Cloned (Comment) | Organism |
---|---|
recombinant expression of enzyme mutant K236E/E328A as N-terminally His6-SUMO2-Gly-tagged enzyme in Escherichia coli strain Rosetta2 (DE3) from plasmid DNA PLQ7619 | Escherichia coli |
Crystallization (Comment) | Organism |
---|---|
purified Gly-GluRS K236E/E328A, hanging drop vapour diffusion method, mixing of 0.001 ml of 20 mg/ml protein in 20 mM HEPES, pH 7.5, 50 mM NaCl, 10 mM 2-mercaptoethanol, and 50 mM ZnCl2, with 0.0012 ml of crystallization solution containing 0.1 M MOPS/HEPES-Na, pH 7.7, 0.02 M each of L-Glu.Na, DL-Ala, DL-Lys-HCl, Gly and DL-Ser, 14% w/v PEG 8000, 22% v/v ethylene glycol, 2 weeks, method optimization, X-ray diffraction structure determination and analysis at 3.5 A resolution, molecular replacement | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
K236E/E328A | by mapping crystal contacts of the homologous GluRS from Bacillus thailandensis, PDB ID 4g6z, onto the Escherichia coli GluRS sequence, two surface residues are identified that might be hindering crystallization attempts. Accordingly, these two residues are mutated and crystallization of the double mutant is attempted | Escherichia coli |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Escherichia coli | |
Zn2+ | C-x-C-xn-C-x-H zinc-binding motif | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-glutamate + tRNAGlu | Escherichia coli | - |
AMP + diphosphate + L-glutamyl-tRNAGlu | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His6-SUMO2-Gly-tagged K236E/E328A mutant enzyme from Escherichia coli strain Rosetta2 (DE3) by nickel affinity chromatography and dialysis, tag cleavage by SENP2 protease and another step of nickel affinity chromatography to remove the tag, to over 95% purity, followed by ultrafiltration | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-glutamate + tRNAGlu | - |
Escherichia coli | AMP + diphosphate + L-glutamyl-tRNAGlu | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 53600, about, sequence calculation | Escherichia coli |
More | enzyme secondary-structure analysis | Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
GluRS | - |
Escherichia coli |
Glutamyl-tRNA synthetase | - |
Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Escherichia coli |
General Information | Comment | Organism |
---|---|---|
evolution | many bacterial GluRS are capable of recognizing two tRNA substrates: tRNAGlu and tRNAGln, e.g. GluRS from such as Bacillus subtilis, Thermosynechococcus elongatus, and Mycobacterium tuberculosis. In bacteria such as Escherichia coli and Thermus thermophilus that possess glutaminyl-tRNA synthetase (GlnRS), the cognate aminoacylating enzyme for tRNAGln, GluRS exclusively glutamylates tRNAGlu. tRNA-GluRS interaction in bacteria is also associated with phylum-specific idiosyncrasies, structure-function analysis, overview | Escherichia coli |