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Literature summary for 6.3.1.2 extracted from

  • Murray, D.S.; Chinnam, N.; Tonthat, N.K.; Whitfill, T.; Wray, L.V.; Fisher, S.H.; Schumacher, M.A.
    Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism (2013), J. Biol. Chem., 288, 35801-35811.
    View publication on PubMedView publication on EuropePMC

Application

Application Comment Organism
drug development the GSI-alpha-specific regulatory network can be exploited for inhibitor design against Gram-positive pathogens Bacillus subtilis

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli Bacillus subtilis

Crystallization (Commentary)

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

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

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 [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

Metals/Ions Comment Organism Structure
Mg2+ required Bacillus subtilis
Mn2+ required for gamma-glutamylhydroxamate synthetase activity Bacillus subtilis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
600000
-
isozyme GSI-alpha Bacillus subtilis

Natural Substrates/ Products (Substrates)

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

Organism UniProt Comment Textmining
Bacillus subtilis P12425 isozyme GSI-alpha
-
Bacillus subtilis 168 P12425 isozyme GSI-alpha
-

Purification (Commentary)

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

Reaction Comment Organism Reaction ID
ATP + L-glutamate + NH3 = ADP + phosphate + L-glutamine structure-function relationship and catalytic mechanism, overview Bacillus subtilis

Substrates and Products (Substrate)

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

Subunits Comment Organism
dodecamer isozyme GSI-alpha Bacillus subtilis

Synonyms

Synonyms Comment Organism
GluA
-
Bacillus subtilis
Glutamine synthetase
-
Bacillus subtilis

pH Optimum

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

Cofactor Comment Organism Structure
ATP
-
Bacillus subtilis

General Information

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