Characterization of deoxyuridine 5′‐triphosphate nucleotidohydrolase from Trypanosoma cruzi¹

Abstract

We report the cloning and kinetic characterization of Trypanosoma cruzi deoxyuridine 5P P-triphosphate nucleotidohydrolase (dUTPase) whose coding sequence was isolated by genetic complementation in Escherichia coli. The deduced amino acid sequence was similar to Leishmania major dUTPase although it exhibits an amino acid insertion which is sensitive to protease inactivation. The catalytically active species of the enzyme is a dimer and a detailed kinetic characterization showed that it is highly speci¢c for dUTP and dUDP. The general observation that dUTPases from the Trypanosomatidae di¡er in sequence, conformation and substrate speci¢city suggests that a di¡erent family of dUTPases exists in certain organisms, which may be exploited as drug targets against infectious diseases. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Trypanosoma cruzi Contains a Single Detectable Uracil-DNA Glycosylase and Repairs Uracil Exclusively Via Short Patch Base Excision Repair

“…Thus, proteins serving DNAprotective functions in T. cruzi are considered potential targets for drug therapy. Two such enzymes encoded by the T. cruzi genome are deoxyuridine pyrophosphatase (TcdUTPase) 5 and uracil-DNA glycosylase (TcUNG) 6 which are both involved in protection against uracil in DNA. The former is responsible for the hydrolysis of dUTP to dUMP and pyrophosphate, and thereby eliminates the triphosphate form of deoxyuridine, and erroneous incorporation of dUMP into DNA.…”

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].…”

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

“…Data of the multiple-turnover hydrolysis of dUTP by C. jejuni dUTPase was fitted 13.1 T.cruzi [9] dimer 0.5 2. Using dUTP concentrations between 5 and 100 mM, the real K m value obtained was 0.66 mM.…”

“…dUTPases are present in a variety of different oligomeric forms: monomeric forms are found in herpes virus [5]; homodimers in the Trypanosomatidae and certain bacteria [6,7]; and homotrimers in bacteria and mammals. The homodimeric enzymes lack sequence and structure conservation with the trimeric group of enzymes [8,9]. Campylobacter jejuni, is a microaerophilic, Gramnegative, flagellate, spiral bacterium closely related to the gastric pathogen Helicobacter pylori.…”