Cloned (Comment) | Organism |
---|---|
cloning and expression of soluble wild-type and mutant enzymes in Escherichia coli strain XL1-Blue | [Clostridium] symbiosum |
Protein Variants | Comment | Organism |
---|---|---|
A242G | site-directed mutagenesis, the mutant shows A242G showed a decreased overall catalytic efficiency for NADH at all pH values of pH 6.0-8.0 after Ala replacement with Gly compared to the wild-type enzyme, the mutation had a severe effect on the overall catalytic efficiency with NADPH as coenzyme | [Clostridium] symbiosum |
D263K | site-directed mutagenesis, the D263K mutation produces remarkably little change in the kinetic parameters for NADH at pH 6.0-8.0 compared to the wild-type enzyme, with NADPH at all three pH values the kcat for the mutant is much higher than for wild-type GDH, and this factor increases from pH 6.0 to pH 7.0 and pH 8.0 | [Clostridium] symbiosum |
F238S | site-directed mutagenesis, the mutant shows markedly increased catalytic efficiency with NADPH, especially at pH 8.0 in the range of pH 6.0-8.0 | [Clostridium] symbiosum |
F238S/P262S | site-directed mutagenesis, the mutant shows markedly increased catalytic efficiency with NADPH, especially at pH 8.0 in the range of pH 6.0-8.0 | [Clostridium] symbiosum |
P262S | site-directed mutagenesis, the mutant shows markedly increased catalytic efficiency with NADPH, especially at pH 8.0 in the range of pH 6.0-8.0 | [Clostridium] symbiosum |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | enzyme kinetics of wild-tyype and mutant enzymes with NADPH, overview | [Clostridium] symbiosum | |
0.018 | - |
NADH | pH 6.0, 25°C, mutant P262S | [Clostridium] symbiosum | |
0.02 | - |
NADH | pH 7.0, 25°C, recombinant wild-type enzyme | [Clostridium] symbiosum | |
0.022 | - |
NADH | pH 6.0, 25°C, mutant D263K | [Clostridium] symbiosum | |
0.023 | - |
NADH | pH 7.0, 25°C, mutant D263K | [Clostridium] symbiosum | |
0.025 | - |
NADH | pH 7.0, 25°C, mutant A242G | [Clostridium] symbiosum | |
0.028 | - |
NADH | pH 6.0, 25°C, mutant F238S | [Clostridium] symbiosum | |
0.034 | - |
NADH | pH 6.0, 25°C, mutant A242G | [Clostridium] symbiosum | |
0.035 | - |
NADH | pH 7.0, 25°C, mutant F238S/P262S | [Clostridium] symbiosum | |
0.036 | - |
NADH | pH 6.0, 25°C, recombinant wild-type enzyme | [Clostridium] symbiosum | |
0.04 | - |
NADH | pH 7.0, 25°C, mutant F238S | [Clostridium] symbiosum | |
0.047 | - |
NADH | pH 7.0, 25°C, mutant P262S | [Clostridium] symbiosum | |
0.048 | - |
NADH | pH 6.0, 25°C, mutant F238S/P262S | [Clostridium] symbiosum | |
0.078 | - |
NADH | pH 8.0, 25°C, recombinant wild-type enzyme | [Clostridium] symbiosum | |
0.085 | - |
NADH | pH 8.0, 25°C, mutant A242G | [Clostridium] symbiosum | |
0.091 | - |
NADH | pH 8.0, 25°C, mutant D263K | [Clostridium] symbiosum | |
0.142 | - |
NADH | pH 8.0, 25°C, mutant P262S | [Clostridium] symbiosum | |
0.204 | - |
NADH | pH 8.0, 25°C, mutant F238S/P262S | [Clostridium] symbiosum | |
0.23 | - |
NADH | pH 8.0, 25°C, mutant F238S | [Clostridium] symbiosum |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-glutamate + H2O + NAD+ | [Clostridium] symbiosum | - |
2-oxoglutarate + NH3 + NADH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
[Clostridium] symbiosum | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain XL1-Blue | [Clostridium] symbiosum |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-glutamate + H2O + NAD+ | - |
[Clostridium] symbiosum | 2-oxoglutarate + NH3 + NADH + H+ | - |
r |
Synonyms | Comment | Organism |
---|---|---|
NAD+-specific GDH | - |
[Clostridium] symbiosum |
NAD-dependent glutamate dehydrogenase | - |
[Clostridium] symbiosum |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | [Clostridium] symbiosum |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
11.2 | - |
NADH | pH 6.0, 25°C, mutant A242G | [Clostridium] symbiosum | |
13.6 | - |
NADH | pH 6.0, 25°C, mutant F238S | [Clostridium] symbiosum | |
13.9 | - |
NADH | pH 6.0, 25°C, mutant F238S/P262S | [Clostridium] symbiosum | |
14.4 | - |
NADH | pH 6.0, 25°C, mutant P262S | [Clostridium] symbiosum | |
14.6 | - |
NADH | pH 6.0, 25°C, mutant D263K | [Clostridium] symbiosum | |
19.9 | - |
