Information on EC 6.1.1.24 - glutamate-tRNAGln ligase

Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Mark a special word or phrase in this record:
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)


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

EC NUMBER
COMMENTARY
6.1.1.24
-
RECOMMENDED NAME
GeneOntology No.
glutamate-tRNAGln ligase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ATP + L-glutamate + tRNAGlx = AMP + diphosphate + glutamyl-tRNAGlx
show the reaction diagram
-
-
-
-
ATP + L-glutamate + tRNAGlx = AMP + diphosphate + L-glutamyl-tRNAGlx
show the reaction diagram
-
-
-
-
ATP + L-glutamate + tRNAGlx = AMP + diphosphate + L-glutamyl-tRNAGlx
show the reaction diagram
active site structure and substrate recognition
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Aminoacylation
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
-
Metabolic pathways
-
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.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Glu-Q-RS
-
-
GluRS
-
-
-
-
GluRS1
Acidithiobacillus ferrooxidans GluRS1, Acidithiobacillus ferrooxidans GluRS2
-
-
-
GluRS2
Acidithiobacillus ferrooxidans GluRS1, Acidithiobacillus ferrooxidans GluRS2
-
-
-
GluRS2
Helicobacter pylori GluRS1
-
;
-
GluRS2
Helicobacter pylori GluRS2
-
-
-
glutamyl-queuosine tRNAAsp synthetase
-
-
Glutamyl-tRNA synthetase
Q9X2I8
-
ND-GluRS
Q9X2I8
-
non-discriminating glutamyl-tRNA synthetase
-
-
non-discriminating glutamyl-tRNA synthetase
-
-
nondiscriminating GluRS
-
-
nondiscriminating GluRS
Q9X2I8
-
nondiscriminating glutamyl-tRNA synthetase
-
-
-
-
nondiscriminating glutamyl-tRNA synthetase
Q9X2I8
-
TM1875
Q9X2I8
; only TM1875 functions as a nondiscriminating GluRS
CAS REGISTRY NUMBER
COMMENTARY
9068-76-2
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
Acidithiobacillus ferrooxidans GluRS1
GluRS1
-
-
Manually annotated by BRENDA team
Acidithiobacillus ferrooxidans GluRS2
GluRS2
-
-
Manually annotated by BRENDA team
Helicobacter pylori GluRS2
GluRS2
Uniprot
Manually annotated by BRENDA team
Helicobacter pylori GluRS2
GluRS2
-
-
Manually annotated by BRENDA team
mutant C-2A
-
-
Manually annotated by BRENDA team
; TM1875
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
evolution
-
ND-GluRS is a class I aminoacyl-tRNA synthetase
physiological function
Q9X2I8, -
ND-GluRS produces the intermediate Glu-tRNAGln, which is converted to Gln-tRNAGln by Glu-tRNAGln amidotransferase. GluRS avoids competition with Glu-tRNAGln amidotransferase GatCAB and glutamylates tRNAGln
physiological function
-
for synthesis of Gln-tRNAGln, a two-step process is required: the non-discriminating glutamyl-tRNA synthetase, ND-GluRS, forms Glu-tRNAGln, while the heterodimeric amidotransferase GatDE converts this mischarged tRNA to Gln-tRNAGln. A similar complex for Gln-tRNAGln formation in Methanothermobacter thermautotrophicus that allows the mischarged Glu-tRNAGln made by the tRNA synthetase to be channeled to the amidotransferase. The association of archaeal ND-GluRS with GatDE sequesters the tRNA synthetase for Gln-tRNAGln formation, with GatDE reducing the affinity of ND-GluRS for tRNAGlu by at least 13fold. The archaeal complex does not require tRNA for its formation, is not stable through product (Gln-tRNAGln) formation, and has no major effect on the kinetics of tRNAGln glutamylation nor transamidation
evolution
-
evolution in the GlxRS family, overview. Two pathways exist for the formation of Gln-tRNAGln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase, ND-GluRS, that can form both Glu-tRNAGlu and Glu-tRNAGln. The Glu-tRNAGln is then converted to Gln-tRNAGln by an amidotransferase. ND-GluRS is the evolutionary predecessor of both the glutaminyl-tRNA synthetase, GlnRS, and the eukaryotic discriminating GluRS
additional information
-
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
additional information
-
ND-GluRS:GatDE complex formation and structure, detailed overview
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
-
-
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
-
-
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
-
-
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
-
GluRS1 charges tRNAGln(CUG)
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
O25360
GluRS2
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
-
GluRS2 is specific solely for tRNAGln
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
-
GluRS2 preferetially charges tRNAGln(UUG)
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
Q9X2I8, -
the glutamylation ability of tRNAGln by ND-GluRS is measured in the presence of the bacterial Glu-tRNAGln amidotransferase GatCAB. Glutamylation efficiency is not affected even in the presence of excess GatCAB
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
Acidithiobacillus ferrooxidans GluRS1
-
GluRS1 charges tRNAGln(CUG), GluRS2 preferetially charges tRNAGln(UUG)
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
Helicobacter pylori GluRS1
-
GluRS2
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
Helicobacter pylori GluRS1, Helicobacter pylori GluRS2
-
GluRS2 is specific solely for tRNAGln
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
Acidithiobacillus ferrooxidans GluRS2
-
GluRS1 charges tRNAGln(CUG), GluRS2 preferetially charges tRNAGln(UUG)
-
-
?
