Properties of Leishmania major dUTP nucleotidohydrolase, a distinct nucleotide-hydrolysing enzyme in kinetoplastids.
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.
Camacho
A
A
Instituto de Parasitología y Biomedicina 'López-Neyra', Consejo Superior de Investigaciones Científicas, Calle Ventanilla 11, 18001-Granada, Spain.
Hidalgo-Zarco
F
F
Bernier-Villamor
V
V
Ruiz-Pérez
L M
LM
González-Pacanowska
D
D
D000818:Animals; D002412:Cations / Q000494:pharmacology; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D019281:Dimerization; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D018087:Plastids / Q000201:enzymology*; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012969:Sodium Fluoride / Q000494:pharmacology; D013379:Substrate Specificity; D013816:Thermodynamics
D002412:Cations; D003856:Deoxyuracil Nucleotides; D011756:Diphosphates; D009711:Nucleotides; D010710:Phosphates; D011994:Recombinant Proteins; C027078:deoxyuridine triphosphate; D012969:Sodium Fluoride; C007267:2'-deoxyuridylic acid; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase