases [36], including several distinct enzyme families (dUTPases in trypanosomatides, Campylobacter jejuni and dCTP/dUTPases in some Gram-negative bacteria and T4-like phages, as well as dUTPases in various Gram-positive bacteria and their phages) [89][90][91].…”

Kinetic properties and inhibition of the dimeric dUTPase-dUDPase fromCampylobacter jejuni

“…The soluble crude extract was obtained by sonication and centrifugation at 11000xg. The purification protocol developed consisted in three chromatographic steps (Table I), hydroxyapatite, anion exchange chromatography in DEAE-cellulose, and mono-Q chromatography as previously described for dimeric enzymes [15,16]. This protocol gives 20 mg of protein per liter of culture and the protein is more than 95% free of impurities as judged by SDS-PAGE analysis with Coomassie blue staining of overloaded gels.…”