Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) RIL | Thermococcus kodakarensis |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme Tk-PTP(form I) Tk-PTP(form II) Tk-PTP(G95A), sitting-drop vapor diffusion method, for enzyme form I: mixing 0.001 ml of 10 mg/ml protein solution with 0.001 ml of precipitant solution containing 0.1 M sodium citrate, pH 5.4, 8% w/v PEG 10000, and 14% v/v dioxane, for enzyme form II: mixing of mixing 0.001 ml of 10 mg/ml protein solution with 0.001 ml of precipitant solution containing 0.03 M citric acid, 0.07 M Bis-Tris propane, pH 7.6, 8% w/v PEG 3350, and 0.08 M spermine tetrahydrochloride, and for mutant Tk-PTP(G95A): mixing of 0.001 ml of 10 mg/ml protein solution with 0.001 ml of precipitant solution containing 0.1 M Bis-Tris, pH 6.25, and 0.8 M magnesium formate dehydrate, all at 18°C, X-ray diffraction structure determination and analysis at 1.7-2.3 A resolution, molecular replacement method, model building | Thermococcus kodakarensis |
Protein Variants | Comment | Organism |
---|---|---|
C93S | site-directed mutagenesis, inactive mutant | Thermococcus kodakarensis |
D63A | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Thermococcus kodakarensis |
D63N | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Thermococcus kodakarensis |
D63N/E132A | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Thermococcus kodakarensis |
E132A | site-directed mutagenesis, the mutant shows about 1.5fold increased activity compared to wild-type | Thermococcus kodakarensis |
E132L | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Thermococcus kodakarensis |
E132Q | site-directed mutagenesis, the mutant shows about 2.5fold increased activity compared to wild-type | Thermococcus kodakarensis |
G95A | site-directed mutagenesis, the mutant shows about 10fold increased activity compared to wild-type | Thermococcus kodakarensis |
Q136A | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Thermococcus kodakarensis |
R124A | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Thermococcus kodakarensis |
R124E | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type | Thermococcus kodakarensis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
Sodium vanadate | a PTP inhibitor targeting the catalytic site pocket. Vanadate is anchored in the active site of Tk-PTP(form II), stabilized by electrostatic interaction with the guanidinium group of Arg109 and by its oxygen atom-mediated hydrogen bonds with the main chain amides of Met94, Gly95, Leu97, Gly98, and Arg99. The Tk-PTP(form II) P-loop is structurally similar to those of catalytically active DUSP proteins | Thermococcus kodakarensis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.023 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant E132A | Thermococcus kodakarensis | |
0.0274 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant D63A | Thermococcus kodakarensis | |
0.0658 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant E132A | Thermococcus kodakarensis | |
0.0666 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant D63A | Thermococcus kodakarensis | |
0.0682 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant G95A | Thermococcus kodakarensis | |
0.106 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant G95A | Thermococcus kodakarensis | |
0.135 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant Q136A | Thermococcus kodakarensis | |
0.15 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant E132Q | Thermococcus kodakarensis | |
0.223 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant D63N/E132A | Thermococcus kodakarensis | |
0.251 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant E132L | Thermococcus kodakarensis | |
0.252 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant D63N | Thermococcus kodakarensis | |
0.258 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant R124E | Thermococcus kodakarensis | |
0.276 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, wild-type enzyme | Thermococcus kodakarensis | |
0.301 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant R124A | Thermococcus kodakarensis | |
0.307 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant E132Q | Thermococcus kodakarensis | |
0.363 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant Q136A | Thermococcus kodakarensis | |
0.363 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant R124E | Thermococcus kodakarensis | |
0.466 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant D63N/E132A | Thermococcus kodakarensis | |
0.56 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant D63N | Thermococcus kodakarensis | |
0.611 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant R124A | Thermococcus kodakarensis | |
0.719 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, wild-type enzyme | Thermococcus kodakarensis | |
0.862 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant E132L | Thermococcus kodakarensis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
[a protein]-serine/threonine phosphate + H2O | Thermococcus kodakarensis | - |
[a protein]-serine/threonine + phosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermococcus kodakarensis | Q8X270 | i.e. Pyrococcus kodakaraensis | - |
Thermococcus kodakarensis ATCC BAA-918 | Q8X270 | i.e. Pyrococcus kodakaraensis | - |
Thermococcus kodakarensis JCM 12380 | Q8X270 | i.e. Pyrococcus kodakaraensis | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) RIL by nickel affinity chromatography, tag cleavage by thrombin, and gel filtration | Thermococcus kodakarensis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
6,8-difluoro-4-methylumbelliferyl phosphate + H2O | - |
Thermococcus kodakarensis | 6,8-difluoro-4-methylumbelliferone + phosphate | - |
? | |
6,8-difluoro-4-methylumbelliferyl phosphate + H2O | - |
Thermococcus kodakarensis JCM 12380 | 6,8-difluoro-4-methylumbelliferone + phosphate | - |
? | |
6,8-difluoro-4-methylumbelliferyl phosphate + H2O | - |
Thermococcus kodakarensis ATCC BAA-918 | 6,8-difluoro-4-methylumbelliferone + phosphate | - |
? | |
additional information | the enzyme shows dual specificity, protein tyrosine/serine/threonine phosphatase activity | Thermococcus kodakarensis | ? | - |
? | |
additional information | the enzyme shows dual specificity, protein tyrosine/serine/threonine phosphatase activity | Thermococcus kodakarensis JCM 12380 | ? | - |
? | |
additional information | the enzyme shows dual specificity, protein tyrosine/serine/threonine phosphatase activity | Thermococcus kodakarensis ATCC BAA-918 | ? | - |
