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Information on EC 6.1.1.24 - glutamate-tRNAGln ligase and Organism(s) Escherichia coli and UniProt Accession P00962

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IUBMB Comments
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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Escherichia coli
UNIPROT: P00962
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Word Map
The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
glu-q-rs, nd-glurs, non-discriminating glutamyl-trna synthetase, glxrs, nondiscriminating glurs, tm1875, non-discriminating glurs, nondiscriminating glutamyl-trna synthetase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Glu-Q-RS
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GluRS
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glutamyl-queuosine tRNAAsp synthetase
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nondiscriminating glutamyl-tRNA synthetase
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Aminoacylation
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PATHWAY SOURCE
PATHWAYS
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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.
CAS REGISTRY NUMBER
COMMENTARY hide
9068-76-2
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + L-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
ATP + L-glutamine + tRNAGln
AMP + diphosphate + glutaminyl-tRNAGln
show the reaction diagram
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-
-
?
ATP + L-Glu + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
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synthesis of Glu-tRNAGln by engineered, not natural GlnRS, overview
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?
ATP + L-glutamate + tRNAAsp
AMP + diphosphate + L-glutamyl-tRNAAsp
show the reaction diagram
additional information
?
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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
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?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + L-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
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-
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?
ATP + L-glutamate + tRNAAsp
AMP + diphosphate + L-glutamyl-tRNAAsp
show the reaction diagram
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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
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
240
L-glutamate
mutant C229R GlnRS, with tRNAGln
0.21
L-glutamine
mutant C229R GlnRS, with tRNAGln
0.62
ATP
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
5.8
L-Glu
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
0.0076
tRNAGln
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00041
L-glutamate
mutant C229R GlnRS, with tRNAGln
0.0025
L-glutamine
mutant C229R GlnRS, with tRNAGln
0.1
ATP
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
0.09
L-Glu
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
0.04
tRNAGln
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.16
ATP
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
0.0155
L-Glu
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
5.2
tRNAGln
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
assay at room temperature
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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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
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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three-dimensional structure of Glu-QRS complexed to Glu, the enzyme lacks the anticodon-binding domain
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
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L1L2
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site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants
T231L
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site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants
V218S
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site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants
W256Y
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site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants
Y240D/D241F
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site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
presence of 10% glycerol during the purification prevents the precipitation of Glu-Q-RS
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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
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expression of Glu-Q-RS in Escherichia coli strain BL21(DE3)
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
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
J. Mol. Biol.
381
1224-1237
2008
Escherichia coli
Manually annotated by BRENDA team
Bullock, T.L.; Rodriguez-Hernandez, A.; Corigliano, E.M.; Perona, J.J.
A rationally engineered misacylating aminoacyl-tRNA synthetase
Proc. Natl. Acad. Sci. USA
105
7428-7433
2008
Escherichia coli (P00962)
Manually annotated by BRENDA team
Rodriguez-Hernandez, A.; Bhaskaran, H.; Hadd, A.; Perona, J.J.
Synthesis of Glu-tRNAGln by engineered and natural aminoacyl-tRNA synthetases
Biochemistry
49
6727-6736
2010
Escherichia coli, Methanothermobacter thermautotrophicus
Manually annotated by BRENDA team