Functional expression of a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster and its C-terminal deletion mutants
(2000) In The Journal of biological chemistry 275(9). p.6673-6679- 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... (More)
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.
(Less)
- author
- Munch-Petersen, B LU ; Knecht, W LU ; Lenz, C ; Søndergaard, Leif and Piskur, J LU
- publishing date
- 2000-03-03
- type
- Contribution to journal
- publication status
- published
- keywords
- Amino Acids/analysis, Animals, Cloning, Molecular, Deoxyribonucleosides/metabolism, Drosophila melanogaster/enzymology, Escherichia coli, Kinetics, Molecular Sequence Data, Phosphorylation, Phosphotransferases (Alcohol Group Acceptor)/chemistry, Recombinant Fusion Proteins, Sequence Alignment, Sequence Deletion, Substrate Specificity
- in
- The Journal of biological chemistry
- volume
- 275
- issue
- 9
- pages
- 6673 - 6679
- publisher
- American Society for Biochemistry and Molecular Biology
- external identifiers
-
- scopus:0034092788
- pmid:10692477
- ISSN
- 0021-9258
- DOI
- 10.1074/jbc.275.9.6673
- language
- English
- LU publication?
- no
- id
- 4de15fff-238d-4701-8d56-e672315ffba4
- date added to LUP
- 2020-07-22 14:33:38
- date last changed
- 2024-04-03 11:52:19
@article{4de15fff-238d-4701-8d56-e672315ffba4,
abstract = {{<p>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.</p>}},
author = {{Munch-Petersen, B and Knecht, W and Lenz, C and Søndergaard, Leif and Piskur, J}},
issn = {{0021-9258}},
keywords = {{Amino Acids/analysis; Animals; Cloning, Molecular; Deoxyribonucleosides/metabolism; Drosophila melanogaster/enzymology; Escherichia coli; Kinetics; Molecular Sequence Data; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor)/chemistry; Recombinant Fusion Proteins; Sequence Alignment; Sequence Deletion; Substrate Specificity}},
language = {{eng}},
month = {{03}},
number = {{9}},
pages = {{6673--6679}},
publisher = {{American Society for Biochemistry and Molecular Biology}},
series = {{The Journal of biological chemistry}},
title = {{Functional expression of a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster and its C-terminal deletion mutants}},
url = {{http://dx.doi.org/10.1074/jbc.275.9.6673}},
doi = {{10.1074/jbc.275.9.6673}},
volume = {{275}},
year = {{2000}},
}