DESCRIPTION OF A NOVEL EUKARYOTIC DEOXYURIDINE 5'-TRIPHOSPHATE NUCLEOTIDOHYDROLASE IN LEISHMANIA-MAJOR

A Leishmania major full-length cDNA encoding a functional dUTP nucleot idohydrolase (dUTPase; EC 3.6.1.23) was isolated from a cDNA expressio n library by genetic complementation of dUTPase deficiency in Escheric hia coli. The cDNA contained an open reading frame that encoded a prot ein of 269 amino acid residues with a calculated molecular mass of 30. 3 kDa. Although eukaryotic dUTPases exhibit extensive similarity and t here are five amino acid motifs that are common to all currently known dUTPase enzymes, the sequence of the protozoan gene differs significa ntly from its eukaryotic counterparts. None of the characteristic moti fs were readily identifiable and the sequence encoded a larger polypep tide. However, the products of the reaction were dUMP and PPi, competi tion experiments with other deoxyribonucleoside triphosphates showed t hat the reaction is specific for dUTP, and the protozoan gene compleme nted dUTPase deficiency in Escherichia coli. The gene is of single cop y; Northern blots indicated a transcript of the same size as the cDNA isolated in the screening procedure. The enzyme can be efficiently ove rexpressed in a highly active form by using the expression vector pET- 11c. The availability of recombinant enzyme in large quantities will n ow permit detailed mechanistic and structural studies, which might con tribute to a rational design of specifically targeted inhibitors again st dUTPase from L. major.