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Enzymes mediating nucleotide metabolism are potential chemotherapy targets of tumor cells. Deoxyuridine triphosphatase (dUTPase) hydrolyzes dUTP into dUMP and pyrophosphate, playing a crucial role in limiting the misincorporation of uracil into DNA. Uracil incorporated into DNA is subjected to excision, but repeated excision repair leads to DNA strand breaks and cell death. Most dUTPases are homotrimers having three active sites with each active site consisting of motif III of the first subunit, motifs I, II, and IV from the second subunit and motif V from the third subunit. Because the cell biology of the eukaryote Dictyostelium discoideum parallels that of mammalian cells, a study of the enzymology of D. discoideum dUTPase will provide insights into its regulation. The dUTPase gene was synthesized and the recombinant protein was expressed in Escherichia coli. A trimer of 9-kDa segment, lacking motifs I and II, was purified and confirmed by mass spectroscopic analyses and N-terminal sequencing. The enzyme has dUTPase activity with a Km of 0.566 ± 0.02 µM, a specific activity of 0.0259 µmol dUMP/min/µg at pH 8.0 and 25 ˚C, and optimal activity at pH 8.0 and 50 ˚C.
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