Cloned (Comment) | Organism |
---|---|
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain JM109 | Halobacterium salinarum |
Protein Variants | Comment | Organism |
---|---|---|
G114R | wild-type enzyme is a hexamer at 25°C and dissociates to dimer at 35°C in low salt medium. Mutant maintains hexameric structure at both 25 and 35°C. Refolding of heat-denatured wild-type enzyme requires salt concentrations above 2 M. Mutant G114R efficiently refolds in presence of 1 M NaCl. Residue 114 is in close proximity to E155 of the neighboring subunit in the enzyme hexamer. In the mutant, R114 may stabilize the hexameric subunit assembly | Halobacterium salinarum |
G114R | random mutagenesis, the mutant enzyme shows increased thermal stability and refolding after salt treatment compared to the wild-type enzyme, substrate specificity and kinetic of the mutant enzyme, overview | Halobacterium salinarum |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.6 | - |
ADP | wild-type, pH 7.5, 30°C | Halobacterium salinarum | |
0.7 | - |
ADP | mutant G114R, pH 7.5, 30°C | Halobacterium salinarum | |
3 | - |
GTP | wild-type, pH 7.5, 30°C | Halobacterium salinarum | |
3.8 | - |
GTP | wild-type, pH 7.5, 30°C, presence of 3.8 M NaCl | Halobacterium salinarum | |
4.4 | - |
GTP | mutant G114R, pH 7.5, 30°C | Halobacterium salinarum | |
4.8 | - |
UTP | mutant G114R, pH 7.5, 30°C | Halobacterium salinarum | |
5.5 | - |
GTP | mutant G114R, pH 7.5, 30°C, presence of 3.8 M NaCl | Halobacterium salinarum | |
6 | - |
UTP | mutant G114R, pH 7.5, 30°C, presence of 3.8 M NaCl | Halobacterium salinarum | |
6.1 | - |
UTP | wild-type, pH 7.5, 30°C | Halobacterium salinarum | |
8.1 | - |
UTP | wild-type, pH 7.5, 30°C, presence of 3.8 M NaCl | Halobacterium salinarum |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Halobacterium salinarum |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
18160 | - |
2 * 18160, calculated. Wild-type enzyme is a hexamer at 25°C and dissociates to dimer at 35°C in low salt medium | Halobacterium salinarum |
18160 | - |
2 * 18160, calculated. Wild-type enzyme is a hexamer at 25°C and dissociates to dimer at 35°C in low salt medium. Mutant G114R maintains hexameric structure at both 25 and 35°C | Halobacterium salinarum |
36600 | - |
gel filtration of wild-type, 35°C | Halobacterium salinarum |
106800 | - |
gel filtration of wild-type, 35°C, of mutant G114R at 25 and 35°C | Halobacterium salinarum |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
NTP + ADP | Halobacterium salinarum | - |
NDP + ATP | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Halobacterium salinarum | P61136 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by affinity chromatography, cleavage of the His-tag by thrombin | Halobacterium salinarum |
Renatured (Comment) | Organism |
---|---|
refolding of heat-denatured wild-type enzyme requires salt concentrations above 2 M. Mutant G114R efficiently refolds in presence of 1 M NaCl | Halobacterium salinarum |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
CTP + ADP | - |
Halobacterium salinarum | CDP + ATP | - |
? | |
GTP + ADP | - |
Halobacterium salinarum | GDP + ATP | - |
? | |
additional information | substrate specificities of wild-type and G114R mutant enzymes, overview | Halobacterium salinarum | ? | - |
? | |
NTP + ADP | - |
Halobacterium salinarum | NDP + ATP | - |
? | |
TTP + ADP | - |
Halobacterium salinarum | TDP + ATP | - |
? | |
UTP + ADP | - |
Halobacterium salinarum | UDP + ATP | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | 2 * 18160, calculated. Wild-type enzyme is a hexamer at 25°C and dissociates to dimer at 35°C in low salt medium | Halobacterium salinarum |
hexamer | 2 * 18160, calculated. Wild-type enzyme is a hexamer at 25°C and dissociates to dimer at 35°C in low salt medium. Mutant G114R maintains hexameric structure at both 25 and 35°C | Halobacterium salinarum |
More | mutant G114R runs faster on SDS-PAGE than wild-type | Halobacterium salinarum |
Synonyms | Comment | Organism |
---|---|---|
NDK | - |
Halobacterium salinarum |
nucleoside diphosphate kinase | - |
Halobacterium salinarum |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Halobacterium salinarum |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
35 | - |
wild-type, inactivation in low salt medium | Halobacterium salinarum |
35 | - |
wild-type enzyme, 0.2 M NaCl, inactivation | Halobacterium salinarum |
40 | - |
mutant G114R, low salt medium, retains full activity | Halobacterium salinarum |
40 | - |
mutant G114R, 0.2 M NaCl, stable and active up to | Halobacterium salinarum |
70 | - |
wild-type, presence of 3.8 M NaCl, stable up to | Halobacterium salinarum |
70 | - |
wild-type enzyme, 3.8 M NaCl, stable and active up to | Halobacterium salinarum |
80 | - |
mutant G114R, presence of 3.8 M NaCl, stable up to | Halobacterium salinarum |
80 | - |
mutant G114R, 3.8 M NaCl, stable and active up to | Halobacterium salinarum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
asay at | Halobacterium salinarum |