Activating Compound | Comment | Organism | Structure |
---|---|---|---|
formate | the mutant enzyme does not have any activity with the substrate 2-nitrophenyl-beta-D-glucopyranose. However, in the presence of 3 M sodium formate the mutant is reactivated, 10fold increment between 0.1 M and 3 M sodium formate | Pyrococcus horikoshii |
Application | Comment | Organism |
---|---|---|
synthesis | in the presence of sodium formate buffer pH 4.0 at 75°C the E324G mutant acts as a hyperthermophilic glycosynthase. Though the yield of the reaction does not exceed 10%, it is demonstrated that this could be a general strategy for the preparation of hyperthermophilic glycosynthase. The peculiar specificity of the enzyme for alkyl-glycosides makes the resulting glycosynthase a promising tool for biocatalysis | Pyrococcus horikoshii |
Cloned (Comment) | Organism |
---|---|
mutant enzyme E324G is expressed in Escherichia coli BL21(DE3) RIL | Pyrococcus horikoshii |
the mutant enzyme E324G is prepared as a fusion with an amino-terminal His-tag and expressed in Escherichia coli BL21(DE3) RIL strain | Pyrococcus horikoshii |
Protein Variants | Comment | Organism |
---|---|---|
E324G | mutant enzyme does not have any activity with the substrate 2-nitrophenyl-beta-D-glucopyranose. However, in the presence of 3 M sodium formate the mutant is reactivated. acts as a hyperthermophilic glycosynthase. kcat/KM of the mutant enzyme is more than 5-fold higher at pH 4.0 than that at pH 6.5. The hydrolytic activity of the mutant at 90°C at pH 6.5 in 3 M sodium formate is about 600fold lower than that of the wild type assayed on 4-nitrophenyl-glucose at 90°C in 50 mM sodium phosphate buffer pH 6.0, 0.1% Triton X-100, and 0.3 M NaCl | Pyrococcus horikoshii |
E324G | mutation completely eliminates the activity of the enzyme. Activity can be reactivated in sodium formate at pH 4.0. The enzyme acts as a glycosynthase. The peculiar specificity of the mutant enzyme E324G for alkyl-glycosides makes the glycosynthase a promising tool for biocatalysis | Pyrococcus horikoshii |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
15.11 | - |
2-nitrophenyl beta-D-glucopyranoside | pH 6.5, 50 mM sodium phosphate buffer + 3 M sodium formate, 75°C | Pyrococcus horikoshii | |
30.55 | - |
2-nitrophenyl beta-D-glucopyranoside | pH 4.0, 50 mM sodium phosphate buffer, 75°C | Pyrococcus horikoshii | |
39.47 | - |
2-nitrophenyl beta-D-glucopyranoside | pH 4.0, 200 mM sodium phosphate buffer, 75°C | Pyrococcus horikoshii |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
membrane | bound to | Pyrococcus horikoshii | 16020 | - |
membrane | membrane bound | Pyrococcus horikoshii | 16020 | - |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pyrococcus horikoshii | O58104 | - |
- |
Pyrococcus horikoshii OT-3 | O58104 | - |
- |
Purification (Comment) | Organism |
---|---|
- |
Pyrococcus horikoshii |
mutant enzyme E324G | Pyrococcus horikoshii |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-nitrophenyl beta-D-glucopyranoside + H2O | - |
Pyrococcus horikoshii | 2-nitrophenol + beta-D-glucopyranose | - |
? | |
2-nitrophenyl beta-D-glucopyranoside + H2O | wide substrate specificity belonging to family 1 of glycoside hydrolases classification. The mutant enzyme E324G does not have any activity with the substrate 2-nitrophenyl-beta-D-glucopyranose. However, in the presence of 3 M sodium formate the mutant is reactivated | Pyrococcus horikoshii | 2-nitrophenol + beta-D-glucopyranose | - |
? | |
2-nitrophenyl beta-D-glucopyranoside + H2O | - |
Pyrococcus horikoshii OT-3 | 2-nitrophenol + beta-D-glucopyranose | - |
? | |
2-nitrophenyl beta-D-glucopyranoside + H2O | wide substrate specificity belonging to family 1 of glycoside hydrolases classification. The mutant enzyme E324G does not have any activity with the substrate 2-nitrophenyl-beta-D-glucopyranose. However, in the presence of 3 M sodium formate the mutant is reactivated | Pyrococcus horikoshii OT-3 | 2-nitrophenol + beta-D-glucopyranose | - |
? | |
additional information | in the presence of sodium formate buffer pH 4.0 at 75°C the E324G mutant acts as a hyperthermophilic glycosynthase. Though the yield of the reaction does not exceed 10%, it is demonstrated that this could be a general strategy for the preparation of hyperthermophilic glycosynthase | Pyrococcus horikoshii | ? | - |
? | |
additional information | in the presence of sodium formate buffer pH 4.0 at 75°C the E324G mutant acts as a hyperthermophilic glycosynthase. Though the yield of the reaction does not exceed 10%, it is demonstrated that this could be a general strategy for the preparation of hyperthermophilic glycosynthase | Pyrococcus horikoshii OT-3 | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
monomer | - |
Pyrococcus horikoshii |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
75 | - |
assay at | Pyrococcus horikoshii |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.092 | - |
2-nitrophenyl beta-D-glucopyranoside | pH 6.5, 50 mM sodium phosphate buffer + 3 M sodium formate, 75°C | Pyrococcus horikoshii | |
0.592 | - |
2-nitrophenyl beta-D-glucopyranoside | pH 4.0, 50 mM sodium phosphate buffer, 75°C | Pyrococcus horikoshii | |
1.302 | - |
2-nitrophenyl beta-D-glucopyranoside | pH 4.0, 200 mM sodium phosphate buffer, 75°C | Pyrococcus horikoshii |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
4 | - |
assay at | Pyrococcus horikoshii |
4 | - |
assay at, optimally active under acidic conditions | Pyrococcus horikoshii |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.006 | - |
2-nitrophenyl beta-D-glucopyranoside | pH 6.5, 50 mM sodium phosphate buffer + 3 M sodium formate, 75°C | Pyrococcus horikoshii | |
0.019 | - |
2-nitrophenyl beta-D-glucopyranoside | pH 4.0, 50 mM sodium phosphate buffer, 75°C | Pyrococcus horikoshii | |
0.033 | - |
2-nitrophenyl beta-D-glucopyranoside | pH 4.0, 200 mM sodium phosphate buffer, 75°C | Pyrococcus horikoshii |