Information on EC 6.1.1.24 - glutamate-tRNAGln ligase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
6.1.1.24
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RECOMMENDED NAME
GeneOntology No.
glutamate-tRNAGln ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-glutamate + tRNAGlx = AMP + diphosphate + glutamyl-tRNAGlx
show the reaction diagram
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ATP + L-glutamate + tRNAGlx = AMP + diphosphate + L-glutamyl-tRNAGlx
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Aminoacylation
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
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glutamate and glutamine metabolism
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glutaminyl-tRNAgln biosynthesis via transamidation
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Metabolic 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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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GluRS1
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Manually annotated by BRENDA team
GluRS2
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Manually annotated by BRENDA team
mutant C-2A
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
Methanothermobacter thermautotrophicum
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Manually annotated by BRENDA team
strain BP-1
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Manually annotated by BRENDA team
; TM1875
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
ATP + Glu + tRNAGlU
AMP + diphosphate + L-glutamyl-tRNAGlU
show the reaction diagram
ATP + L-Glu + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
ATP + L-glutamate + tRNAAsp
AMP + diphosphate + L-glutamyl-tRNAAsp
show the reaction diagram
ATP + L-glutamate + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
ATP + L-glutamate + tRNAGln
AMP + diphosphate + glutamyl-tRNAGln
show the reaction diagram
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-
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + Glu-tRNAGlu
show the reaction diagram
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + L-glutamyl-tRNAGlu
show the reaction diagram
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + Glu-tRNAGlx
show the reaction diagram
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + glutamyl-tRNAGlx
show the reaction diagram
Methanothermobacter thermautotrophicum
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-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + L-Glu + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
Methanothermobacter thermautotrophicum
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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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?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + glutamyl-tRNAGln
show the reaction diagram
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?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + L-glutamyl-tRNAGlu
show the reaction diagram
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-
-
-
?
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + Glu-tRNAGlx
show the reaction diagram
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + glutamyl-tRNAGlx
show the reaction diagram
Methanothermobacter thermautotrophicum
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-
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?
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
GatDE
Methanothermobacter thermautotrophicum
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a heterodimeric amidotransferase. In the presence of GatDE at 1.8 mM, the Glu-tRNAGlu activity of ND-GluRS decreases almost in half
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.62 - 3.1
ATP
5.8 - 6.2
L-Glu
240
L-glutamate
mutant C229R GlnRS, with tRNAGln
0.21
L-glutamine
mutant C229R GlnRS, with tRNAGln
0.000038 - 0.0076
tRNAGln
0.00079 - 0.0014
tRNAGlu
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1
ATP
0.09
L-Glu
0.00041
L-glutamate
Escherichia coli
P00962
mutant C229R GlnRS, with tRNAGln
0.0025
L-glutamine
Escherichia coli
P00962
mutant C229R GlnRS, with tRNAGln
0.036 - 0.1
tRNAGln
0.05 - 0.1
tRNAGlu
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.16
ATP
Escherichia coli
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
4
0.0155
L-Glu
Escherichia coli
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
202
5.2
tRNAGln
Escherichia coli
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recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
2875
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
GatDE
Methanothermobacter thermautotrophicum
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apparent KI of GatDE for ND-GluRS in the Glu-tRNAGlu reaction is 31 nM
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2 - 7.5
Methanothermobacter thermautotrophicum
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
assay at room temperature
37
Methanothermobacter thermautotrophicum
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assay at
