Cloned (Comment) | Organism |
---|---|
gene glnS, expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 | Escherichia coli |
Crystallization (Comment) | Organism |
---|---|
GlnRS-tRNAGln complex, 6.6 mg/ml protein in 10 mM PIPES, pH 7.5, 10 mM MgCl2, and 1.8-5.4 mM tRNA. The tRNA/analog solution is then mixed with equal volumes of a 6.3 mg/ml solution of GlnRS, containing 5mM PIPES, pH 7.0, and 5 mM 2-mercaptoethanol, X-ray diffraction structure determination and analysis at 2.6 A resolution | Escherichia coli |
purified recombinant GlnRS C229R-tRNAGln complex, a protein solution containing 6.3mg/ml GlnRS prepared in 5 mM PIPES, pH 7.0, 5 mM 2-mercaptoethanol, is mixed with the tRNAGln solution, X-ray diffraction structure determination and analysis at 2.6 A resolution | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
C229R | site-directed mutagenesis, transplanting the conserved arginine residue from glutamyl-tRNA synthetase, EC 6.1.1.17, to glutaminyltRNA synthetase improves the KM of GlnRS for noncognate glutamate | Escherichia coli |
C229R/Q255I | site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows no activity with L-Gln, but weakly with L-Glu | Escherichia coli |
C229R/Q255I/S227A/F233Y | site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows no activity with L-Gln, but activity with L-Glu | Escherichia coli |
additional information | cumulative replacement of other primary binding site residues than Cys229 in GlnRS, with those of GluRS, only slightly improves the ability of the GlnRS active site to accommodate glutamate. Introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in C229R, improves the capacity of Escherichia coli GlnRS to synthesize misacylated Glu-tRNAGln by 16000fold. This hybrid enzyme recapitulates the function of misacylating GluRS enzymes found in organisms that synthesize Gln-tRNAGln by an alternative pathway, overview | Escherichia coli |
additional information | the engineered mutant hybrid C229R Gln-RS, EC 6.1.1.18, shows activity with L-glutamine or L-glutamate and tRNAGln like the nondiscriminating enzyme, EC 6.1.1.24. Introduction of 22 amino acid replacements and one deletion, including substitution of the entire primary binding site and two surface loops adjacent to the region disrupted in the mutant C229R, improves the capacity of the mutant enzyme to synthesize misacylated Glu-tRNAGln by 16000fold, overview | Escherichia coli |
R30A | site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows no activity with L-Glu | Escherichia coli |
R30K | site-directed mutagenesis, comparison of mutant activity with glutamate and glutamine to charge tRNAGln to the wild-type activity, the mutant shows weak activity with L-Glu | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics of the mutant enzyme compared to wild-type GlnRS, EC 6.1.1.18, overview | Escherichia coli | |
additional information | - |
additional information | no KM value for L-glutamate with the wild-type enzyme due to no saturation, kinetics of wild-type and mutant enzymes, overview. Kinetics of extended-loop GlnRS mutants, overview | Escherichia coli | |
0.21 | - |
L-glutamine | mutant C229R GlnRS, with tRNAGln | Escherichia coli | |
0.26 | - |
L-glutamine | pH 7.5, 22°C, wild-type enzyme | Escherichia coli | |
240 | - |
L-glutamate | mutant C229R GlnRS, with tRNAGln | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | - |
Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-glutamate + tRNAGln | Escherichia coli | - |
AMP + diphosphate + glutamyl-tRNAGln | - |
? | |
ATP + L-glutamine + tRNAGln | Escherichia coli | - |
AMP + diphosphate + L-glutaminyl-tRNAGln | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Escherichia coli | P00962 | gene glnS | - |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-glutamate + tRNAGln | primary binding pocket structure, overview | Escherichia coli | AMP + diphosphate + L-glutamyl-tRNAGln | - |
? | |
ATP + L-glutamate + tRNAGln | - |
Escherichia coli | AMP + diphosphate + glutamyl-tRNAGln | - |
? | |
ATP + L-glutamate + tRNAGln | activity also with Gln-RS, EC 6.1.1.18, mutant C229R | Escherichia coli | AMP + diphosphate + glutamyl-tRNAGln | - |
? | |
ATP + L-glutamine + tRNAGln | - |
Escherichia coli | AMP + diphosphate + L-glutaminyl-tRNAGln | - |
? | |
ATP + L-glutamine + tRNAGln | primary binding pocket structure, overview | Escherichia coli | AMP + diphosphate + L-glutaminyl-tRNAGln | - |
? | |
additional information | wild-type GlnRS catalyzes Glu-tRNAGln synthesis 1000000fold less efficiently than the cognate reaction | Escherichia coli | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
GlnRS | - |
Escherichia coli |
glutaminyltRNA synthetase | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at room temperature | Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00041 | - |
L-glutamate | mutant C229R GlnRS, with tRNAGln | Escherichia coli | |
0.0025 | - |
L-glutamine | mutant C229R GlnRS, with tRNAGln | Escherichia coli | |
0.046 | - |
L-glutamate | pH 7.5, 22°C, wild-type enzyme | Escherichia coli | |
3.2 | - |
L-glutamine | pH 7.5, 22°C, wild-type enzyme | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Escherichia coli |