Abstract
Deoxyuridine 5’-triphosphate nucleotide hydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. It is an enzyme in the pyrimidine- nucleotide metabolism. Enzymes in the DNA metabolism have been used as targets for drugs against cancer and virus-infected diseases. Recent investigations have shown that dUTPase might be included among these targets. Characterization of dUTPase of H. pylori strain 26695, structurally and kinetically, would be a prerequisite for the design of such drugs. The Hp0865 gene encoding dUTPase in H. pylori strain 26695 was cloned into the pQE30 vector and overexpressed in E. coli strain SG13009. The resulting rec-Hp0865 protein with N-terminal 6x-His-tag was purified by Ni-NTA column at a yield of 25 mg/L of bacteria culture. The Rec-Hp0865 protein stored in eluting buffer (20 mM Tris-HCl, 300 mM NaCl, 200 mM imidazole, pH 8.0) would not aggregate. The molecular weight of rec-Hp0865 was determined to be 17 KD by SDS-PAGE. A trimer structure in native protein was suggested from the results of a major peak at 51 kDa characterized by the analytical ultracentrifugation. Purified rec-Hp0865 protein as antigen was used to produce polyclonal antibodies which would detect endogenous Hp0865 protein level in H. pylori. No change of endogenous Hp0865 protein level was observed from bacteria under acidic stress or in different culture times . Spectrophotometric assays showed that 5 ug rec-HP0865 protein would hydrolyze most of 60 uM dUTP at 7 min in reaction buffer including 1 mM MgCl2 at pH 8.0 and 25 oC.. The determination of released PPi by colorimetric assay revealed that 94.6 % dUTP was hydrolyzed in above mentioned condition. The kinetic parameters of this dUTPase have been determined to have a Km of 0.4 uM and a kcat of 0.7 s-1 for dUTP substrate in the presence of 1 mM Mg+2. The product, PPi, causes competitive inhibition with a Kip value of 11.8 uM.. Another product, dUMP, has less inhibition than PPi on enzyme activity. In addition, neither NaF (phosphatase inhibitor) nor resveratrol affected the enzymetic activity of rec-Hp0865. The enzymetic activity of rec-Hp0865 was highly specific for dUTP as substrate. The rec-Hp0865 protein could display ~78% dUTPase activity after one month. Reduced enzyme activity was observed in Hp0865 after pre-incubation at above 60 oC for 15 min. The enzyme activity was not sensitive to pH within the range (pH 7 - 9.5). Dose-dependent increase on enzyme activity appeared in reaction containing 0.1-1 mM MgCl2. The enzyme showed much activity in the presence of Co2+ or Zn2+ instead of Mg2+ at the same concentration. CD spectra changes in the 200–240-nm wavelength range were observed by the substitution of Mg2+ to Zn2+ or Co2+ at a 1 mM. The product inhibition of PPi at low concentration and Zn2+, Co2+ preference instead of Mg2+ for dUTPase activity are novel discovery for rec-Hp0865 protein.