Information on EC 6.1.1.17 - glutamate-tRNA ligase

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

EC NUMBER
COMMENTARY hide
6.1.1.17
-
RECOMMENDED NAME
GeneOntology No.
glutamate-tRNA ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-glutamate + tRNAGlu = AMP + diphosphate + L-glutamyl-tRNAGlu
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Aminoacylation
esterification
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
tetrapyrrole biosynthesis I (from glutamate)
-
-
tRNA charging
-
-
heme metabolism
-
-
Porphyrin and chlorophyll metabolism
-
-
Aminoacyl-tRNA biosynthesis
-
-
Metabolic pathways
-
-
Biosynthesis of secondary metabolites
-
-
Microbial metabolism in diverse environments
-
-
SYSTEMATIC NAME
IUBMB Comments
L-glutamate:tRNAGlu ligase (AMP-forming)
-
CAS REGISTRY NUMBER
COMMENTARY hide
9068-76-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
Mo7 biological clone
UniProt
Manually annotated by BRENDA team
Trp-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Caesalpinia bondue
-
-
-
Manually annotated by BRENDA team
non-discriminating enzyme form
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Helicobacter pylori ATCC 700392 / 26695
isozyme 2
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Methanobacterium thermoautotrophicus
-
UniProt
Manually annotated by BRENDA team
Methanococcus thermoautotrophicum
-
-
-
Manually annotated by BRENDA team
enzyme has dual substrate specificity for L-glutamate and L-proline, enzyme is part of a large aminoacyl-tRNA synthetases complex
-
-
Manually annotated by BRENDA team
Pseudomonas aeruginosa ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
-
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Saccharomyces cerevisiae ATCC 204508 / S288c
-
SwissProt
Manually annotated by BRENDA team
several strains and cultivars, overview, gene gltX
-
-
Manually annotated by BRENDA team
yellow pigment mutant C-2A'
-
-
Manually annotated by BRENDA team
Thermus thermophilus HB8 / ATCC 27634 / DSM 579
-
-
-
Manually annotated by BRENDA team
Tolypothrix sp.
strain PCC 7601, gene gltX
-
-
Manually annotated by BRENDA team
enzyme forms: GuRSP and GluRSE
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
EPRS-haploid (Eprs+/-) mice show enhanced viremia and inflammation and delayed viral clearance
metabolism
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 + tRNAAsp
AMP + diphosphate + L-glutamyl-tRNAAsp
show the reaction diagram
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 + glutamate + chloroplastic tRNAGln
AMP + diphosphate + gluamyl-tRNAGln
show the reaction diagram
ATP + L-glutamate + tRNAAsp
AMP + diphosphate + L-glutamyl-tRNAAsp
show the reaction diagram
-
adB gene encodes a truncated GluRS that lacks the C-terminal third of the protein and, consequently the anticodon binding domain. The YadB protein transfers Glu onto tRNAAsp. Neither tRNAGlu nor tRNAGln are substrates
-
-
?
ATP + L-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
ATP + L-glutamate + tRNAGln(CUG)
AMP + diphosphate + L-glutamyl-tRNAGln(CUG)
show the reaction diagram
-
the enzyme shows a significant catalytic preference for tRNAGln(CUG) compared to the less active tRNAGln(UUG)
-
-
?
ATP + L-glutamate + tRNAGln(UUG)
AMP + diphosphate + L-glutamyl-tRNAGln(UUG)
show the reaction diagram
-
the enzyme shows a significant catalytic preference for tRNAGln(CUG) compared to the less active tRNAGln(UUG)
-
-
?
ATP + L-glutamate + tRNAGlu
?
show the reaction diagram
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + L-glutamyl-tRNAGlu
show the reaction diagram
ATP + L-glutamate + tRNAGlu mutant C36G
AMP + diphosphate + L-glutamyl-tRNAGlu mutant C36G
show the reaction diagram
mutant R358Q, low activity with the wild-type enzyme
-
?
ATP + L-glutamate + tRNAGlu wild-type
AMP + diphosphate + L-glutamyl-tRNAGlu wild-type
show the reaction diagram
enzyme is specific for tRNAGlu
-
?
ATP + L-proline + tRNAGlu
AMP + diphosphate + L-prolyl-tRNAGlu
show the reaction diagram
-
enzyme has dual substrate specificity for L-glutamate and L-proline
-
?
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-glutamate + tRNAGln
AMP + diphosphate + L-glutamyl-tRNAGln
show the reaction diagram
ATP + L-glutamate + tRNAGln(CUG)
AMP + diphosphate + L-glutamyl-tRNAGln(CUG)
show the reaction diagram
-
the enzyme shows a significant catalytic preference for tRNAGln(CUG) compared to the less active tRNAGln(UUG)
-
-
?
ATP + L-glutamate + tRNAGln(UUG)
AMP + diphosphate + L-glutamyl-tRNAGln(UUG)
show the reaction diagram
-
the enzyme shows a significant catalytic preference for tRNAGln(CUG) compared to the less active tRNAGln(UUG)
-
-
?
ATP + L-glutamate + tRNAGlu
?
show the reaction diagram
ATP + L-glutamate + tRNAGlu
AMP + diphosphate + L-glutamyl-tRNAGlu
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mn2+
-
can partially replace Mg2+ in activation, maximal efficiency at 5 mM is 71% of Mg2+-activation
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(1R,2R)-1-(4-methylsulfonylphenyl)-2-(L-methionylsulfone-amido)-1,3-propanediol
-
-
(1R,2R)-1-(4-nitrophenyl)-2-(L-ethionyl-sulfoneamido)-1,3-propanediol
-
competitive inhibition with respect to Asp-tRNAAsn
(1R,2R)-1-phenyl-2-(L-methionyl-sulfone-amido)-1,3-propanediol
-
-
(1R,2S)-1-(4-nitrophenyl)-2-(L-methionyl-sulfoneamido)-1,3-propanediol
-
-
(1S,2R)-1-(4-nitrophenyl)-2-(L-methionyl-sulfoneamido)-1,3-propanediol
-
-
(1S,2S)-1-(4-nitrophenyl)-2-(L-methionyl-sulfone-amido)-1,3-propanediol
-
-
1,10-phenanthroline
-
ATP protects the enzyme against zinc removal
5'-O-(N'-(L-pyroglutamyl)-sulfamoyl)adenosine
-
weak
5'-O-(N-(L-glutamyl)-sulfamoyl)adenosine
5'-O-[N-(L-glutamyl)sulfamoyl]adenosine
-
i.e. glutamyl-sulfamoyl-adenosine or Glu-AMS, tRNAGlu increases the affinity of glutamyl-tRNA synthetase for its inhibitor glutamyl-sulfamoyl-adenosine, an analogue of the aminoacylation reaction intermediate glutamyl-AMP, thermodynamics of the enzyme-inhibitor interactions, overview. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated. The large negative enthalpy is the dominant contribution to DELTAGb in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNAPhe, but the dissociation constant Kd is decreased 50fold in the presence of tRNAGlu. Presence of an H-bond between Glu-AMS and the 3'-OH oxygen of the 3'-terminal ribose of tRNAGlu in the Glu-AMS/GluRS/tRNAGlu complex, molecular dynamics study
-
5'-O-[N-(Lglutamyl)sulfamoyl]adenosine
-
competitive inhibitor with respect to L-glutamate
amino levulinic acid
-
indirect inhibition, growth of Acidithiobacillus ferrooxidans in aminolevulic acid inhibits the activity of GluRS1, the reduced activity of GluRS1 is the result of an interaction of the enzyme with heme or any other intermediate tetrrapyrrole, amino levulic acid added to the reaction mixture has no effect in the activity of GluRSs
Chloramphenicol
-
-
diphosphate
erythro-4-Hydroxy-DL-glutamic acid
-
glutamate transfer to tRNA
erythro-Methyl-L-glutamic acid
-
glutamate transfer to tRNA
-
gamma-Globulin
-
-
-
Glu-AMS
-
-
glutaminyl-beta-ketophosphonate-adenosine
-
i.e. Gln-KPA, competitive inhibition, non-cognate, binds at one site on the monomeric enzyme
glutamol-AMP
glutamyl adenylate
-
-
glutamyl cytidylate
-
weak inhibition
glutamyl dihydrocytidylate
-
very weak inhibition
glutamyl N6-benzoyladenylate
-
-
glutamyl uridylate
-
very weak inhibition
glutamyl-beta-ketophosphonate-adenosine
-
i.e. Glu-KPA, selective, competitive inhibition of GluRS, binds at one site on the monomeric enzyme
H2O2
-
GluRS1 activity is reversibly inactivated upon oxidation by hydrogen peroxide, the enzyme loses 90% activity after 10 min at 0.3 mM H2O2. tRNAGlu is able to protect GluRS1 against oxidative inactivation by hemin plus hydrogen peroxide. GluRS1 is the main enzyme responsible for supplying Glu-tRNAGlu for heme biosynthesis. Partial recovery of the enzymatic activity by treatment with DTT or 2-mercaptoethanol
Hemin
LiCl
-
100 mM, 22% inhibition of ATP-diphosphate exchange, 500 mM, 73% inhibition