ATP + Glu + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
-
-
-
-
?
ATP + Glu + tRNAGlU
AMP + diphosphate + L-glutamyl-tRNAGlU
show the reaction diagram
Acidithiobacillus ferrooxidans, Acidithiobacillus ferrooxidans GluRS1, Acidithiobacillus ferrooxidans GluRS2
-
GluRS1
-
-
?
ATP + L-Glu + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
-
-
-
-
?
ATP + L-Glu + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
-
synthesis of Glu-tRNAGln by engineered, not natural GlnRS, overview
-
-
?
ATP + L-glutamate + tRNAAsp
AMP + diphosphate + L-glutamyl-tRNAAsp
show the reaction diagram
-
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
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
-
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
-
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
-
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
-
-
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
-
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
-
the enzyme efficiently charges E. coli tRNAGlu and both tRNAGlu and tRNAGln from chloroplasts, no activity with the two E. coli tRNAGln species
-
-
-
ATP + L-glutamate + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
-
the enzyme is capable of mischarging plastidal tRNAGln from barley with glutamate as well as regularly charges the plastidal tRNAGlu from Scenedesmus. The mischarged glutamyl-tRNAGln is subsequently amidated by glutamyl-tRNA amidotransferase to form the glutaminyl-tRNAGln required for plastidal protein biosynthesis
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + Glu-tRNAGln
show the reaction diagram
-
the enzyme aminoacylates tRNAGLU and tRNASGln in Bacillus subtilis and efficiently misacylates E. coli tRNA1Gln in vitro
-
-
-
ATP + L-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
P00962
-
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
-
tRNAGln is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
P00962
activity also with Gln-RS, EC 6.1.1.18, mutant C229R
-
-
?
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + L-glutamyl-tRNAGlu
show the reaction diagram
-
-, preferred tRNA substrate
-
-
?
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + Glu-tRNAGlu
show the reaction diagram
-
-
-
?
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + Glu-tRNAGlu
show the reaction diagram
-
-
-
?
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + Glu-tRNAGlu
show the reaction diagram
-
-
-
?
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + Glu-tRNAGlu
show the reaction diagram
-
the enzyme efficiently charges E. coli tRNAGlu and both tRNAGlu and tRNAGln from chloroplasts, no activity with the two E. coli tRNAGln species
-
?
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + Glu-tRNAGlu
show the reaction diagram
-
the enzyme aminoacylates tRNAGlu and tRNASGln in Bacillus subtilis and efficiently misacylates E. coli tRNA1Gln in vitro, not tRNA2Gln from E. coli or tRNAGlu from E. coli
-
?
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + Glu-tRNAGlu
show the reaction diagram
-
the enzyme is capable of mischarging plastidal tRNAGln from barley with glutamate as well as regularly charges the plastidal tRNAGlu from Scenedesmus. The mischarged glutamyl-tRNAGln is subsequently amidated by glutamyl-tRNA amidotransferase to form the glutaminyl-tRNAGln required for plastidal protein biosynthesis
-
?
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + Glu-tRNAGlu
show the reaction diagram
-
the enzyme interacts with the G64-C50 or G64-U50 in the Tpsi stem of its tRNA substrate
-
?
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + Glu-tRNAGlu
show the reaction diagram
-
major recognition element for the enzyme is U at the 34th position of both tRNA1Gln from Bacillus subtilis and tRNA1Gln from E. coli as a modified form
-
?
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + Glu-tRNAGlx
show the reaction diagram
-
-
-
?
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + Glu-tRNAGlx
show the reaction diagram
-
the enzyme is capable of mischarging plastidal tRNAGln from barley with glutamate as well as regularly charges the plastidal tRNAGlu from Scenedesmus. The mischarged glutamyl-tRNAGln is subsequently amidated by glutamyl-tRNA amidotransferase to form the glutaminyl-tRNAGln required for plastidal protein biosynthesis
-
?
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + Glu-tRNAGlx
show the reaction diagram
-
the enzyme aminoacylates both tRNAGlu and tRNAGln because Rhizobium meliloti contains no glutaminyl-tRNAGln ligase
-
?
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + Glu-tRNAGlx
show the reaction diagram
-
the enzyme is responsible for the in vivo aminoacylation of both tRNAGlu and tRNAGln in Bacillus subtilis
-
?
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + glutamyl-tRNAGlx
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
the non-discriminating enzyme charges both tRNAGlu and tRNAGln with glutamate, anticodons of tRNAGlu, 34C/UUC36, and tRNAGln, 34C/UUG36, differ only in base 36, residue Gly366 is responsible for allowing both cytosine and the bulkier purine base G36 of tRNAGln to be tolerated, glutamate recognition structure, overview
-
-
-
additional information
?
-
-
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
-
-
-
additional information
?
-
-
ND-GluRS recognizes both tRNAGlu and tRNAGln without significantly discriminating between them, tRNA discrimination module, overview. The first point of significant distinction between the GlnRS and ND-GluRS tRNA recognition involves residues contacting the G36 nucleobase. A second distiction involves a beta-hairpin module in the CP domain
-
-
-
additional information
?
-
Q9X2I8, -
ND-GluRS TM1875 glutamylates both tRNAGlu and tRNAGln
-
-
-
additional information
?
-
Q9X2I8, -
TM1875 glutamylates both the tRNAGlu and tRNAGln transcripts
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
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
-
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
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
P00962
-
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
-
tRNAGln is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase
-
-
?
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 + Glu-tRNAGlx
show the reaction diagram
-
the enzyme is capable of mischarging plastidal tRNAGln from barley with glutamate as well as regularly charges the plastidal tRNAGlu from Scenedesmus. The mischarged glutamyl-tRNAGln is subsequently amidated by glutamyl-tRNA amidotransferase to form the glutaminyl-tRNAGln required for plastidal protein biosynthesis
-
?
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + Glu-tRNAGlx
show the reaction diagram
-
the enzyme aminoacylates both tRNAGlu and tRNAGln because Rhizobium meliloti contains no glutaminyl-tRNAGln ligase
-
?
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + Glu-tRNAGlx
show the reaction diagram
-
the enzyme is responsible for the in vivo aminoacylation of both tRNAGlu and tRNAGln in Bacillus subtilis
-
?
ATP + L-glutamate + tRNAGlx
AMP + diphosphate + glutamyl-tRNAGlx
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
ND-GluRS recognizes both tRNAGlu and tRNAGln without significantly discriminating between them, tRNA discrimination module, overview. The first point of significant distinction between the GlnRS and ND-GluRS tRNA recognition involves residues contacting the G36 nucleobase. A second distiction involves a beta-hairpin module in the CP domain
-
-
-
additional information
?
-
Q9X2I8, -
ND-GluRS TM1875 glutamylates both tRNAGlu and tRNAGln
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
ATP
-
binding site pocket structure, overview
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Mg2+
Q9X2I8, -
; required
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
GatDE
-
a heterodimeric amidotransferase. In the presence of GatDE at 1.8 mM, the Glu-tRNAGlu activity of ND-GluRS decreases almost in half
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.62
-
ATP
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
3.1
-
ATP
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
5.8
-
L-Glu
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
6.2
-
L-Glu
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
240
-
L-glutamate
P00962
mutant C229R GlnRS, with tRNAGln
0.000038
-
tRNAGln
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
0.00045
-
tRNAGln
-
homologous tRNAGln, in absence of regulatory factor beta
0.00065
-
tRNAGln
-
homologous tRNAGln, in presence of regulatory factor beta
0.0015
-
tRNAGln
-
tRNAGln
0.0076
-
tRNAGln
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
0.00083
-
tRNAGlu
-
homologous tRNAGlu, in absence of regulatory factor beta
0.0014
-
tRNAGlu
-
homologous tRNAGlu, in presence of regulatory factor beta
0.21
-
L-glutamine
P00962
mutant C229R GlnRS, with tRNAGln
additional information
-
additional information
-
kinetics
-
additional information
-
additional information
-
kinetics and binding constants
-
additional information
-
additional information
P00962
kinetics of the mutant enzyme compared to wild-type GlnRS, EC 6.1.1.18, overview
-
additional information
-
additional information
-
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
-
additional information
-
additional information
-
detailed comparison of kinetic parameters between Methanothermobacter thermautotrophicus GluRSND and Escherichia coli recombinant hybrid GlnRS mutants, which are also capable of Glu-tRNAGln synthesis, overview
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.1
-
ATP
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
0.09
-
L-Glu
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
0.00041
-
L-glutamate
P00962
mutant C229R GlnRS, with tRNAGln
0.0025
-
L-glutamine
P00962
mutant C229R GlnRS, with tRNAGln
0.04
-
tRNAGln
-
homologous tRNAGln
0.04
-
tRNAGln
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
0.07
-
tRNAGln
-
tRNAGln from E. coli
0.1
-
tRNAGln
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
0.05
-
tRNAGlu
-
homologous tRNAGlu
kcat/KM VALUE [1/mMs-1]
kcat/KM VALUE [1/mMs-1] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.16
-
ATP
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
22040
0.0155
-
L-Glu
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
32532
5.2
-
tRNAGln
-
recombinant hybrid GlnRS S1/L1/L2, pH not specified in the publication, temperature not specified in the publication
17505
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
additional information
-
GatDE
-
apparent KI of GatDE for ND-GluRS in the Glu-tRNAGlu reaction is 31 nM
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.5
-
P00962
assay at
7.5
-
Q9X2I8, -
assay at
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
22
-
P00962
assay at room temperature
50
-
Q9X2I8, -
assay at
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 54166, calculation from nucleotide sequence
?
-
x * 63237, sequence calculation
additional information
-
structure comparison of non-discriminating GluRS versus the discriminating GluRS, EC 6.1.1.17, overview
additional information
-
three-dimensional structure of Glu-QRS complexed to Glu, the enzyme lacks the anticodon-binding domain
additional information
Q9X2I8, -
Thermotoga maritima ND-GluRS contains a characteristic structure in the connective-peptide domain, which is inserted into the catalytic Rossmann-fold domain, structure, overview
additional information
-
ND-GluRS:GatDE complex formation and structure, detailed overview
additional information
-
structure analysis, and comparison with the structure of the Escherichia coli GlnRS-tRNAGln complex and with GluRS structure, structural determinants responsible for specific tRNAGln recognition, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
P00962
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
-
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 50mM 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
-
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
-
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
Q9X2I8, -
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
presence of 10% glycerol during the purification prevents the precipitation of Glu-Q-RS
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
-
native ND-GluRS by gel filtration
-
recombinant GluRSND from Escherichia coli pLysS Rosetta cells
-
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
-
recombinant TM1875 from Escherichia coli strain Rosetta2 (DE3) by anion exchange chromatography and gel filtration; using ion-exchange column chromatography and gel-filtration
Q9X2I8, -
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expression in Escherichia coli
-
Bacillus subtilis GluRS-dependent Glu-tRNAGln formation may cause growth inhibition in the transformed Escherichia coli strain, possibly due to abnormal protein synthesis
-
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
-
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
-
expression of Glu-Q-RS in Escherichia coli strain BL21(DE3)
-
expression in Escherichia coli
-
expression of GluRSND using a pCYB1 vector in Escherichia coli pLysS Rosetta cells
-
ND-GluRS, phylogenetic analysis
-
expression of C-terminally and N-terminally His6-tagged ND-GluRS in Escherichia coli strain BL21
-
expressed in Escherichia coli; TM1875, sequence comparisons, expression in Escherichia coli strain Rosetta2(DE3)
Q9X2I8, -
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
T231L
-
site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants
V218S
-
site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants
W256Y
-
site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants
Y240D/D241F
-
site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants
E334R/G417T/
O25360
mutant of GluRS2 specifically and more robustly aminoacylates tRNAGlu1 instead of tRNAGln
G417T
O25360
mutant GluRS2 shows weak activity towards tRNAGlu1
E334R/G417T/
Helicobacter pylori GluRS1
-
mutant of GluRS2 specifically and more robustly aminoacylates tRNAGlu1 instead of tRNAGln
-
G417T
Helicobacter pylori GluRS1
-
mutant GluRS2 shows weak activity towards tRNAGlu1
-
L1L2
-
site-directed mutagenesis, construction of a hybrid enzyme, kinetic comparison to wild-type enzyme and other hybrid enzyme mutants
additional information
P00962
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
additional information
-
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