Functional expression of a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster and its C-terminal deletion mutants

The occurrence of a deoxyribonucleoside kinase in Drosophila melanogaster ( Dm-dNK) with remarkably broad substrate specificity has recently been indic ated (Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) J. Biol. C hem. 273, 3926-3931). To prove that the capacity to phosphorylate all four deoxyribonucleosides is in fact associated to one polypeptide chain, partia lly sequenced cDNA clones, originating from the Berkeley Drosophila genome sequencing project, were searched for homology with human deoxyribonucleosi de kinases. The total sequence of one cDNA clone and the corresponding geno mic 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 ribo nucleosides and many therapeutical nucleoside analogs. Dm-dNK has apparentl y the same origin as the mammalian kinases, thymidine kinase 2, deoxycytidi ne kinase, deoxyguanosine kinase, and the herpesviral thymidine kinases, bu t it has a unique C terminus that seems to be important for catalytic activ ity and specificity. The C-terminal-20 amino acids were dispensable for pho sphorylation of deoxyribonucleosides but necessary for full activity with p urine ribonucleosides. Removal of the C-terminal 20 amino acids increased t he specific activity 2-fold, but 99% of the activity was lost after removal of the C-terminal 30 amino acids.