N'-(3-chlorophenyl)-N-(5,5-dimethyl-3-[(E)-[(2E)-3-(5-nitrofuran-2-yl)prop-2-en-1-ylidene]amino]-2-sulfanylidene-1,3-thiazolidin-4-yl)-N-hydroxyurea
competitive with glutamic acid but noncompetitive with ATP
-
N-(2,4-difluorophenyl)-N'-[4-[1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]phenyl]urea
noncompetitive with both ATP and glutamic acid
-
N6-Benzoyl-L-glutamyl AMP
-
specific for glutamyl-tRNA synthetase, does not inhibit glutaminyl-tRNA synthetase
NaCl
-
100 mM, 40% inhibition of ATP-diphosphate exchange, 500 mM, 91% inhibition
NH4Cl
-
100 mM, 31% inhibition of ATP-diphosphate exchange, 500 mM, 82% inhibition of ATP-diphosphate exchange
p-hydroxymercuribenzoate
-
-
threo-4-Hydroxy-L-glutamic acid
threo-4-Methyl-D-glutamic acid
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Arc1p
-
Arc1p-N
-
residues 1-122 of Arc1p, recombinantly expressed, the GluRS-N–Arc1p-N complex represents an unusual mode of interaction
-
bovine serum albumin
-
optimal activity at 0.25 mg/ml
-
Cytoplasmic protein Arc1p
-
forms a complex with glutamyl-tRNA synthetase, facilitates the delivery of tRNA molecules to the Arc1p-associated aminoacyl-tRNA synthetase
-
Diphosphatase
-
the addition of diphosphatase dramatically stimulates the reaction rate
-
tRNA
-
GluRS is one of the aminoacyl-tRNA synthetases that require the cognate tRNA for specific amino acid recognition and activation, tRNA serves as the enzyme activator in the first step, and as the substrate in the second step of aminoacylation, overview, On the other hand, the main chain of the glutamate is immature glutamate-binding site in the absence of tRNA
additional information
-
under high heme requirement for respiration increased levels of GluRS occur
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00235 - 0.32
ATP
0.043 - 0.055
C36G mutant tRNAGlu
-
0.0023 - 3
Glu
0.07 - 0.12
L-Glu
0.001401 - 20
L-glutamate
0.00042
tRNA1Glu
-
-
0.00046
tRNA2Glu
-
-
0.00024
tRNA3Glu
-
-
-
0.00015
tRNAAsp
-
-
0.0000767 - 0.0017
tRNAGln
0.00133
tRNAGln(CUG)
-
wild type enzyme, pH and temperature not specified in the publication
0.00539
tRNAGln(UUG)
-
wild type enzyme, pH and temperature not specified in the publication
0.0000032 - 0.7
tRNAGlu
0.0047 - 0.085
wild-type tRNAGlu
-
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.272 - 2.2
ATP
0.18 - 1.4
C36G mutant tRNAGlu
-
2.2
Glu
-
-
0.3 - 3.4
Glu-tRNA
-
0.8
GlutRNA
-
-
1.39 - 2.15
L-glutamate
0.42 - 2.11
tRNAGln
0.041
tRNAGln(CUG)
-
wild type enzyme, pH and temperature not specified in the publication
0.006
tRNAGln(UUG)
-
wild type enzyme, pH and temperature not specified in the publication
0.0058 - 3.61
tRNAGlu
1.5 - 2.1
wild-type tRNAGlu
-
additional information
additional information
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
704
ATP
-
in 100 mM HEPES-KOH, pH 7.2, 30 mM KOH, 12 mM MgCl2, 2 mM dithiothreitol, at 37°C
0.824 - 990
L-glutamate
560 - 23170
tRNAGln
310
tRNAGln(CUG)
-
wild type enzyme, pH and temperature not specified in the publication
1
tRNAGln(UUG)
-
wild type enzyme, pH and temperature not specified in the publication
10 - 112700
tRNAGlu
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.12
(1R,2R)-1-(4-methylsulfonylphenyl)-2-(L-methionylsulfone-amido)-1,3-propanediol
-
pH 7.0, temperature not specified in the publication
0.027
(1R,2R)-1-(4-nitrophenyl)-2-(L-ethionyl-sulfoneamido)-1,3-propanediol
-
pH 7.0, temperature not specified in the publication
0.4
(1R,2R)-1-phenyl-2-(L-methionyl-sulfone-amido)-1,3-propanediol
-
pH 7.0, temperature not specified in the publication
0.37
(1R,2S)-1-(4-nitrophenyl)-2-(L-methionyl-sulfoneamido)-1,3-propanediol
-
pH 7.0, temperature not specified in the publication
2.8
(1S,2R)-1-(4-nitrophenyl)-2-(L-methionyl-sulfoneamido)-1,3-propanediol
-
pH 7.0, temperature not specified in the publication
0.16
(1S,2S)-1-(4-nitrophenyl)-2-(L-methionyl-sulfone-amido)-1,3-propanediol
-
pH 7.0, temperature not specified in the publication
0.015
5'-O-(N'-(L-pyroglutamyl)-sulfamoyl)adenosine
