Literature summary for 6.1.1.17 extracted from

Chongdar, N.; Dasgupta, S.; Datta, A.B.; Basu, G.
Preliminary X-ray crystallographic analysis of an engineered glutamyl-tRNA synthetase from Escherichia coli (2014), Acta Crystallogr. Sect. F, 70, 922-927 .

Cloned(Commentary)

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 (Commentary)

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

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

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

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Escherichia coli
Zn2+	C-x-C-xn-C-x-H zinc-binding motif	Escherichia coli

Natural Substrates/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Escherichia coli	-	-	-

Purification (Commentary)

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 and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + L-glutamate + tRNAGlu	-	Escherichia coli	AMP + diphosphate + L-glutamyl-tRNAGlu	-	?

Subunits

Subunits	Comment	Organism
?	x * 53600, about, sequence calculation	Escherichia coli
More	enzyme secondary-structure analysis	Escherichia coli

Synonyms

Synonyms	Comment	Organism
GluRS	-	Escherichia coli
Glutamyl-tRNA synthetase	-	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Escherichia coli

General Information

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