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Literature summary for 6.3.1.2 extracted from

  • Zhang, S.; Han, Y.; Kumar, A.; Gao, H.; Liu, Z.; Hu, N.
    Characterization of an L-phosphinothricin resistant glutamine synthetase from Exiguobacterium sp. and its improvement (2017), Appl. Microbiol. Biotechnol., 101, 3653-3661 .
    View publication on PubMed
Search for more literature for 6.3.1.2

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of GST-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Exiguobacterium sp.

Protein Variants

Protein Variants Comment Organism
E60A site-directed mutagenesis, the E60A mutant has about 2.2fold increased activity compared to wild-type Exiguobacterium sp.
E60A/R64G site-directed mutagenesis, no significant change is observed in the activity of E60A/R64G mutant Exiguobacterium sp.
F239Y site-directed mutagenesis, the mutant has slightly reduced activity compared to wild-type Exiguobacterium sp.
P135S site-directed mutagenesis, the mutant has slightly reduced activity compared to wild-type Exiguobacterium sp.
R64G site-directed mutagenesis, the binding pocket undergoes dramatic changes when Arg site of 64 is substituted by Gly, thus promoting the rapid capture of substrates and leading to increase in activity and PPT-resistance of mutant R64G. Kinetic analysis shows that the kcat of R64G mutant is increased by 8.10, 7.25 and 7.63fold for ADP, glutamine and hydroxylamine, respectively, the mutant has about 1.9fold increased activity compared to wild-type Exiguobacterium sp.
V241D site-directed mutagenesis, the mutant has slightly increased activity compared to wild-type Exiguobacterium sp.
Y305F site-directed mutagenesis, the mutant has reduced activity compared to wild-type Exiguobacterium sp.
Y375P site-directed mutagenesis, the mutant has slightly increased activity compared to wild-type Exiguobacterium sp.

Inhibitors

Inhibitors Comment Organism Structure
Co2+ inhibits the activity of the wild-type enzyme at 1 mM Exiguobacterium sp.
Cu2+ Cu2+ strongly inhibits the activity of wild-type and mutant enzymes at 1 mM Exiguobacterium sp.
L-phosphinothricin the glutamine synthetase from Exiguobacterium sp. is L-phosphinothricin resistant. Molecular docking analysis indicates that the substitution of residues Glu60 and Arg64 may lead to significant changes in binding pocket Exiguobacterium sp.
Li+ inhibits the activity of the wild-type enzyme at 1 mM Exiguobacterium sp.
NH4+ inhibits the activity of the wild-type enzyme at 1 mM Exiguobacterium sp.
Ni2+ inhibits the activity of the wild-type enzyme at 1 mM Exiguobacterium sp.
Zn2+ inhibits the activity of the wild-type enzyme at 1 mM Exiguobacterium sp.

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics Exiguobacterium sp.
0.00091
-
 ADP pH 6.0, 35°C, recombinant mutant R64G Exiguobacterium sp.
0.00461
-
 ADP pH 6.0, 35°C, recombinant mutant E60A Exiguobacterium sp.
0.00673
-
 L-glutamine pH 6.0, 35°C, recombinant mutant E60A/R64G Exiguobacterium sp.
0.00865
-
 ADP pH 6.0, 35°C, recombinant wild-type enzyme Exiguobacterium sp.
0.00888
-
 L-glutamine pH 6.0, 35°C, recombinant mutant E60A Exiguobacterium sp.
0.00982
-
 L-glutamine pH 6.0, 35°C, recombinant mutant R64G Exiguobacterium sp.
0.0113
-
 ADP pH 6.0, 35°C, recombinant mutant E60A/R64G Exiguobacterium sp.
0.012
-
 L-glutamine pH 6.0, 35°C, recombinant wild-type enzyme Exiguobacterium sp.

Metals/Ions

Metals/Ions Comment Organism Structure
Ba2+ increases the activity of wild-type and mutant enzymes at 1 mM Exiguobacterium sp.
Co2+ increases the activity of the mutant enzymes at 1 mM Exiguobacterium sp.
Fe3+ increases the activity of wild-type and mutant enzymes at 1 mM Exiguobacterium sp.
K+ increases the activity of wild-type and mutant enzymes at 1 mM Exiguobacterium sp.
Li+ increases the activity of the mutant enzymes at 1 mM Exiguobacterium sp.
Mg2+ required, increases the activity of wild-type and mutant enzymes at 1 mM Exiguobacterium sp.
Na+ increases the activity of wild-type and mutant enzymes at 1 mM Exiguobacterium sp.
NH4+ increases the activity of the mutant enzymes at 1 mM Exiguobacterium sp.
Ni2+ increases the activity of the mutant enzymes at 1 mM Exiguobacterium sp.
Zn2+ increases the activity of the mutant enzymes at 1 mM Exiguobacterium sp.

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + L-glutamate + NH3 Exiguobacterium sp.
-
 ADP + phosphate + L-glutamine
-
 r
ATP + L-glutamate + NH3 Exiguobacterium sp. ATCC BAA-1283
-
 ADP + phosphate + L-glutamine
-
 r

Organism

Organism UniProt Comment Textmining
Exiguobacterium sp. C4L2T5
-
-
Exiguobacterium sp. ATCC BAA-1283 C4L2T5
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant GST-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography, tag cleavage by 3C protease, and Exiguobacterium sp.

