Abstract
A Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) was reported to phosphorylate all four natural deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases. We have in the present study cloned the Dm-dNK cDNA, expressed the 29-kDa protein in Escherichia coli, and characterized the recombinant enzyme for the phosphorylation of nucleosides and clinically important nucleoside analogs. The recombinant enzyme preferentially phosphorylated the pyrimidine nucleosides dThd, dCyd, and dUrd, but phosphorylation of the purine nucleosides dAdo and dGuo was also efficiently catalyzed. Dm-dNK is closely related to human and herpes simplex virus deoxyribonucleoside kinases. The highest level of sequence similarity was noted with human mitochondrial thymidine kinase 2, and these enzymes also share many substrates. The cDNA cloning and characterization of Dm-dNK will be the basis for studies on the use of this multisubstrate nucleoside kinase as a suicide gene in combined gene/chemotherapy of cancer.
Highlights
Deoxyribonucleoside kinases catalyze the phosphorylation of 2Ј-deoxyribonucleosides to 2Ј-deoxyribonucleoside monophosphates
We have identified, cloned, and recombinantly expressed the cDNA of the multisubstrate deoxyribonucleoside kinase of D. melanogaster
The pyrimidine nucleosides dCyd, dThd, and dUrd are the preferred substrates of the recombinant enzyme, it catalyzed the phosphorylation of the purine nucleosides dAdo and dGuo
Summary
The oligonucleotides were used in a polymerase chain reaction, and the amplified DNA fragment was cloned in the EcoRI-SalI sites of the pGEX-5X-1 plasmid vector (Amersham Pharmacia Biotech). The cDNAs of TK1, TK2, dCK, dGK, and HSV-1 TK were inserted in the pGEX-5X-1 vector, expressed as fusion proteins to glutathione S-transferase, and purified as described [7]. The activity of the purified recombinant nucleoside kinases were assayed in a 50-l reaction mixture containing 50 mM Tris-HCl, pH 8.0 (22 °C), 2.5 mM MgCl2, 10 mM dithiothreitol, 0.5 mM CHAPS, 3 mg/ml bovine serum albumin, 2.5 mM ATP, indicated concentrations of [methyl-3H]dThd, [5-3H]dCyd, [8-3H]dGuo, or [8-3H]dAdo, and recombinant Dm-dNK. Insertions and deletions were taken into account, and bootstrap analysis was performed with value 100. The neighbor-joining method with bootstrap was used to interfere the tree topology
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