-
-
0.0000028 - 0.00007
5'-O-(N-(L-glutamyl)-sulfamoyl)adenosine
0.0000028
5'-O-[N-(L-glutamyl)sulfamoyl]adenosine
-
pH and temperature not specified in the publication
-
0.0007
5'-O-[N-(Lglutamyl)sulfamoyl]adenosine
-
in 100 mM HEPES-KOH, pH 7.2, 30 mM KOH, 12 mM MgCl2, 2 mM dithiothreitol, at 37°C
1.85
Chloramphenicol
-
pH 7.0, temperature not specified in the publication
0.027 - 0.101
diphosphate
0.0000028
Glu-AMS
-
pH 7.2, 37°C, versus L-glutamate
2.9
glutaminyl-beta-ketophosphonate-adenosine
-
pH 7.2, 37°C, versus L-glutamate
0.0012 - 0.0039
glutamol-AMP
0.003
glutamyl adenylate
-
37°C
0.63
glutamyl cytidylate
-
37°C
16.7
glutamyl dihydrocytidylate
-
37°C
0.06
glutamyl N6-benzoyladenylate
-
37°C
2.75
glutamyl uridylate
-
37°C
0.018
glutamyl-beta-ketophosphonate-adenosine
-
pH 7.2, 37°C, versus L-glutamate
additional information
additional information
-
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0249
N'-(3-chlorophenyl)-N-(5,5-dimethyl-3-[(E)-[(2E)-3-(5-nitrofuran-2-yl)prop-2-en-1-ylidene]amino]-2-sulfanylidene-1,3-thiazolidin-4-yl)-N-hydroxyurea
Pseudomonas aeruginosa;
Q9XCL6
pH 7.5, 37°C
-
0.0219
N-(2,4-difluorophenyl)-N'-[4-[1-phenyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]phenyl]urea
Pseudomonas aeruginosa;
Q9XCL6
pH 7.5, 37°C
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0148
Caesalpinia bondue
-
-
0.036
-
wheat germ enymes
0.0396
-
-
0.06
-
chloroplastic enzyme
0.16
-
purified recombinant enzyme expressed in Bacillus subtilis
0.79
-
strain HRE-600
1.071
-
-
1.31
-
overproducing strain HS7611
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2
-
tRNAGlu-dependent ATP-diphosphate exchange
7.8 - 8.2
-
ATP-diphosphate exchange
8 - 8.5
-
aminoacylation
8 - 9
-
aminoacylation, in presence of 5 mM Mg2+
8.2
-
glutamyl-tRNA formation
8.6
-
glutamyl-tRNA formation
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8.2
-
7: rapid drop of activity below, 7.8-8.2: maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
47
-
aminoacylation
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35 - 55
-
35°C: about 70% of maximal activity, 55°C: about 60% of maximal activity
50 - 80
-
about 25% of maximal activity at 50°C and 80°C
additional information
-
microcalorimetry analysis at 20°C, 30°C and 37°C, thermodynamics in presence and absence of tRNAGlu and inhibitor Glu-AMS and non-cognate aa-AMP
PDB
SCOP
CATH
UNIPROT
ORGANISM
O51345
Borrelia burgdorferi (strain ATCC 35210 / B31 / CIP 102532 / DSM 4680);
Q2SX36
Burkholderia thailandensis (strain ATCC 700388 / DSM 13276 / CIP 106301 / E264);
A0A077E909
Elizabethkingia anophelis NUHP1;
B5Z6J9
Helicobacter pylori (strain G27);
Homo sapiens;
O26157
Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H);
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv);
Q9XCL6
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1);
Saccharomyces cerevisiae (strain ATCC 204508 / S288c);
Q8DLI5
Thermosynechococcus elongatus (strain BP-1);
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099);
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579);
Q5H2R3
Xanthomonas oryzae pv. oryzae (strain KACC10331 / KXO85);
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32500
-
2 * 32500, SDS-PAGE
46000
-
1 * 56000 (catalytic subunit) + 1 * 46000 (no detectable enzymatic activity, protective function), SDS-PAGE
53901
-
x * 53901, calculation from nucleotide sequence
55671
-
x * 55671, calculation from nucleotide sequence
60000
-
HPLC gel filtration
63000
-
x * 63000, SDS-PAGE
65500
-
1 * 65500, enzyme copurifies with a 46000 MW polypeptide which increases the affinity for glutamate and ATP, and stabilizes it against heat inactivation, SDS-PAGE
68400
-
x * 68400, calculated from amino acid sequence
72000
x * 72000, calculated from amino acid sequence
79000
-
2 * 79000, SDS-PAGE, enzyme form GluRSP
81000
x * 81000, recombinant protein, SDS-PAGE
83000
-
2 * 83000, SDS-PAGE, enzyme form GluRSE
102000
-
calculation from amino acid composition
105000
-
gel filtration
110000 - 112000
-
non-denaturing PAGE, gel filtration, chloroplastic enzyme
111000
-
gel filtration
155000 - 160000
-
gel filtration, non-denaturing PAGE, enzyme form GluRSP
164000 - 165000
-
non-denaturing PAGE, gel filtration, enzyme form GluRSE
172000
-
-
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
lipoprotein
-
high molecular weight aminoacyl-tRNA synthetase complex contains lipid. Delipidation does not affect the size or activity of the complex, but a variety of functional and structural properties of individual synthetases in the complex are altered: sensitivity to salts plus detergents, temperature inactivation, hydrophobicity, sensitivity to protease digestion
phosphoprotein
additional information
-
GluRS is substrate of DsbA, a protein involved in the restoration of the reduced state of cysteines in proteins upon oxidation
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
-
purified recombinant GluRS-N-Arc1p-N complex, hanging drop vapour diffusion method, 0.002 ml protein solution containing 15 mg/ml protein in 20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, pH 7.2 with NaOH, is mixed with 0.002 ml reservoir solution containing 30-35% PEG 3350, 300-500 mM NaSCN, X-ray diffraction structure determination and analysis at 2.05 A resolution
-
purified recombinant truncated enzyme, 0.002 ml of 20 mg/ml protein in 20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, pH 7.2 with NaOH, mixed with 0.002 ml reservoir solution at 20°C, equilibration against 0.075 ml reservoir solution, containing 1.7-1.8 M (NH4)2SO4, 200 mM KSCN for selenomethionine-substituted crystals and 1.8-1.9 M (NH4)2SO4, 200 mM NaI for native crystals, dispersion with selenomethionine, X-ray diffraction structure determination and analysis at 2.5 A resolution, modeling
-
architectures of class-defining and specific domains
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crystallization of complexes: 1. GluRS and L-Glu, 2. GluRS, tRNAGlu, and L-Glu, 3. GluRS, tRNAGlu, ATP, and L-glutamol, 4. GluRS, tRNAGlu, and L-glutamyl-sulfamoyl adenosine, by hanging drop vapour diffusion method, 5.0 mg/ml enzyme in 10 mM MOPS-Na buffer, pH 6.5, MgCl2, 5 mM 2-mercaptoethanol, 1% PEG 6000, and 2 mM L-glutamate, equilibration against a 1 ml reservoir solution containing 10% PEG at 4°C, ERS/tRNA/Glu and ERS/tRNA/ESA crystals are prepared by diffusing 1 mM L-glutamate and 0.5 mM glutamyl-sulfamoyl adenosine, i.e. ESA, respectively, into the ERS/tRNA binary complex crystals, ERS/tRNA/ATP/Eol crystals are obtained by adding both 1 mM ATP and 1 mM L-glutamol, i.e. Eol, to drops containing the ERS/tRNA binary complex, X-ray diffraction structure determination and analysis at 1.98 A, 2.4 A, 2.2 A, and 2.69 A resolution, respectively
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crystallization of the enzyme in different complexes: 1. non-productively complexed with ATP and L-glutamate, 2. with ATP, 3. with tRNAGlu and ATP, 4. with tRNAGlu and the glutamyl-AMP analogue glutamol-AMP, hanging-drop method, 0.008 ml of 5.0 mg/ml protein in 10 mM Na-MOPS, pH 6.5, 5 mM MgCl2, 2.5 mM 2-mercaptoethanol, 1% PEG 6000, 1-2 mM ATP and/or 2 mM glutamate and/or 0.5 mM glutamol-AMP, plus 1 ml reservoir solution containing 10% PEG 6000 at 4 or 20°C, 3 days or more, X-ray diffraction structure determination at 1.8 A resolution, molecular replacement, and analysis
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molecular modeling, internal pKa calculations, and molecular dynamics simulations for consideration of distinct, mechanistically relevant post-transfer states with charged tRNA bound to glutamyl-tRNA synthetase. The transfer of amino acid to tRNA is accompanied by the protonation of AMP to H-AMP. Subsequent migration of proton to water reduces the stability of the complex and loosens the interface both in the presence and in the absence of AMP. The subsequent undocking of AMP or tRNA then proceeds along thermodynamically competitive pathways. Release of the tRNA acceptor stem is further accelerated by the deprotonation of the alpha-ammonium group on the charging amino acid. The proposed general base is Glu41
