Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 2.7.4.6 extracted from

  • Ishibashi, M.; Tatsuda, S.; Izutsu, K.; Kumeda, K.; Arakawa, T.; Tokunaga, M.
    A single Gly114Arg mutation stabilizes the hexameric subunit assembly and changes the substrate specificity of halo-archaeal nucleoside diphosphate kinase (2007), FEBS Lett., 581, 4073-4079.
    View publication on PubMed

Cloned(Commentary)

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

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 [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

Metals/Ions Comment Organism Structure
Mg2+
-
Halobacterium salinarum

Molecular Weight [Da]

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/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
NTP + ADP Halobacterium salinarum
-
NDP + ATP
-
?

Organism

Organism UniProt Comment Textmining
Halobacterium salinarum P61136
-
-

Purification (Commentary)

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 (Commentary)

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 and Products (Substrate)

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

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

Synonyms Comment Organism
NDK
-
Halobacterium salinarum
nucleoside diphosphate kinase
-
Halobacterium salinarum

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Halobacterium salinarum

Temperature Stability [°C]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
asay at Halobacterium salinarum