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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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full-length ND-GluRS encompassing residues 1-552, including four cysteines and 18 methionine residues, hanging-drop method by mixing 0.001 ml of reservoir buffer containing 50 mM sodium cacodylate, pH 7.0, 50 mM calcium chloride and 8% PEG 6000, with 0.001 ml of protein solution at 20 mg/ml, 20C, 5 days, X-ray diffraction structure determination and analysis, single-wavelength anomalous dispersion method, using sulfur anomalous dispersion, modelling
Methanothermobacter thermautotrophicum
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GluRS in complex with glutamate, hanging drop vapor diffusion at 20C, 0.003 ml protein solution containing 3 mg/ml protein in 20 mM HEPES, pH 7.9, 20 mM NaCl, 10 mM DTT, 0.25 mM zinc acetate and 0.25 mM MgCl2, is mixed with 0.003 ml reservoir solution containing 740 mM sodium citrate, 140 mM citric acid, pH 5.8, and 10 mM DTT, 1-2 weeks, cryoprotection using 25% v/v of a 50% w/v trehalose solution, X-ray diffraction structure determination and analysis at 2.45 A resolution
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the crystal structure of the Thermotoga maritima ND-GluRS, TM1875, is determined in complex with a Glu-AMP analogue at 2.0 A resolution. ND-GluRS contains a characteristic structure in the connective-peptide domain, which is inserted into the catalytic Rossmann-fold domain; TM1875 in complex with a Glu-AMP analogue, mixing of 0.0018 ml of 15 mg/ml protein in 20 mM Tris-HCl, pH 7.0, containing 5 mM MgCl2, 10 mM 2-mercaptoethanol, 50 mM NaCl, and 1 mM L-glutamylsulfamoyl adenosine, with 0.0018 ml reservoir solution containing 100 mM HEPES-NaOH, pH 7.5, 8% ethylene glycol and 15% PEG 8000, and 0.0004 ml of 30% D-sorbitol, equilibration against 0.5 ml reservoir solution, 20C, 1 week, X-ray diffraction structure determination and analysis at 2.0 A resolution
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
native ND-GluRS by gel filtration
Methanothermobacter thermautotrophicum
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recombinant C-terminally and N-terminally His6-tagged ND-GluRS from Escherichia coli strain BL21 by nickel affinity and anion exchange chromatography, and gel filtration to homogeneity
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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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recombinant GluRSND from Escherichia coli pLysS Rosetta cells
Methanothermobacter thermautotrophicum
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recombinant TM1875 from Escherichia coli strain Rosetta2 (DE3) by anion exchange chromatography and gel filtration; using ion-exchange column chromatography and gel-filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Bacillus subtilis GluRS-dependent Glu-tRNAGln formation may cause growth inhibition in the transformed Escherichia coli strain, possibly due to abnormal protein synthesis
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expressed in Escherichia coli; TM1875, sequence comparisons, expression in Escherichia coli strain Rosetta2(DE3)
expression in Escherichia coli
expression of C-terminally and N-terminally His6-tagged ND-GluRS in Escherichia coli strain BL21
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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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expression of GluRSND using a pCYB1 vector in Escherichia coli pLysS Rosetta cells
Methanothermobacter thermautotrophicum
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ND-GluRS, phylogenetic analysis
Methanothermobacter thermautotrophicum
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the gene is cloned with its sigmaA promoter and a downstream region including a rho-independent terminator in the shuttle vector pRB394 for Escherichia coli and bacillus subtilis. Transformation of Bacillus subtilis with this recombinant plasmidleads to a 30fold increase of glutamyl-tRNA synthetase specific activity in crude extracts. Transformation of Escherichia coli with this plasmid gives no recombinants. The presence of Bacillus subtilis glutamyl-tRNA synthetase is lethal for Escherichia coli, probably because this enzyme glutamylates tRNA1Gln in vivo as it does in vitro
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ENGINEERING
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
E334R/G417T/
mutant of GluRS2 specifically and more robustly aminoacylates tRNAGlu1 instead of tRNAGln
G417T
mutant GluRS2 shows weak activity towards tRNAGlu1
E334R/G417T/
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mutant of GluRS2 specifically and more robustly aminoacylates tRNAGlu1 instead of tRNAGln
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G417T
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mutant GluRS2 shows weak activity towards tRNAGlu1
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additional information