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purified recombinant enzyme in complex with tRNAGlu, hanging-drop vapour diffusion method, precipitant solution contains 37 mM Na-MOPS, pH 6.7, 22% PEG 1500, 37 mM ammonium sulfate, 1% 2-methyl-2,4-pentanediol, 10 mM MgCl2, 5 mM 2-mercaptoethanol, X-ray diffraction structure determination at 2.4 A resolution, and analysis
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42
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stable up to, without addition of substrate
50
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50% loss of activity, when temperature is increased gradually at the rate of 1 C per min, without addition of substrate
60
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complete loss of activity, when temperature is increased gradually at the rate of 1°C per min, without addition of substrate
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Mycobacterium tuberculosis GluRS is significantly more sensitive than the Escherichia coli form to tryptic and chymotryptic limited proteolysis. Chymotrypsin-sensitive sites are found in the predicted tRNA stem contact domain next to the ATP binding site. Enzyme is fully protected from proteolysis by ATP and glutamol-AMP
no stabilization by various sulfydryl reagents, glycerol and glutamic acid
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tRNAGlu in presence of Mg2+ protects against heat inactivation, Mg2+ alone is much less effective
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C or -70°C, 20 mM sodium Hepes, pH 7.2, 0.1 mM EDTA, 0.5 mM DTT, 65% glycerol, stable for at least 1 year
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-20°C, 10 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 20 mM 2-mercaptoethanol, 0.1 mM PMSF, 50% glycerol, stable for several months
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85% loss of activity after 24 h
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
50fold, recombinant enzyme from overexpression in Bacillus subtilis, to homogeneity
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Ni-NTA column chromatography and gel filtration
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partial, recombinant enzyme from Escherichia coli with or without N-terminal His-tag
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recombinant enzyme to over 98% homogeneity
recombinant GST-GluRS1 by glutathione affinity chromatography, followed by removal of GST
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recombinant His-tagged chimeric mutant enzyme or catalytic domain of GluRS from strain BL21(DE3) or the temperature sensitive strain JP1449(DE3) by nickel affinity chromatography
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recombinant His-tagged ERS from strain BL21(DE3) by nickel affinity chromatography
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recombinant His-tagged GluRS from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
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recombinant His6-tagged GluRS-N from Escherichia coli strain BL21(DE3) to homogeneity by nickel affinity chromatography and gel filtration
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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
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recombinant tagged wild-type and mutant enzymes from Escherichia coli strain Escherichia coli BL21-CodonPlus (DE3)-RIPL by affinity chromatography and gel filtration
recombinant TM1351 from Escherichia coli strain Rosetta2 (DE3) by anion exchange chromatography and gel filtration
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six-h procedure
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