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Abstract
The occurrence of a deoxyribonucleoside kinase in Drosophila melanogaster (Dm-dNK) with remarkably broad substrate specificity has recently been indicated (Munch-Petersen, B., Piskur, J., and Søndergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). To prove that the capacity to phosphorylate all four deoxyribonucleosides is in fact associated to one polypeptide chain, partially sequenced cDNA clones, originating from the Berkeley Drosophila genome sequencing project, were searched for homology with human deoxyribonucleoside kinases. The total sequence of one cDNA clone and the corresponding genomic DNA was determined and expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified and thrombin cleaved recombinant protein phosphorylated the four deoxyribonucleosides with high turnover and K(m) values similar to those of the native Dm-dNK, as well as the four ribonucleosides and many therapeutical nucleoside analogs. Dm-dNK has apparently the same origin as the mammalian kinases, thymidine kinase 2, deoxycytidine kinase, deoxyguanosine kinase, and the herpes viral thymidine kinases, but it has a unique C terminus that seems to be important for catalytic activity and specificity. The C-terminal 20 amino acids were dispensable for phosphorylation of deoxyribonucleosides but necessary for full activity with purine ribonucleosides. Removal of the C-terminal 20 amino acids increased the specific activity 2-fold, but 99% of the activity was lost after removal of the C-terminal 30 amino acids.
Highlights
DNA is made of four deoxyribonucleoside triphosphates, provided by the de novo and salvage pathways
We have isolated a deoxyribonucleoside kinase from Drosophila melanogaster and named it D. melanogaster deoxyribonucleoside kinase (Dm-dNK) [17]
Sequencing of an EST Clone Containing the Putative cDNA for Dm-dNK—Deposited EST sequences belonging to D. melanogaster were searched for homology to human deoxyribonucleoside kinases
Summary
Materials—Glutathione-Sepharose, pGEX-2T vector, E. coli strain BL21(DE3)pLysS, thrombin, and molecular weight standards for SDSpolyacrylamide gel electrophoresis were from Amersham Pharmacia Biotech. The PCR fragments were cut with BamHI and EcoRI and subcloned into the multiple cloning site of the pGEX-2T plasmid vector, allowing expression of the protein encoded by the open reading frame fused to the glutathione S-transferase (GST) gene. Bound contaminating proteins were removed by recirculating twice for 1 h each time at room temperature, each time with two column volumes of Buffer A containing 3 mM ATP and 10 mM MgCl2. The expressed protein was cleaved from glutathione S-transferase by recirculation for 2 h at room temperature with 4 column volumes of Buffer B containing 400 units of thrombin. The standard assay conditions were as follows: 50 mM Tris/HCl (pH 7.8, 22 °C), 5 mM MgCl2, 100 mM KCl, 0.5 mM phosphoenolpyruvate, 2.2 units/ml pyruvate kinase, 3.1 units/ml lactate dehydrogenase, 0.15 mM NADH, and 0.15 mM ATP in a total volume of 1 ml. Pairwise and Multiple Sequence Alignments—Pairwise sequence alignments were performed using the Smith-Waterman algorithm [20, 21], and multiple alignments were performed with PILEUP and CLUSTAL W [22]
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