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 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.
Munch-Petersen
B
B
Department of Life Sciences and Chemistry, Roskilde University, P. O. Box 260, DK 4000 Roskilde, Denmark. bmp@ruc.dk
Knecht
W
W
Lenz
C
C
Søndergaard
L
L
Piskur
J
J
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003001:Cloning, Molecular; D003853:Deoxyribonucleosides / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology*; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion; D013379:Substrate Specificity
D000596:Amino Acids; D003853:Deoxyribonucleosides; D011993:Recombinant Fusion Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases