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Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D

Welin, M.; Skovgaard, T.; Knecht, W.; Zhu, C.; Berenstein, D.; Munch-Petersen, B.; Piskur, J.; Eklund, H.; FEBS J. 272, 3733-3742 (2005)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
2.7.1.145
expression of the wild-type and mutant enzymes in enzyme-deficient Escherichia coli strain KY895
Drosophila melanogaster
Crystallization (Commentary)
EC Number
Crystallization
Organism
2.7.1.145
purified recombinant mutant N64D in complex with dT or dTTP, counter diffusion and vapour diffusion methods, 20 mg/ml enzyme with 10 mM dT, is mixed with cyrstallization solution containing 0.15 M MES, pH 6.5, 0.3 M lithium sulfate, and 27,5% w/v PEG 2000 monomethylether, or 10 mg/ml enzyme with 5 mM dTTP is mixed with 0.1 M MES, pH 6.5, 0.16 M lithium sulfate, and 25% v/v PEG 2000 monomethylether, 2 weeks, 15°C, X-ray diffraction structure determination and analysis at 3.1 A and 2.2 A resolution, respectively
Drosophila melanogaster
Engineering
EC Number
Amino acid exchange
Commentary
Organism
2.7.1.145
additional information
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
Drosophila melanogaster
2.7.1.145
N45D
site-directed mutagenesis, decreased activity with natural substrates
Drosophila melanogaster
2.7.1.145
N45D/N64D
decreased activity with natural substrates, decreased feedback inhibition by dTTP compared to the wild-type enzyme
Drosophila melanogaster
2.7.1.145
N64D
site-directed mutagenesis, highly decreased activity with natural substrates, decreased feedback inhibition by dTTP compared to the wild-type enzyme
Drosophila melanogaster
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
2.7.1.145
dTTP
competitive versus ATP, feedback inhibition, binding at the active site, binding structure of mutant N64D, modelling
Drosophila melanogaster
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.7.1.145
0.0009
-
2'-deoxycytidine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
0.0009
-
2'-deoxythymidine
recombinant mutants N45D
Drosophila melanogaster
2.7.1.145
0.0111
-
3'-azido-2',3'-didehydro-3'-deoxythymidine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.0117
-
3'-azido-2',3'-didehydro-3'-deoxythymidine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
0.0232
-
2'-deoxythymidine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.118
-
2'-deoxycytidine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.119
-
2'-deoxyadenosine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
0.412
-
2'-deoxyguanosine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
3.82
-
2'-deoxyadenosine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
20.35
-
2'-deoxyguanosine
recombinant mutant N64D
Drosophila melanogaster
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
2.7.1.145
Mg2+
-
Drosophila melanogaster
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.7.1.145
ATP + 2'-deoxyadenosine
Drosophila melanogaster
-
ADP + 2'-deoxyadenosine 5'-phosphate
-
-
?
2.7.1.145
ATP + 2'-deoxycytidine
Drosophila melanogaster
-
ADP + 2'-deoxycytidine 5'-phosphate
-
-
?
2.7.1.145
ATP + 2'-deoxyguanosine
Drosophila melanogaster
-
ADP + 2'-deoxyguanosine 5'-phosphate
-
-
?
2.7.1.145
ATP + 2'-deoxythymidine
Drosophila melanogaster
-
ADP + 2'-deoxythymidine 5'-phosphate
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.7.1.145
Drosophila melanogaster
-
-
-
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
2.7.1.145
additional information
-
-
Drosophila melanogaster
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.7.1.145
ATP + 2'-deoxyadenosine
-
661702
Drosophila melanogaster
ADP + 2'-deoxyadenosine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 2'-deoxycytidine
-
661702
Drosophila melanogaster
ADP + 2'-deoxycytidine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 2'-deoxyguanosine
-
661702
Drosophila melanogaster
ADP + 2'-deoxyguanosine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 2'-deoxythymidine
-
661702
Drosophila melanogaster
ADP + 2'-deoxythymidine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 2'-deoxythymidine
substrate binding structure of mutant N64D
661702
Drosophila melanogaster
ADP + 2'-deoxythymidine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 3'-azido-2',3'-didehydro-3'-deoxythymidine
-
661702
Drosophila melanogaster
ADP + 3'-azido-2',3'-didehydro-3'-deoxythymidine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + aciclovir
-
661702
Drosophila melanogaster
ADP + aciclovir phosphate
-
-
-
?
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.7.1.145
0.03
-
3'-azido-2',3'-didehydro-3'-deoxythymidine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
0.037
-
3'-azido-2',3'-didehydro-3'-deoxythymidine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.12
-
2'-deoxyguanosine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.4
-
2'-deoxyadenosine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.82
-
2'-deoxythymidine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
1.3
-
2'-deoxyguanosine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
1.6
-
2'-deoxycytidine
recombinant mutants N45D and N64D
Drosophila melanogaster
2.7.1.145
1.83
-
2'-deoxyadenosine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
2.6
-
2'-deoxythymidine
recombinant mutant N45D
Drosophila melanogaster
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
2.7.1.145
ATP
-
Drosophila melanogaster
Ki Value [mM]
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
2.7.1.145
0.0047
-
dTTP
uncompetitive versus 2'-deoxythymidine, recombinant wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0163
-
dTTP
competitive versus ATP, recombinant wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.52
-
dTTP
uncompetitive versus 2'-deoxythymidine, recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.829
-
dTTP
competitive versus ATP, recombinant mutant N64D
Drosophila melanogaster
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
2.7.1.145
expression of the wild-type and mutant enzymes in enzyme-deficient Escherichia coli strain KY895
Drosophila melanogaster
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
2.7.1.145
ATP
-
Drosophila melanogaster
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
2.7.1.145
purified recombinant mutant N64D in complex with dT or dTTP, counter diffusion and vapour diffusion methods, 20 mg/ml enzyme with 10 mM dT, is mixed with cyrstallization solution containing 0.15 M MES, pH 6.5, 0.3 M lithium sulfate, and 27,5% w/v PEG 2000 monomethylether, or 10 mg/ml enzyme with 5 mM dTTP is mixed with 0.1 M MES, pH 6.5, 0.16 M lithium sulfate, and 25% v/v PEG 2000 monomethylether, 2 weeks, 15°C, X-ray diffraction structure determination and analysis at 3.1 A and 2.2 A resolution, respectively
Drosophila melanogaster
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
2.7.1.145
additional information
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
Drosophila melanogaster
2.7.1.145
N45D
site-directed mutagenesis, decreased activity with natural substrates
Drosophila melanogaster
2.7.1.145
N45D/N64D
decreased activity with natural substrates, decreased feedback inhibition by dTTP compared to the wild-type enzyme
Drosophila melanogaster
2.7.1.145
N64D
site-directed mutagenesis, highly decreased activity with natural substrates, decreased feedback inhibition by dTTP compared to the wild-type enzyme
Drosophila melanogaster
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
2.7.1.145
dTTP
competitive versus ATP, feedback inhibition, binding at the active site, binding structure of mutant N64D, modelling
Drosophila melanogaster
Ki Value [mM] (protein specific)
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
2.7.1.145
0.0047
-
dTTP
uncompetitive versus 2'-deoxythymidine, recombinant wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0163
-
dTTP
competitive versus ATP, recombinant wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.52
-
dTTP
uncompetitive versus 2'-deoxythymidine, recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.829
-
dTTP
competitive versus ATP, recombinant mutant N64D
Drosophila melanogaster
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.7.1.145
0.0009
-
2'-deoxycytidine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
0.0009
-
2'-deoxythymidine
recombinant mutants N45D
Drosophila melanogaster
2.7.1.145
0.0111
-
3'-azido-2',3'-didehydro-3'-deoxythymidine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.0117
-
3'-azido-2',3'-didehydro-3'-deoxythymidine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
0.0232
-
2'-deoxythymidine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.118
-
2'-deoxycytidine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.119
-
2'-deoxyadenosine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
0.412
-
2'-deoxyguanosine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
3.82
-
2'-deoxyadenosine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
20.35
-
2'-deoxyguanosine
recombinant mutant N64D
Drosophila melanogaster
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
2.7.1.145
Mg2+
-
Drosophila melanogaster
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.7.1.145
ATP + 2'-deoxyadenosine
Drosophila melanogaster
-
ADP + 2'-deoxyadenosine 5'-phosphate
-
-
?
2.7.1.145
ATP + 2'-deoxycytidine
Drosophila melanogaster
-
ADP + 2'-deoxycytidine 5'-phosphate
-
-
?
2.7.1.145
ATP + 2'-deoxyguanosine
Drosophila melanogaster
-
ADP + 2'-deoxyguanosine 5'-phosphate
-
-
?
2.7.1.145
ATP + 2'-deoxythymidine
Drosophila melanogaster
-
ADP + 2'-deoxythymidine 5'-phosphate
-
-
?
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
2.7.1.145
additional information
-
-
Drosophila melanogaster
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.7.1.145
ATP + 2'-deoxyadenosine
-
661702
Drosophila melanogaster
ADP + 2'-deoxyadenosine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 2'-deoxycytidine
-
661702
Drosophila melanogaster
ADP + 2'-deoxycytidine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 2'-deoxyguanosine
-
661702
Drosophila melanogaster
ADP + 2'-deoxyguanosine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 2'-deoxythymidine
-
661702
Drosophila melanogaster
ADP + 2'-deoxythymidine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 2'-deoxythymidine
substrate binding structure of mutant N64D
661702
Drosophila melanogaster
ADP + 2'-deoxythymidine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 3'-azido-2',3'-didehydro-3'-deoxythymidine
-
661702
Drosophila melanogaster
ADP + 3'-azido-2',3'-didehydro-3'-deoxythymidine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + aciclovir
-
661702
Drosophila melanogaster
ADP + aciclovir phosphate
-
-
-
?
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.7.1.145
0.03
-
3'-azido-2',3'-didehydro-3'-deoxythymidine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
0.037
-
3'-azido-2',3'-didehydro-3'-deoxythymidine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.12
-
2'-deoxyguanosine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.4
-
2'-deoxyadenosine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
0.82
-
2'-deoxythymidine
recombinant mutant N64D
Drosophila melanogaster
2.7.1.145
1.3
-
2'-deoxyguanosine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
1.6
-
2'-deoxycytidine
recombinant mutants N45D and N64D
Drosophila melanogaster
2.7.1.145
1.83
-
2'-deoxyadenosine
recombinant mutant N45D
Drosophila melanogaster
2.7.1.145
2.6
-
2'-deoxythymidine
recombinant mutant N45D
Drosophila melanogaster