Cloned (Comment) | Organism |
---|---|
sequence comparisons, expression of wild-type and mutant enzymes | Escherichia coli |
Crystallization (Comment) | Organism |
---|---|
purified GDH in the absence of reactants, hanging drop vapour diffusion method, mixing of 10 mg/ml protein in 20 mM Tris-HCl, pH 7.6, with 15-25% PEG 3350, Tris/HEPES, pH 70-8.0, and 0.2 M NaCl, 18°C, 2 days, X-ray diffraction structure determination and analysis at 2.5 A resolution, modelling | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
K286Q | site-directed mutagenesis, the mutant shows increased KM for NADP+ compared to the wild-type enzyme | Escherichia coli |
K286Q/R289Q/R292Q | site-directed mutagenesis, the mutant shows highly increased KM for NADP+ compared to the wild-type enzyme | Escherichia coli |
K286Q/R289Q/R292Q/S264L | site-directed mutagenesis, the mutant shows highly increased KM for NADP+ compared to the wild-type enzyme | Escherichia coli |
K286Q/R289Q/R292Q/S264L/S240A | site-directed mutagenesis, the mutant shows highly increased KM for NADP+ compared to the wild-type enzyme | Escherichia coli |
R289Q | site-directed mutagenesis, the mutant shows increased KM for NADP+ compared to the wild-type enzyme | Escherichia coli |
R292Q | site-directed mutagenesis, the mutant shows increased KM for NADP+ compared to the wild-type enzyme | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0184 | - |
NADP+ | wild-type enzyme, pH 8.0, temperature not specified in the publication | Escherichia coli | |
0.0328 | - |
NADP+ | mutant K289Q, pH 8.0, temperature not specified in the publication | Escherichia coli | |
0.0819 | - |
NADP+ | mutant K292Q, pH 8.0, temperature not specified in the publication | Escherichia coli | |
0.129 | - |
NADP+ | mutant K286Q, pH 8.0, temperature not specified in the publication | Escherichia coli | |
0.929 | - |
NADP+ | mutant K286Q/R289Q/R292Q , pH 8.0, temperature not specified in the publication | Escherichia coli | |
18.3 | - |
NADP+ | mutant K286Q/R289Q/R292Q/S264L/S240A, pH 8.0, temperature not specified in the publication | Escherichia coli | |
19.06 | - |
NADP+ | mutant K286Q/R289Q/R292Q/S264L, pH 8.0, temperature not specified in the publication | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-glutamate + H2O + NADP+ | Escherichia coli | - |
2-oxoglutarate + NH3 + NADPH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P00370 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-glutamate + H2O + NADP+ | - |
Escherichia coli | 2-oxoglutarate + NH3 + NADPH + H+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
hexamer | EcGDH is a hexamer with 32 symmetry, an assembly. Protomer F (EcGDH-F) is the only molecule in the hexamer that adopts an open conformation that is sufficiently wide for diffusion of both substrate and cofactor, structure comparison to the enzyme from Clostridium symbiosum and other GDHs, overview | Escherichia coli |
More | modelling of NADP+ in domain II reveals the potential contribution of positively charged residues from a neighbouring alpha-helical hairpin to phosphate recognition, sequence-structure relationship, overview | Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
NADP+-dependent GDH | - |
Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.5 | - |
NADP+ | mutant K286Q/R289Q/R292Q/S264L, pH 8.0, temperature not specified in the publication | Escherichia coli | |
6.9 | - |
NADP+ | mutant K286Q/R289Q/R292Q/S264L/S240A, pH 8.0, temperature not specified in the publication | Escherichia coli | |
24 | - |
NADP+ | mutant K286Q/R289Q/R292Q, pH 8.0, temperature not specified in the publication | Escherichia coli | |
34.2 | - |
NADP+ | mutant K286Q, pH 8.0, temperature not specified in the publication | Escherichia coli | |
37.3 | - |
NADP+ | wild-type enzyme, pH 8.0, temperature not specified in the publication | Escherichia coli | |
37.6 | - |
NADP+ | mutant K289Q, pH 8.0, temperature not specified in the publication | Escherichia coli | |
47.3 | - |
NADP+ | mutant K292Q, pH 8.0, temperature not specified in the publication | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADP+ | binding structure involving Asp263 and Ser264, residues P7 and P8, molecular modelling of NADP+ onto the free enzyme structure, overview | Escherichia coli | |
NADPH | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
additional information | modelling of NADP+ in domain II reveals the potential contribution of positively charged residues from a neighbouring alpha-helical hairpin to phosphate recognition, sequence-structure relationship, overview. A single sequence accommodates both coenzymes in the dual-specificity GDHs of animals | Escherichia coli |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00000013 | - |
NADP+ | mutant K286Q/R289Q/R292Q/S264L, pH 8.0, temperature not specified in the publication | Escherichia coli | |
0.00000038 | - |
NADP+ | mutant K286Q/R289Q/R292Q/S264L/S240A, pH 8.0, temperature not specified in the publication | Escherichia coli | |
0.0000258 | - |
NADP+ | mutant K286Q/R289Q/R292Q, pH 8.0, temperature not specified in the publication | Escherichia coli | |
0.000115 | - |
NADP+ | mutant K289Q, pH 8.0, temperature not specified in the publication | Escherichia coli | |
0.000265 | - |
NADP+ | mutant K286Q, pH 8.0, temperature not specified in the publication | Escherichia coli | |
0.000578 | - |
NADP+ | mutant K292Q, pH 8.0, temperature not specified in the publication | Escherichia coli | |
0.00203 | - |
NADP+ | wild-type enzyme, pH 8.0, temperature not specified in the publication | Escherichia coli |