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ADP + L-glutamine + hydroxylamine
-
 Exiguobacterium sp. gamma-glutamylhydroxamate + ?
-
 r
ADP + L-glutamine + hydroxylamine
-
 Exiguobacterium sp. ATCC BAA-1283 gamma-glutamylhydroxamate + ?
-
 r
ADP + phosphate + L-glutamine
-
 Exiguobacterium sp. ATP + L-glutamate + NH3
-
 r
ADP + phosphate + L-glutamine
-
 Exiguobacterium sp. ATCC BAA-1283 ATP + L-glutamate + NH3
-
 r
ATP + L-glutamate + NH3
-
 Exiguobacterium sp. ADP + phosphate + L-glutamine
-
 r
ATP + L-glutamate + NH3
-
 Exiguobacterium sp. ATCC BAA-1283 ADP + phosphate + L-glutamine
-
 r

Subunits

Subunits Comment Organism
? x * 50100, wild-type enzyme, SDS-PAGE, x * 79600, recombinant GST-tagged wild-type enzyme, SDS-PAGE Exiguobacterium sp.

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
35
-
 wild-type enzyme Exiguobacterium sp.
40
-
 enzyme mutant R64G Exiguobacterium sp.
45
-
 enzyme mutant E60A Exiguobacterium sp.

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
50
-
 purified wild-type and mutant enzymes are completely stable for at least 3 h Exiguobacterium sp.
60
-
 purified the wild-type enzyme shows 50% reduction in activity after 1 h Exiguobacterium sp.
70
-
 purified mutant enzymes R64G and E60A are 4.3 and 5.7times, respectively, more stable at 70°C than the wild-type, wild-type enzyme shows 50% reduction in activity after 0.5 h, the E60A and R64G mutants retain approximately 40% of their original activities after 3 h, but a rapid decrease occurs in the activities of wild-type and mutant E60A/R64G Exiguobacterium sp.

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.05
-
 ADP pH 6.0, 35°C, recombinant wild-type enzyme Exiguobacterium sp.
1.62
-
 L-glutamine pH 6.0, 35°C, recombinant wild-type enzyme Exiguobacterium sp.
4
-
 ADP pH 6.0, 35°C, recombinant mutant E60A/R64G Exiguobacterium sp.
4.67
-
 ADP pH 6.0, 35°C, recombinant mutant E60A Exiguobacterium sp.
8.96
-
 L-glutamine pH 6.0, 35°C, recombinant mutant E60A/R64G Exiguobacterium sp.
9.27
-
 L-glutamine pH 6.0, 35°C, recombinant mutant E60A Exiguobacterium sp.
9.53
-
 ADP pH 6.0, 35°C, recombinant mutant R64G Exiguobacterium sp.
13.4
-
 L-glutamine pH 6.0, 35°C, recombinant mutant R64G Exiguobacterium sp.

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
 assay at Exiguobacterium sp.

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Exiguobacterium sp.

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
2.43
-
 L-phosphinothricin pH 6.0, 35°C, recombinant wild-type enzyme Exiguobacterium sp.
3.75
-
 L-phosphinothricin pH 6.0, 35°C, recombinant E60A/R64G Exiguobacterium sp.
4.14
-
 L-phosphinothricin pH 6.0, 35°C, recombinant E60A Exiguobacterium sp.
4.91
-
 L-phosphinothricin pH 6.0, 35°C, recombinant R64G Exiguobacterium sp.

General Information

General Information Comment Organism
additional information structure-function relationships of wild-type and mutant enzymes, ligand molecular docking, homology structure modeling using the crystal structure of the enzyme from Bacillus subtilis, PDB ID 4LNF, as a template, overview Exiguobacterium sp.
physiological function glutamine synthetase is an important enzyme that catalyzes the conversion of L-glutamate into L-glutamine and ammonia in an energy dependent reaction with the simultaneous hydrolysis of ATP to ADP. In the cellular system, the enzyme plays an important role in nitrogen metabolism under ammonia-limiting conditions Exiguobacterium sp.

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.104
-
 L-glutamine pH 6.0, 35°C, recombinant mutant E60A Exiguobacterium sp.
0.135
-
 L-glutamine pH 6.0, 35°C, recombinant wild-type enzyme Exiguobacterium sp.
1.33
-
 L-glutamine pH 6.0, 35°C, recombinant mutant E60A/R64G Exiguobacterium sp.
1.36
-
 L-glutamine pH 6.0, 35°C, recombinant mutant R64G Exiguobacterium sp.
121
-
 ADP pH 6.0, 35°C, recombinant wild-type enzyme Exiguobacterium sp.
350
-
 ADP pH 6.0, 35°C, recombinant mutant E60A/R64G Exiguobacterium sp.
1030
-
 ADP pH 6.0, 35°C, recombinant mutant E60A Exiguobacterium sp.
10500
-
 ADP pH 6.0, 35°C, recombinant mutant R64G Exiguobacterium sp.