Viral dUTPases, Recombinant Expression, Purification and Characterization

Sammanfattning: The enzyme deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate. The reaction suppresses misincorporation of uracil into DNA and provides dUMP for the de novo synthesis of dTTP. dUTPase is a widespread enzyme and the fact that many viruses encode a dUTPase of their own suggests the enzyme to be important for the viral life cycle and thereby a potential target for development of new antiviral drugs. Most of the known dUTPases are homotrimers with the active sites located at the subunit interfaces. The amino acid residues, which form the active site, are conserved and clustered into five regions (motifs) of the polypeptide chain. dUTPases from the herpes virus family have 2-3 times longer polypeptide chains and display a rearranged order of the conserved motifs compared to the trimeric enzymes. dUTPase encoded by herpes simplex virus type 1 (HSV-1) has been found to be monomeric. dUTPase from the retrovirus mouse mammary tumor virus (MMTV) shows another special feature by being amino-terminally fused to the retroviral nucleocapsid protein. In this thesis, the dUTPases from HSV-1 and from the retroviruses MMTV and equine infectious anemia virus (EIAV) as well as the nucleocapsid protein from MMTV were overexpressed in E. coli by using the T7 RNA polymerase expression system. The recombinant proteins and HSV-1 dUTPase from infected Vero (green monkey kidney) cells were purified by phosphocellulose chromatography. The final yields of purified recombinant enzyme differ, from about 45 mg per liter of bacterial culture for the EIAV dUTPase to 10 mg in the case of HSV-1 dUTPase. Gel-filtration experiments suggest the two retroviral enzymes to be trimers. The specific enzyme activities range from 8 micromol/min mg for the MMTV dUTPase to 120 micromol/min mg for the EIAV enzyme. The kinetic properties of HSV-1 dUTPase were determined and compared to those of dUTPase from the bacterium Escherichia coli. The HSV-1 dUTPase shows a specificity constant (kcat/KM) of the same order of magnitude (10exp7) as the highly specific bacterial enzyme. KM for dUTP was found to be 0.3 microM and the inhibitor 2’-deoxyuridine 5’-(alpha,beta-imido)triphosphate has an inhibition constant of 0.9 microM. Experiments with racemic 2’-deoxyuridine 5’-(alpha-thio)triphosphate led to the conclusion that the catalytic mechanism involves interaction of a divalent metal ion with the alpha-phosphate of the substrate.