NADH | pH 6.0, 25°C, recombinant wild-type enzyme | [Clostridium] symbiosum | |
65.1 | - |
NADH | pH 7.0, 25°C, mutant A242G | [Clostridium] symbiosum | |
80 | - |
NADH | pH 7.0, 25°C, mutant D263K | [Clostridium] symbiosum | |
105 | - |
NADH | pH 7.0, 25°C, mutant F238S/P262S | [Clostridium] symbiosum | |
130.2 | - |
NADH | pH 7.0, 25°C, recombinant wild-type enzyme | [Clostridium] symbiosum | |
133 | - |
NADH | pH 8.0, 25°C, mutant F238S/P262S | [Clostridium] symbiosum | |
139 | - |
NADH | pH 7.0, 25°C, mutant F238S | [Clostridium] symbiosum | |
150 | - |
NADH | pH 8.0, 25°C, mutant D263K | [Clostridium] symbiosum | |
160 | - |
NADH | pH 7.0, 25°C, mutant P262S | [Clostridium] symbiosum | |
177 | - |
NADH | pH 8.0, 25°C, mutant A242G | [Clostridium] symbiosum | |
232 | - |
NADH | pH 8.0, 25°C, recombinant wild-type enzyme | [Clostridium] symbiosum | |
402 | - |
NADH | pH 8.0, 25°C, mutant F238S | [Clostridium] symbiosum | |
490 | - |
NADH | pH 8.0, 25°C, mutant P262S | [Clostridium] symbiosum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | 8 | assay at | [Clostridium] symbiosum |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | in the wild-type enzyme there is a strong pH dependence in the level of discrimination between NADH and NADPH | [Clostridium] symbiosum | |
NAD+ | the core fingerprint for coenzyme binding is the conserved sequence Gly-X-Gly-X-X-Gly, which consists of residues that form a hydrophobic core between the beta-strands and the alpha-helix, and glycine residues that allow for a tight turn between the first beta-strand and the following alpha-helix. In all these enzymes, the last residue in helix alphaA has to be a Gly, the first Gly of the consensus, because any larger residue would interfere with the adenine ribose. Coenzyme binding structure, overview | [Clostridium] symbiosum | |
NADH | the core fingerprint for coenzyme binding is the conserved sequence Gly-X-Gly-X-X-Gly, which consists of residues that form a hydrophobic core between the beta-strands and the alpha-helix, and glycine residues that allow for a tight turn between the first beta-strand and the following alpha-helix. In all these enzymes, the last residue in helix alphaA has to be a Gly, the first Gly of the consensus, because any larger residue would interfere with the adenine ribose. Coenzyme binding structure, overview | [Clostridium] symbiosum |
General Information | Comment | Organism |
---|---|---|
additional information | at position 242, P6 of the core fingerprint, where NAD+- and NADP+-dependent enzymes normally have Gly or Ala, respectively, clostridial GDH already has Ala. Replacement with Gly produced negligible shift in coenzyme specificity | [Clostridium] symbiosum |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
289 | - |
NADH | pH 6.0, 25°C, mutant F238S/P262S | [Clostridium] symbiosum | |
329 | - |
NADH | pH 6.0, 25°C, mutant A242G | [Clostridium] symbiosum | |
485 | - |
NADH | pH 6.0, 25°C, mutant F238S | [Clostridium] symbiosum | |
553 | - |
NADH | pH 6.0, 25°C, recombinant wild-type enzyme | [Clostridium] symbiosum | |
655 | - |
NADH | pH 8.0, 25°C, mutant F238S/P262S | [Clostridium] symbiosum | |
663 | - |
NADH | pH 6.0, 25°C, mutant D263K | [Clostridium] symbiosum | |
800 | - |
NADH | pH 6.0, 25°C, mutant P262S | [Clostridium] symbiosum | |
1648 | - |
NADH | pH 8.0, 25°C, mutant D263K | [Clostridium] symbiosum | |
1747 | - |
NADH | pH 8.0, 25°C, mutant F238S | [Clostridium] symbiosum | |
2082 | - |
NADH | pH 8.0, 25°C, mutant A242G | [Clostridium] symbiosum | |
2604 | - |
NADH | pH 7.0, 25°C, mutant A242G | [Clostridium] symbiosum | |
2974 | - |
NADH | pH 8.0, 25°C, recombinant wild-type enzyme | [Clostridium] symbiosum | |
3000 | - |
NADH | pH 7.0, 25°C, mutant F238S/P262S | [Clostridium] symbiosum | |
3404 | - |
NADH | pH 7.0, 25°C, mutant P262S | [Clostridium] symbiosum | |
3450 | - |
NADH | pH 8.0, 25°C, mutant P262S | [Clostridium] symbiosum | |
3475 | - |
NADH | pH 7.0, 25°C, mutant F238S | [Clostridium] symbiosum | |
3478 | - |
NADH | pH 7.0, 25°C, mutant D263K | [Clostridium] symbiosum | |
6500 | - |
NADH | pH 7.0, 25°C, recombinant wild-type enzyme | [Clostridium] symbiosum |