Information on EC 6.1.1.17 - glutamate-tRNA ligase

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

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
Aminoacyl-tRNA biosynthesis
-
-
Biosynthesis of secondary metabolites
-
-
heme metabolism
-
-
Metabolic pathways
-
-
Porphyrin and chlorophyll metabolism
-
-
tetrapyrrole biosynthesis I (from glutamate)
-
-
tRNA charging
-
-
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
yellow pigment mutant C-2A'
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
Mo7 biological clone
UniProt
Manually annotated by BRENDA team
strain 168 Trp-
-
-
Manually annotated by BRENDA team
strain 168T
-
-
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
strain 168T
-
-
Manually annotated by BRENDA team
strain 168T
-
-
Manually annotated by BRENDA team
strain 26695, isozyme 2
-
-
Manually annotated by BRENDA team
GluRS1
-
-
Manually annotated by BRENDA team
-
-
-
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
Methanothermobacter thermautotrophicum
-
-
-
Manually annotated by BRENDA team
-
-
-
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
-
-
-
Manually annotated by BRENDA team
strain RS453
-
-
Manually annotated by BRENDA team
several strains and cultivars, overview, gene gltX
-
-
Manually annotated by BRENDA team
-
-
-
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
metabolism
-
the sensitivity to oxidation of GluRS1 might provide a means to regulate tetrapyrrole and protein biosynthesis in response to extreme changes in both the redox and heme status of the cell via a single enzyme. The glutamate moiety of Glu-tRNAGlu is transformed to glutamate semialdehyde by the glutamyl-tRNA reductase and is subsequently transformed to 4-aminolevulinic acid, the universal precursor of tetrapyrroles, by the glutamate semialdehyde amidotransferase
physiological function
additional information
-
targets for oxidation-based inhibition are cysteines from a SWIM zinc-binding motif located in the tRNA acceptor helix-binding domain. Oxidation of the metal-binding site cysteine of GluRS1 significantly impaired catalysis. Also, binding of ATP or tRNA protects the distant cysteines of the SWIM motif
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
Methanothermobacter thermautotrophicum
-
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
Methanothermobacter thermautotrophicum
-
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
Methanothermobacter thermautotrophicum
-
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
Methanothermobacter thermautotrophicum
-
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
Zn2+
-
bound at the active site, Zn2+ is essential for the proper binding of glutamate to GluRS. GluRS1 contains one Zn2+ ion per enzyme molecule, determination of the Zn2+ content of GluRS1 by mass spectrometry, overview. A relatively canonical SWIM motif, C-X-C-X24-C-X-E, forms a Zn2+-binding site
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-(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
LiCl
-
100 mM, 22% inhibition of ATP-diphosphate exchange, 500 mM, 73% inhibition
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-N
-
residues 1-122 of Arc1p, recombinantly expressed, the GluRS-NArc1p-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 - 2.7
L-glutamate
0.00042
tRNA1Glu
-
-
0.00046
tRNA2Glu
-
-
0.00024
tRNA3Glu
-
-
-
0.00015
tRNAAsp
-
-
0.0000767 - 0.0017
tRNAGln
0.00133
tRNAGln(CUG)
Methanothermobacter thermautotrophicum
-
wild type enzyme, pH and temperature not specified in the publication
0.00539
tRNAGln(UUG)
Methanothermobacter thermautotrophicum
-
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
Escherichia coli
-
-
0.3 - 3.4
Glu-tRNA
0.8
GlutRNA
Escherichia coli
-
-
1.39 - 2.15
L-glutamate
0.42 - 2.11
tRNAGln
0.041
tRNAGln(CUG)
Methanothermobacter thermautotrophicum
-
wild type enzyme, pH and temperature not specified in the publication
0.006
tRNAGln(UUG)
Methanothermobacter thermautotrophicum
-
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
Plasmodium falciparum
-
in 100 mM HEPES-KOH, pH 7.2, 30 mM KOH, 12 mM MgCl2, 2 mM dithiothreitol, at 37C
4
990
L-glutamate
Plasmodium falciparum
-
in 100 mM HEPES-KOH, pH 7.2, 30 mM KOH, 12 mM MgCl2, 2 mM dithiothreitol, at 37C
41
560 - 23170
tRNAGln
310
tRNAGln(CUG)
Methanothermobacter thermautotrophicum
-
wild type enzyme, pH and temperature not specified in the publication
136829
1
tRNAGln(UUG)
Methanothermobacter thermautotrophicum
-
wild type enzyme, pH and temperature not specified in the publication
136830
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
0.0007
5'-O-[N-(Lglutamyl)sulfamoyl]adenosine
-
in 100 mM HEPES-KOH, pH 7.2, 30 mM KOH, 12 mM MgCl2, 2 mM dithiothreitol, at 37C
-
1.85
Chloramphenicol
-
pH 7.0, temperature not specified in the publication
0.027 - 0.101
diphosphate
0.0000028
Glu-AMS
-
pH 7.2, 37C, versus L-glutamate
2.9
glutaminyl-beta-ketophosphonate-adenosine
-
pH 7.2, 37C, versus L-glutamate
0.0012 - 0.0039
glutamol-AMP
0.003
glutamyl adenylate
-
37C
0.63
glutamyl cytidylate
-
37C
16.7
glutamyl dihydrocytidylate
-
37C
0.06
glutamyl N6-benzoyladenylate
-
37C
2.75
glutamyl uridylate
-
37C
0.018
glutamyl-beta-ketophosphonate-adenosine
-
pH 7.2, 37C, versus L-glutamate
additional information
additional information
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
7.5
-
assay at
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
-
35C: about 70% of maximal activity, 55C: about 60% of maximal activity
50 - 80
-
about 25% of maximal activity at 50C and 80C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
2 enzyme forms: GluRSP and GluRSE
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Borrelia burgdorferi (strain ATCC 35210 / B31 / CIP 102532 / DSM 4680)
Burkholderia thailandensis (strain E264 / ATCC 700388 / DSM 13276 / CIP 106301)
Methanothermobacter thermautotrophicus (strain ATCC 29096 / DSM 1053 / JCM 10044 / NBRC 100330 / Delta H)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Thermosynechococcus elongatus (strain BP-1)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
58000
-
