Cloned (Comment) | Organism |
---|---|
gene gadB, expression of C-terminally His-tagged wild-type and mutant enzyme in Escherichia coli strain BL21(DE3), cloning in Escherichia coli strain DH5alpha | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
additional information | improvement of the thermostability of GadB through structural optimization ofits N-terminal interdomain. Residues Gln5, Val6, and Thr7 are potential mutational target sites for the optimization of inter- and intra-molecular interactions of the triple-helix bundle. Generation of a deletion mutant of GadB lacking residues 1-14 at the N-terminus, GadBDELTA1-14 | Escherichia coli |
Q5D/V6I/T7E | site-directed mutagenesis the mutant shows higher thermostability and increased melting temperature compared to the wild-type, but shows no reduction of catalytic activity | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.32 | - |
L-glutamate | pH 4.6, 37°C, recombinant untagged wild-type enzyme | Escherichia coli | |
20.31 | - |
L-glutamate | pH 4.6, 37°C, recombinant C-terminally His-tagged wild-type enzyme | Escherichia coli | |
25.09 | - |
L-glutamate | pH 4.6, 37°C, recombinant deletion mutant GadBDELTA1-14 | Escherichia coli | |
26.02 | - |
L-glutamate | pH 4.6, 37°C, recombinant mutant Q5D/V6I/T7E | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Cl- | binding of chloride ions to the N-terminus could stabilizes the triple-helix bundle | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-glutamate | Escherichia coli | - |
4-aminobutanoate + CO2 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P69910 | gene gadB | - |
Purification (Comment) | Organism |
---|---|
recombinant C-terminally His-tagged wild-type and mutant enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography and ultrafiltration | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-glutamate | - |
Escherichia coli | 4-aminobutanoate + CO2 | - |
? |
Subunits | Comment | Organism |
---|---|---|
homohexamer | - |
Escherichia coli |
More | homohexameric GadB forms a triple-helix bundle interdomain at acidic pH | Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
GadB | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Escherichia coli |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
additional information | - |
homohexameric GadB forms a triple-helix bundle interdomain at acidic pH and contributes to the thermostability of GadB | Escherichia coli |
55 | - |
purified recombinant wild-type enzyme, without chloride ions, 10 min, 71% activity remaining at pH 4.6, 2% remaining at pH 8.0, the deletion mutant GadB DELTA1-14 exhibits similar thermostabilityat pH 4.6 and pH 8.0 | Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
59.26 | - |
L-glutamate | pH 4.6, 37°C, recombinant C-terminally His-tagged wild-type enzyme | Escherichia coli | |
73.35 | - |
L-glutamate | pH 4.6, 37°C, recombinant deletion mutant GadBDELTA1-14 | Escherichia coli | |
75.41 | - |
L-glutamate | pH 4.6, 37°C, recombinant mutant Q5D/V6I/T7E | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
4.6 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
additional information | the N-terminal fourteen residues (1-14) of homohexameric GadB forms a triple-helix bundle interdomain at acidic pH and contributes to the thermostability of GadB as the pH shifts from pH 7.6 to pH 4.6 | Escherichia coli |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.88 | - |
L-glutamate | pH 4.6, 37°C, recombinant mutant Q5D/V6I/T7E | Escherichia coli | |
2.92 | - |
L-glutamate | pH 4.6, 37°C, recombinant C-terminally His-tagged wild-type enzyme and deletion mutant GadBDELTA1-14 | Escherichia coli |