When this enzyme acts on tRNAGlu, it catalyses the same reaction as EC 6.1.1.17, glutamate---tRNA ligase. It has, however, diminished discrimination, so that it can also form glutamyl-tRNAGln. This relaxation of specificity has been found to result from the absence of a loop in the tRNA that specifically recognizes the third position of the anticodon . This accounts for the ability of this enzyme in, for example, Bacillus subtilis, to recognize both tRNA1Gln (UUG anticodon) and tRNAGlu (UUC anticodon) but not tRNA2Gln (CUG anticodon). The ability of this enzyme to recognize both tRNAGlu and one of the tRNAGln isoacceptors derives from their sharing a major identity element, a hypermodified derivative of U34 (5-methylaminomethyl-2-thiouridine). The glutamyl-tRNAGln is not used in protein synthesis until it is converted by EC 6.3.5.7, glutaminyl-tRNA synthase (glutamine-hydrolysing), into glutaminyl-tRNAGln.
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SYSTEMATIC NAME
IUBMB Comments
L-glutamate:tRNAGlx ligase (AMP-forming)
When this enzyme acts on tRNAGlu, it catalyses the same reaction as EC 6.1.1.17, glutamate---tRNA ligase. It has, however, diminished discrimination, so that it can also form glutamyl-tRNAGln. This relaxation of specificity has been found to result from the absence of a loop in the tRNA that specifically recognizes the third position of the anticodon [1]. This accounts for the ability of this enzyme in, for example, Bacillus subtilis, to recognize both tRNA1Gln (UUG anticodon) and tRNAGlu (UUC anticodon) but not tRNA2Gln (CUG anticodon). The ability of this enzyme to recognize both tRNAGlu and one of the tRNAGln isoacceptors derives from their sharing a major identity element, a hypermodified derivative of U34 (5-methylaminomethyl-2-thiouridine). The glutamyl-tRNAGln is not used in protein synthesis until it is converted by EC 6.3.5.7, glutaminyl-tRNA synthase (glutamine-hydrolysing), into glutaminyl-tRNAGln.
Glu-Q-RS binds the noncognate amino acids L-Gln and D-Glu fourfold and sixfold, respectively, weaker than L-Glu. Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties, selection of the cognate amino acid and by the mechanism of its activation, overview. Structural basis of the reaction mechanism, overview
the L-glutamyl-queuosine tRNAAsp synthetase, Glu-Q-RS from Escherichia coli is a paralogue of the catalytic core of glutamyl-tRNA synthetase, GluRS, that catalyzes glutamylation of queuosine in the wobble position of tRNAAsp
the L-glutamyl-queuosine tRNAAsp synthetase, Glu-Q-RS from Escherichia coli is a paralogue of the catalytic core of glutamyl-tRNA synthetase, GluRS, that catalyzes glutamylation of queuosine in the wobble position of tRNAAsp. Activation of Glu to form Glu-AMP, the intermediate of tRNA aminoacylation, in the absence of tRNA. Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNAAsp. A C in position 38 is crucial for glutamylation of Q34
the L-glutamyl-queuosine tRNAAsp synthetase, Glu-Q-RS from Escherichia coli is a paralogue of the catalytic core of glutamyl-tRNA synthetase, GluRS, that catalyzes glutamylation of queuosine in the wobble position of tRNAAsp
detailed comparison of kinetic parameters between recombinant hybrid GlnRS S1/L1/L2 and other recombinant hybrid mutant enzymes, with the naturally occurring Methanothermobacter thermautotrophicus GluRSND, which is also capable of Glu-tRNAGln synthesis, overview. Both kcat and Km for glutamate are recapitulated in the engineered enzyme, but Km for tRNA is 200fold higher
a hybrid enzyme, in which 23 amino acids from the catalytic domain of Escherichia coli glutaminyl-tRNA synthetase, GlnRS, are replaced with the corresponding residues of human glutamyl-tRNA synthetase, GluRS, synthesizes Glu-tRNAGln over 104fold more efficiently than GlnRS. Identification of residues involved in improving complementarity for glutamate and in communicating between amino acid and tRNA binding sites, overview
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
Glu-QRS complexed to Glu, sitting drop vapour diffusion method, mixing of 0.002 ml of protein solution, containing 9.7 mg/ml protein in 20 mM Na-HEPES buffer, pH 7.2, and 50 mM NaCl, with 0.002 ml of reservoir solution containing 0.1 M Mg-acetate and Na-cacodylate buffer, pH 5.5, 0.2 M KCl, 10% polyethylene glycol 8000, and 2 mM L-Glu, a few days, X-ray diffraction structure determination and analysis at 1.5 A resolution
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
construction of a hybrid enzyme in which 23 amino acids from the catalytic domain of Escherichia coli glutaminyl-tRNA synthetase, GlnRS, are replaced with the corresponding residues of human glutamyl-tRNA synthetase, GluRS. Further introduction of two distal surface loops bridging core secondary structural elements of the Rossmann fold then produces a hybrid enzyme GlnRS S1/L1/L2. The engineered hybrid GlnRS S1/L1/L2 synthesizes Glu-tRNAGln over 104fold more efficiently than GlnRS, overview. The simultaneous optimization of paired amino acid and tRNA binding sites found in a naturally occurring enzyme is not recapitulated in a hybrid that is successfully engineered for amino acid complementarity. Design and characterization of four additional hybrids identify further residues involved in improving complementarity for glutamate and in communicating between amino acid and tRNA binding sites, complementarity for tRNA, mutant enzyme structure, overview. Relationship between tRNA and amino acid binding sites in the hybrid enzymes, overview
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant Glu-Q-RS from Escherichia coli strain BL21(DE3) by anion exchange and hydroxyapatite chromatography, followed by dialysis, presence of 10% glycerol during the purification prevents the precipitation of Glu-Q-RS
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of C-terminally His-tagged mutant enzymes containing an N-terminal signal sequence tag directing the protein to the periplasm, without effect on the steady-state kinetic parameters of GlnRS. The mutants are expressed as N-terminal fusions with the leader sequence of the bacterial fd gene III protein in Escherichia coli
Blaise, M.; Olieric, V.; Sauter, C.; Lorber, B.; Roy, B.; Karmakar, S.; Banerjee, R.; Becker, H.D.; Kern, D.
Crystal structure of glutamyl-queuosine tRNAAsp synthetase complexed with L-glutamate: structural elements mediating tRNA-independent activation of glutamate and glutamylation of tRNAAsp anticodon