electrophoresis in polyacrylamide gels of various concentrations
60000
-
HPLC gel filtration
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
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 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 20C, 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
-
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 4C, 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
-
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 20C, 3 days or more, X-ray diffraction structure determination at 1.8 A resolution, molecular replacement, and analysis
-
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
-
stable up to, without addition of substrate
50
-
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 1C 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
-20C or -70C, 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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-20C, 10 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 20 mM 2-mercaptoethanol, 0.1 mM PMSF, 50% glycerol, stable for several months
-
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
-
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 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 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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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning and overexpression in Escherichia coli, coexpression of recombinant tRNA mutant variants
DNA and amino acid sequence determination and analysis, full length gene, single gene, expression in Escherichia coli BL21 as S-tagged enzyme
expressed in Escherichia coli BL21(DE3) cells
Methanothermobacter thermautotrophicum
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expressed in Escherichia coli KRX cells
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expression in Escherichia coli
expression in Escherichia coli; gene gltX1 and gltX2, expression in Escherichia coli temperature-sensitive mutant strain
-
expression of C-terminally His-tagged, full-length EPRS
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expression of His-tagged ERS in strain BL21(DE3)
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expression of His6-tagged GluRS-N, comprising residues 1-197, 17-207 and 1-207, in Escherichia coli strain BL21(DE3)
-
expression of the His-tagged chimeric mutant enzyme and of the catalytic domain of GluRS in Escherichia coli strain BL21(DE3) or the temperature sensitive strain JP1449(DE3)
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expression of wild-type and mutant His-tagged GluRSs in Escherichia coli strain BL21(DE3)
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gene gltX is encoded in an operon comprising the genes gltX, cysE, and cysS, after transcription the polycistronic mRNA is cleaved into the gltX transcript and the cysE-cysS transcript due to an internal signal structure and probably by self-cleavage, analysis, the gltX mRNA may be stabilized by secondary structures at the 3'-end and the 5'-end, expression in Escherichia coli and in vitro transcription
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gene gltX mutant, mutant enzyme expression in Escherichia coli temperature-sensitive mutant strain
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gene gltX, DNA and amino acid sequence determination and analysis, expression in Escherichia coli
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gene gltX, expression in Escherichia coli is lethal, but a modulated enzyme, lacking a part of the tRNA acceptor stem binding domain, can be recombinantly expressed in Escherichia coli DH5alpha, overexpression in Bacillus subtilis
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gene gltX2, expression in Escherichia coli temperature-sensitive mutant strain
-
overexpression in Escherichia coli
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overexpression of GST-GluRS1
-
overexpression of GST-tagged isozyme GluRS1 in Escherichia coli strain Bl21(DE3), co-expression with Glu-tRNA reductase
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overexpression of the enzyme in Escherichia coli with or without N-terminal His-tag
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phylogenetic and expression analysis, the tnpA gene of the IS element and gene gltX are co-transcribed and their expression is transiently upregulated upon retrieval of the ammonium source irrespective of whether nitrate or no nitrogen source are available, structural elements of the promoter directing the expression of the tnpA-gltX operon are localized within the IS
Tolypothrix sp.
-
TM1351, sequence comparisons, expression in Escherichia coli strain Rosetta2(DE3)
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
construction of an anticodon-binding domain truncated GluRS catalytic domain and a chimeric protein, constructed from the catalytic domain of Escherichia coli GluRS and the anticodon-binding domain of glutaminyl-tRNA synthetase GlnRS. Both proteins discriminate against tRNAGln. In addition to the anticodon-binding domain, tRNAGln discriminatory elements may be present in the catalytic domain in Escherichia coli GluRS as well
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Q373R
-
substrate specificity restricted to tRNAGlu compared to the wild-type which also accepts tRNAGln
H129Q
-
mutants encoding GluRS variants altered in the 98C-138C segment. Thermosensitive mutants H129Q, H131Q, H132Q and C138S. Mutants without glutamyl-tRNA synthetase activity: C100S, C125S. In the mutants C98S and H127Q the activity is 10fold lower than in cells overproducing the wild-type enzyme or the variants H129Q, H131Q, H132Q, and C138S
R358Q
site-directed mutagenesis, exchange of the Arg residue results in a mutant that no longer discriminates between tRNAGlu and tRNAGln anticodons YUC and YUG, respectively
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
-
design of better inhibitors specific for bacterial GluRSs, which are promising targets for antimicrobial therapy
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