Application | Comment | Organism |
---|---|---|
drug development | the GSI-alpha-specific regulatory network can be exploited for inhibitor design against Gram-positive pathogens | Bacillus subtilis |
Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli | Bacillus subtilis |
Crystallization (Comment) | Organism |
---|---|
purified recombinant detagged apo-enzyme, enzyme-glutamate-AMPPCP complex, and enzyme transition state, hanging drop vapor diffusion, for enzyme-glutamate-AMPPCP crystals: mixing of 40 mg/ml protein at a 1:1 ratio with 40% 4-methyl-2,4-pentanediol and 200 mM MgSO4, and inverting the drop over the reservoir solution containing 15% PEG 8000, 0.1 M HEPES, pH 7.5, and 10 mM MgCl2, for enzyme-L-methionine-S-sulfoximine-phosphate-ADP crystals: mixing of 40 mg/ml protein with 5 mM MgCl2, 5 mM ATP, and 5 mM L-methionine-S-sulfoximine,and combining in a 1:1 ratio with crystallization reagent containing 10% PEG 4000, 0.1 M HEPES, pH 7.5, for apo-enzyme crystals: mixing the protein 40 mg/ml at a 1:1 ratio with 40% 4-methyl-2,4-pentanediol and 200 mM MgSO4 and inverting the drop over the reservoir solution, room temperature, X-ray diffraction structure determination and analysis at 3.1 A, 2.87 A, and 2.58 A resolution, respectively | Bacillus subtilis |
Protein Variants | Comment | Organism |
---|---|---|
E304A/A305G | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Bacillus subtilis |
R62A | site-directed mutagenesis, the mutation abrogates Gln feedback inhibition but does not affect catalysis | Bacillus subtilis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
L-glutamine | feedback inhibition of isozymes GSI-alpha and GSI-beta. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-alpha-specific residue, Arg62, from an adjacent subunit. Arg62 must be ejected for proper active site reorganization. An R62A mutation abrogates Gln feedback inhibition but does not affect catalysis | Bacillus subtilis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.18 | - |
NH3 | pH 7.0, temperature not specified in the publication, wild-type enzyme | Bacillus subtilis | |
0.34 | - |
NH3 | pH 7.0, temperature not specified in the publication, mutant R62A | Bacillus subtilis | |
0.68 | - |
hydroxylamine | pH 7.0, temperature not specified in the publication, mutant E304A | Bacillus subtilis | |
0.83 | - |
hydroxylamine | pH 7.0, temperature not specified in the publication, wild-type enzyme | Bacillus subtilis | |
1 | 4 | L-glutamate | pH 7.0, temperature not specified in the publication, mutant R62A | Bacillus subtilis | |
1.2 | - |
ATP | pH 7.0, temperature not specified in the publication, mutant E304A | Bacillus subtilis | |
1.4 | - |
hydroxylamine | pH 7.0, temperature not specified in the publication, mutant R62A | Bacillus subtilis | |
2.3 | - |
ATP | pH 7.0, temperature not specified in the publication, mutant R62A | Bacillus subtilis | |
2.4 | - |
ATP | pH 7.0, temperature not specified in the publication, wild-type enzyme | Bacillus subtilis | |
3.4 | - |
L-glutamate | pH 7.0, temperature not specified in the publication, mutant E304A | Bacillus subtilis | |
27 | - |
L-glutamate | pH 7.0, temperature not specified in the publication, wild-type enzyme | Bacillus subtilis | |
32 | - |
NH3 | pH 7.0, temperature not specified in the publication, mutant E304A | Bacillus subtilis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Bacillus subtilis | |
Mn2+ | required for gamma-glutamylhydroxamate synthetase activity | Bacillus subtilis |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
600000 | - |
isozyme GSI-alpha | Bacillus subtilis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-glutamate + NH3 | Bacillus subtilis | - |
ADP + phosphate + L-glutamine | - |
? | |
ATP + L-glutamate + NH3 | Bacillus subtilis 168 | - |
ADP + phosphate + L-glutamine | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus subtilis | P12425 | isozyme GSI-alpha | - |
Bacillus subtilis 168 | P12425 | isozyme GSI-alpha | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography, urea treatment, tag cleavage by thrombin, again nickel affinity chromatography to remove the tag, and dialysis | Bacillus subtilis |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
ATP + L-glutamate + NH3 = ADP + phosphate + L-glutamine | structure-function relationship and catalytic mechanism, overview | Bacillus subtilis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-glutamate + NH3 | - |
Bacillus subtilis | ADP + phosphate + L-glutamine | - |
? | |
ATP + L-glutamate + NH3 | - |
Bacillus subtilis 168 | ADP + phosphate + L-glutamine | - |
? | |
hydroxylamine + L-glutamine + ATP | gamma-glutamylhydroxamate synthetase activity | Bacillus subtilis | L-gamma-glutamyl-hydroxamate + ammonium + ADP | - |
r | |
hydroxylamine + L-glutamine + ATP | gamma-glutamylhydroxamate synthetase activity | Bacillus subtilis 168 | L-gamma-glutamyl-hydroxamate + ammonium + ADP | - |
r |
Subunits | Comment | Organism |
---|---|---|
dodecamer | isozyme GSI-alpha | Bacillus subtilis |
Synonyms | Comment | Organism |
---|---|---|
GluA | - |
Bacillus subtilis |
Glutamine synthetase | - |
Bacillus subtilis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
gamma-glutamylhydroxamate synthetase forward reaction assay at | Bacillus subtilis |
7.5 | - |
gamma-glutamylhydroxamate synthetase reverse reaction assay at | Bacillus subtilis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Bacillus subtilis |
General Information | Comment | Organism |
---|---|---|
metabolism | glutamine synthetase plays essential roles in nitrogen metabolism | Bacillus subtilis |
additional information | the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Structure-function relationship, overview | Bacillus subtilis |