Description of a novel eukaryotic deoxyuridine 5'-triphosphate nucleotidohydrolase in Leishmania major.
A Leishmania major full-length cDNA encoding a functional dUTP nucleotidohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expression library by genetic complementation of dUTPase deficiency in Escherichia coli. The cDNA contained an open reading frame that encoded a protein of 269 amino acid residues with a calculated molecular mass of 30.3 kDa. Although eukaryotic dUTPases exhibit extensive similarity and there are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significantly from its eukaryotic counterparts. None of the characteristic motifs were readily identifiable and the sequence encoded a larger polypeptide. However, the products of the reaction were dUMP and PPi, competition experiments with other deoxyribonucleoside triphosphates showed that the reaction is specific for dUTP, and the protozoan gene complemented dUTPase deficiency in Escherichia coli. The gene is of single copy; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure. The enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. The availability of recombinant enzyme in large quantities will now permit detailed mechanistic and structural studies, which might contribute to a rational design of specifically targeted inhibitors against dUTPase from L. major.
Camacho
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Instituto de Parasitología y Biomedicina 'López-Neyra', Consejo Superior de Investigaciones Científicas, Calle Ventanilla 11, 18001 Granada, Spain.
Arrebola
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R
Peña-Diaz
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J
Ruiz-Pérez
L M
LM
González-Pacanowska
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D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003854:Deoxyribonucleotides / Q000494:pharmacology; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D003854:Deoxyribonucleotides; D003856:Deoxyuracil Nucleotides; D011994:Recombinant Proteins; C007267:2'-deoxyuridylic acid; D013762:Tetrahydrofolate Dehydrogenase; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase