2.7.1.145: deoxynucleoside kinase
This is an abbreviated version!
For detailed information about deoxynucleoside kinase, go to the full flat file.
Word Map on EC 2.7.1.145
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2.7.1.145
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thymidine
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drosophila
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deoxycytidine
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suicide
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deoxyguanosine
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herpes
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medicine
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deoxyadenosine
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e-5-2-bromovinyl-2'-deoxyuridine
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kinase-deficient
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dttp
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deoxythymidine
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dado
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1-beta-d-arabinofuranosylthymine
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synthesis
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agriculture
- 2.7.1.145
- thymidine
- drosophila
- deoxycytidine
-
suicide
- deoxyguanosine
-
herpes
- medicine
- deoxyadenosine
-
e-5-2-bromovinyl-2'-deoxyuridine
-
kinase-deficient
- dttp
- deoxythymidine
-
dado
- 1-beta-d-arabinofuranosylthymine
- synthesis
- agriculture
Reaction
Synonyms
AGCK, AgdNK, BmdNK, D. melanogaster deoxynucleoside kinase, deoxyribonucleoside kinase, deoxyribonucleoside kinase
ECTree
2.7.1.145 deoxynucleoside kinaseAdvanced search results
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results (Engineering
Engineering on EC 2.7.1.145 - deoxynucleoside kinase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
A110D
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site-directed mutagenesis, altered substrate specificity and kinetics, and highly reduced activity compared to the wild-type enzyme
F114A
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site-directed mutagenesis, active site mutant, altered substrate specificity compared to the wild-type enzyme
F114Y
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site-directed mutagenesis, active site mutant, altered substrate specificity compared to the wild-type enzyme
I102M/N117S/M118V/D208N
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clones harbouring the mutant enzyme are more sensitive to 3'-azido-2',3'-dideoxythymidine and beta-D-arabinofuranosylcytosine
I29H
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site-directed mutagenesis, active site mutant, altered substrate specificity compared to the wild-type enzyme
I29H/F114Y
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site-directed mutagenesis, active site mutant, inactive mutant
I47T/N210D
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clones harbouring the mutant enzyme are more sensitive to 3'-azido-2',3'-dideoxythymidine and beta-D-arabinofuranosylcytosine
M1T/T85A/N121S
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clones harbouring the mutant enzyme are more sensitive to 1-beta-D-arabinofuranosylcytosine and 2',3'-dideoxycytidine
M88R
M88R/A110D
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site-directed mutagenesis, altered substrate specificity and kinetics, and highly reduced activity compared to the wild-type enzyme
N210D/L239P
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clones harbouring the mutant enzyme are more sensitive to 3'-azido-2',3'-dideoxythymidine and beta-D-arabinofuranosylcytosine
N28P
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site-directed mutagenesis, active site mutant, inactive mutant
N28P/I29H
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site-directed mutagenesis, active site mutant, inactive mutant
N28P/I29H/F114Y
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site-directed mutagenesis, active site mutant, inactive mutant
N38D/N64D
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clones harbouring the mutant enzyme are more sensitive specifically to 3'-azido-2',3'-dideoxythymidine
N45D
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site-directed mutagenesis, decreased activity with natural substrates
N45D/N64D
N59D/M118V/Y179H
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clones harbouring the mutant enzyme are more sensitive to 3'-azido-2',3'-dideoxythymidine and beta-D-arabinofuranosylcytosine
N64D
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site-directed mutagenesis, highly decreased activity with natural substrates, decreased feedback inhibition by dTTP compared to the wild-type enzyme
N64S/L68S/M69L
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clones harbouring the mutant enzyme are more sensitive specifically to 3'-azido-2',3'-dideoxythymidine
Q81D
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site-directed mutagenesis, active site mutant, inactive mutant
Q81E
the mutant enzyme shows higher phosphorylating activity with 6-amino-5-nitro-3-(1'-beta-D-2'-deoxyribofuranosyl)-2(1H)-pyridone as compared to the wild type enzyme
Q81N
R247S
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site-directed mutagenesis, mutation abolishes nuclear import of the enzyme, the mutant enzyme is localized in the cytosol
T12A/V84A/N213S
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clones harbouring the mutant enzyme are more sensitive to 1-beta-D-arabinofuranosylcytosine
T85A
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clones harbouring the mutant enzyme are more sensitive to 1-beta-D-arabinofuranosylcytosine and 2',3'-dideoxycytidine
V84A
V84A/M88R
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site-directed mutagenesis of the full length enzyme and of the DELTA20 N-terminally truncated enzyme, highly increased Km for acceptor substrates, altered substrate specificity compared to the wild-type enzyme
V84A/M88R/A110D
V84M
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site-directed mutagenesis, altered substrate specificity and kinetics, and highly reduced activity compared to the wild-type enzyme
V84S
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site-directed mutagenesis, altered substrate specificity and kinetics, and reduced activity compared to the wild-type enzyme
V84S/M88R/A110D
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site-directed mutagenesis, altered substrate specificity and kinetics, and highly reduced activity compared to the wild-type enzyme
Y70W
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mutant enzyme loses activity towards purines. Negative cooperativity towards dThd and dCyd is observed
additional information
M88R
-
site-directed mutagenesis of the full length enzyme and of the DELTA20 N-terminally truncated enzyme, highly increased Km for acceptor substrates, altered substrate specificity compared to the wild-type enzyme
M88R
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site-directed mutagenesis, altered substrate specificity and kinetics, and highly reduced activity compared to the wild-type enzyme
N45D/N64D
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clones harbouring the mutant enzyme are more sensitive to 3'-azido-2',3'-dideoxythymidine and other nucleoside analogs
N45D/N64D
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mutant enzyme with the largest und universal increase in sensitivity of all mutants tested. 316fold increase to 3'-azido-2',3'-dideoxythymidine, more than 11fold to 2',3'-dideoxycytidine, and 3.2fold increase in sensitivity to beta-D-arabinofuranosylcytosine and 2',3'-dideoxyadenosine. Higher Km-values for native substrates than wild-type enzyme and Vmax-values are substantially lower, decrease in feedback inhibition by TTP, Km and Vmax values for 3'-azido-2',3'-dideoxythymidine and Km value for 2',3'-dideoxycytidine are nearly unchanged
N45D/N64D
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decreased activity with natural substrates, decreased feedback inhibition by dTTP compared to the wild-type enzyme
N45D/N64D
site-directed mutagenesis, mutant shows increased drug sensitivity and decreased inhibition by dTTP compared to the wild-type enzyme
N45D/N64D
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enhanced cytotoxicity for parimidine analogs especially azidothymidine
Q81N
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site-directed mutagenesis, active site mutant, altered substrate specificity compared to the wild-type enzyme
Q81N
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mutation shows a 200fold and 100fold increase in Km. kcat is decreased 5fold and 2fold for dThd and dCyd
V84A
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clones harbouring the mutant enzyme are more sensitive to 1-beta-D-arabinofuranosylcytosine
V84A
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site-directed mutagenesis, altered substrate specificity and kinetics, and reduced activity compared to the wild-type enzyme
V84A/M88R/A110D
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site-directed mutagenesis of the full length enzyme and of the DELTA20 N-terminally truncated enzyme, highly increased Km for acceptor substrates, altered substrate specificity compared to the wild-type enzyme
V84A/M88R/A110D
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site-directed mutagenesis, altered substrate specificity and kinetics, and highly reduced activity compared to the wild-type enzyme
additional information
the deletion mutants rDm-dNKDELTAC10 and rDm-dNKDELTAC20 show the same substrate activity pattern as the recombinant wild-type enzyme. Relative phosphorylation of 2'-deoxycytidine and 2-chloro-2'-deoxyadenosine increases with increasing C-terminal truncation. The relative activities of rDm-dNKDELTAC10 and rDm-dNKDELTAC20 with deoxyribonucleosides remains largely unchanged, whereas there is a substantial decrease in the phosphorylation of the purine ribonucleosides adenosine and guanosine, as well as of all dideoxyribonucleosides and 3'-azido-2',3'-dideoxythymidine. The relative activities with the pyrimidine ribonucleosides and 1-beta-D-arabinofuranosylcytosine and 1-beta-D-arabinofuranosylthymine are not affected by the C-terminal deletions
additional information
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the deletion mutants rDm-dNKDELTAC10 and rDm-dNKDELTAC20 show the same substrate activity pattern as the recombinant wild-type enzyme. Relative phosphorylation of 2'-deoxycytidine and 2-chloro-2'-deoxyadenosine increases with increasing C-terminal truncation. The relative activities of rDm-dNKDELTAC10 and rDm-dNKDELTAC20 with deoxyribonucleosides remains largely unchanged, whereas there is a substantial decrease in the phosphorylation of the purine ribonucleosides adenosine and guanosine, as well as of all dideoxyribonucleosides and 3'-azido-2',3'-dideoxythymidine. The relative activities with the pyrimidine ribonucleosides and 1-beta-D-arabinofuranosylcytosine and 1-beta-D-arabinofuranosylthymine are not affected by the C-terminal deletions
additional information
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expression of the enzyme in enzyme-deficient Escherichia coli strain KY895 confers resistnce against 3'-azido-2',3'-didehydro-3'-deoxythymidine at more than 0.1 mM
additional information
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overexpression in cancer cell lines leads to increased cell sensitivity to several cytotoxic nucleoside analogues, expression of wild-type and mutant enzyme in an osteosarcoma cell line utilizing a replication-deficient retroviral vector reveals that the localization of the enzyme in cytosol or nucleus is not important for cell sensitivity and bystander cell killing
additional information
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construction of libraries of hybrid enzymes by non-homologous recombination of the pyrimidinespecific human thymidine kinase 2 and the broad-specificity dNK from Drosophila melanogaster. Identification of chimeras that phosphorylate nucleoside analogs with higher activity than either parental enzyme, and that possess new activity towards the anti-HIV prodrug 2',3'-didehydro-3'-deoxythymidine
additional information
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creation of mutants with increased specificity for several nucleoside analogs. The mutants have a reduced ability to phosphorylate pyrimidines, while the ability to phosphorylate purine analogs is relatively similar to the wild-type enzyme
additional information
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engineering of multisubstrate nucleoside kinase targeted to the mitochondrial matrix and expression in thymidine kinase-1 deficient osteosarcoma cell line. Although the total deoxythymidine phosphorylation activity is similar in cells expressing multisubstrate nullceoside kinase in the nucleus or in the mitochondria, the cells expressing the enzyme in the mitochondria show higher sensitivity to the antiproliferative activity of pyrimidine nucleoside analogs, such as (E)-5-(2-bromovinyl)-2-deoxyuridine, 5-bromo-2-deoxyuridine, and 5-fluoro-2-deoxyuridine. Cells expressing the mitochondrial enzyme have an increased incorporation of [3H]dThd into DNA, due to a higher [3H]dTTP specific activity of the total dTTP pool in the cells in which the enzyme is targeted to the mitochondria
additional information
enzyme DmdNK is adsorbed on a solid ion exchange support (bearing primary amino groups) achieving an expressed activity of over 98%. Upon cross-linking with aldehyde dextran, the expressed activity is 30-40%, both biocatalysts (adsorbed or cross-linked) are stable at pH 10.0 and room temperature for 24 h with about 70% of retained activity. Optimization of the reaction conditions to 50 mM ammonium acetate, a substrate/ATP ratio of 1:1.25, 2 mM MgCl2, 37°C, pH 8.0, results in conversions of up to 90%