? | |
[a protein]-serine/threonine phosphate + H2O | - |
Thermococcus kodakarensis | [a protein]-serine/threonine + phosphate | - |
? |
Synonyms | Comment | Organism |
---|---|---|
dual-specificity phosphatase | - |
Thermococcus kodakarensis |
DUSP | - |
Thermococcus kodakarensis |
More | cf. EC 3.1.3.48 | Thermococcus kodakarensis |
TK0241 | - |
Thermococcus kodakarensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
80 | - |
- |
Thermococcus kodakarensis |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
additional information | - |
the hyperthermostability of Tk-PTP depends on its intramolecular hydrophobic interactions, structure comparisons, overview | Thermococcus kodakarensis |
60 | - |
the enzymatic activity of Tk-PTP is not affected by pre-heating the protein at 60°C for 3 h | Thermococcus kodakarensis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0143 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant D63N | Thermococcus kodakarensis | |
0.0371 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant R124E | Thermococcus kodakarensis | |
0.0415 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant D63A | Thermococcus kodakarensis | |
0.0652 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant D63N/E132A | Thermococcus kodakarensis | |
0.102 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant Q136A | Thermococcus kodakarensis | |
0.188 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant R124E | Thermococcus kodakarensis | |
0.197 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant E132L | Thermococcus kodakarensis | |
0.248 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant R124A | Thermococcus kodakarensis | |
0.46 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant Q136A | Thermococcus kodakarensis | |
0.591 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant E132A | Thermococcus kodakarensis | |
0.688 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant D63N/E132A | Thermococcus kodakarensis | |
1.29 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant D63A | Thermococcus kodakarensis | |
1.59 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant R124A | Thermococcus kodakarensis | |
2.92 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant D63N | Thermococcus kodakarensis | |
2.97 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant E132L | Thermococcus kodakarensis | |
3.83 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant E132A | Thermococcus kodakarensis | |
4.17 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant G95A | Thermococcus kodakarensis | |
4.74 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, wild-type enzyme | Thermococcus kodakarensis | |
5.64 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant E132Q | Thermococcus kodakarensis | |
8.82 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant E132Q | Thermococcus kodakarensis | |
30 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, wild-type enzyme | Thermococcus kodakarensis | |
35.8 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant G95A | Thermococcus kodakarensis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
4.5 | 5 | - |
Thermococcus kodakarensis |
General Information | Comment | Organism |
---|---|---|
additional information | Tk-PTP adopts a common dual-specificity phosphatase (DUSP) fold, but it undergoes an atypical temperature-dependent conformational change in its P-loop and alpha4-alpha5 loop regions, switching between the inactive and active forms. Tk-PTP contains a PTP signature motif HCxxGxxR, HC93MGGLGR99 in Tk-PTP constituting the phosphate binding loop (or simply called P-loop), contains the catalytic cysteine residue (Cys93) that functions as a nucleophile for dephosphorylation and the conserved arginine residue (Arg99) that anchors the phosphate group of the substrate during the enzyme reaction. Tk-PTP contains dual general acid/base residues, Asp63 or Glu132. Structural analysis of the conformation of the P-loop in of Tk-PTP(form I), structure comparisons of PTPs, structure-function analysis of the two enzyme forms, overview. Molecular dynamics simulations | Thermococcus kodakarensis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.102 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant R124E | Thermococcus kodakarensis | |
0.14 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant D63N/E132A | Thermococcus kodakarensis | |
0.229 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant E132L | Thermococcus kodakarensis | |
0.281 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant Q136A | Thermococcus kodakarensis | |
0.406 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant R124A | Thermococcus kodakarensis | |
0.623 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant D63A | Thermococcus kodakarensis | |
0.729 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant R124E | Thermococcus kodakarensis | |
3.09 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant D63N/E132A | Thermococcus kodakarensis | |
3.41 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant Q136A | Thermococcus kodakarensis | |
5.28 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant R124A | Thermococcus kodakarensis | |
6.59 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, wild-type enzyme | Thermococcus kodakarensis | |
8.98 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant E132A | Thermococcus kodakarensis | |
11.6 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant D63N | Thermococcus kodakarensis | |
11.8 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant E132L | Thermococcus kodakarensis | |
18.4 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant E132Q | Thermococcus kodakarensis | |
47.1 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant D63A | Thermococcus kodakarensis | |
58.8 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant E132Q | Thermococcus kodakarensis | |
61.1 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant G95A | Thermococcus kodakarensis | |
109 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, wild-type enzyme | Thermococcus kodakarensis | |
167 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant E132A | Thermococcus kodakarensis | |
255 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 20°C, mutant D63N | Thermococcus kodakarensis | |
338 | - |
6,8-difluoro-4-methylumbelliferyl phosphate | pH 5.0, 60°C, mutant G95A | Thermococcus kodakarensis |