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Information on EC 6.1.1.2 - tryptophan-tRNA ligase and Organism(s) Geobacillus stearothermophilus and UniProt Accession P00953
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6.1.1.2 tryptophan-tRNA ligaseSpecify your search results
Word Map
- 6.1.1.2
- synthetases
- aminoacyl-trna
- aminoacylation
- pancreas
- beef
-
anticodon
- stearothermophilus
- tyrrs
- tyrosyl-trna
- indoleamine
-
angiostatic
-
kmsks
-
ido
- tryptamine
-
tryptophan-dependent
-
aarss
- ap4a
- glutaminyl-trna
- hisrs
-
anticodon-binding
- glnrs
- medicine
- synthesis
- biotechnology
The taxonomic range for the selected organisms is: Geobacillus stearothermophilus
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
tryptophanyl-trna synthetase, trprs, wars2, mini-trprs, t2-trprs, tryptophanyl trna synthetase, htrprs, trprs1, trprs ii, trp-rs, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
(Mt)TrpRS
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-
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hWRS
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-
-
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IFP53
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-
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Synthetase, tryptophanyl-transfer ribonucleate
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TrpRS
Tryptophan translase
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Tryptophan--tRNA ligase
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Tryptophanyl ribonucleic synthetase
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Tryptophanyl-transfer ribonucleate synthetase
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Tryptophanyl-transfer ribonucleic acid synthetase
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Tryptophanyl-transfer ribonucleic synthetase
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Tryptophanyl-transfer RNA synthetase
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Tryptophanyl-tRNA synthase
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Tryptophanyl-tRNA synthetase
additional information
-
the enzyme belongs to the class I aminoacyl-tRNA transferases
TrpRS
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-
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Tryptophanyl-tRNA synthetase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + L-tryptophan + tRNATrp = AMP + diphosphate + L-tryptophyl-tRNATrp
random, bi-bi kinetic scheme mechanism, KMSKS motif loop is involved in the catalytic mechanism
ATP + L-tryptophan + tRNATrp = AMP + diphosphate + L-tryptophyl-tRNATrp
a nested, nonlinear model for the sum of metal-free and metal-catalyzed activities and its use in determining metal-free enzyme activity jointly with transition-state metal binding affinity is described, by fitting observed values obtained from Mg2+-depleted assays with increasing EDTA concentrations at known Mg2+ concentrations. Trp activation by TrpRS falls asymptotically to a plateau value 5 orders of magnitude below that observed for the Mg2+-supplemented enzyme at EDTA concentrations that reduce the free metal concentration to below 1 pmolar. The fitted regression model parameters yield a relative rate acceleration of 93000 attributable to the catalytic effect of Mg2+ and an enhanced transition state binding of Mg2+. Factorial analysis indicates that 80% of the reduction in free energy of activation effected by TrpRS arises from protein-ligand interactions
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
esterification
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Aminoacylation
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SYSTEMATIC NAME
IUBMB Comments
L-tryptophan:tRNATrp ligase (AMP-forming)
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CAS REGISTRY NUMBER
COMMENTARY
9023-44-3
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophan-tRNATrp
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-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophanyl-tRNATrp
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-
-
-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophyl-tRNATrp
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-
-
-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophanyl-tRNATrp
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-
-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophanyl-tRNATrp
a two-step reaction of amino acid activation followed by aminoacylation, catalysis of amino acid activation involves three allosteric states: 1.open, 2. closed pre-transition state involving Mg2+ and an active site lysine residue, and 3. closed producs, the interconversions of these states entail significant domain motions driven by ligand binding, molecular dynamic simulations, overview
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-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophyl-tRNATrp
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-
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?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophyl-tRNATrp
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-
-
-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophan-tRNATrp
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-
-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophan-tRNATrp
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-
-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophan-tRNATrp
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-
-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophan-tRNATrp
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?
additional information
?
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enzyme also performs the ATP-diphosphate exchange reaction
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?
additional information
?
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ligand-linked conformational stability changes associated with this catalytic cycle, overview
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?
additional information
?
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ligand-linked conformational stability changes associated with this catalytic cycle, overview
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?
additional information
?
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the enzyme also accepts L-tyrosine as substrate, albeit with lower catalytic efficiency
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?
additional information
?
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the enzyme also accepts L-tyrosine as substrate, albeit with lower catalytic efficiency
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?
additional information
?
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TrpRS MCD catalytic activity verifies a key prediction of the Rodin-Ohno hypothesis, overview
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?
additional information
?
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an ancestral tryptophanyl-tRNA synthetase precursor achieves high catalytic rate enhancement without ordered ground-state tertiary structures
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophan-tRNATrp
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-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophanyl-tRNATrp
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-
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?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophanyl-tRNATrp
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?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophyl-tRNATrp
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-
-
-
?
additional information
?
-
-
an ancestral tryptophanyl-tRNA synthetase precursor achieves high catalytic rate enhancement without ordered ground-state tertiary structures
-
-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophyl-tRNATrp
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-
-
?
ATP + L-tryptophan + tRNATrp
AMP + diphosphate + L-tryptophyl-tRNATrp
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-
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ATP
assemble of the ATP binding site by domain movement, acts as a key allosteric effector on the enzyme, conformational trigger, not requiring tryptophan pocket ligands, enzyme forms high-affinity complexes at low ATP concentration and low-affinity complexes at high ATP concentration
ATP
Mg2+-ATP interactions are coupled to interactions between ATP and active-site lysine side-chains
additional information
indolmycin and ATP form a ternary complex with BsTrpRS
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additional information
-
indolmycin and ATP form a ternary complex with BsTrpRS
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mn2+
replacing Mg2+ with Mn2+ reduces the specificity for L-tryptophan, increasing the relative activation of L-tyrosine by about 3.5fold
Mg2+
Mg2+
Mg2+-ATP interactions are coupled to interactions between ATP and active-site lysine side-chains
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
indolmycin
competitive inhibitor, produced by Streptomyces griseus, causes selective, mechanism-based inhibition of the bacterial enzyme, which preferentially binds indolmycin about 1500fold more tightly than its tryptophan substrate, binding structure analysis overview. Long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg2+ ion in a manner that allows it to assist in transition state stabilization. The Mg2+ ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. This altered coordination stabilizes a ground state Mg2+-ATP-configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin
additional information
indolmycin and ATP form a ternary complex with BsTrpRS
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additional information
-
indolmycin and ATP form a ternary complex with BsTrpRS
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
diphosphate exchange kinetics show non-reciprocal cooperativity between ATP and Trp
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0.000943
L-tryptophan
wild type enzyme, in 0.1 M Tris-HCl (pH 7.5), 0.01 M potassium fluoride, 5 mM MgCl2, at 37°C
0.00527
L-tryptophan
wild type enzyme, in 0.1 M Tris-HCl (pH 7.5), 0.01 M potassium fluoride, 5 mM MnCl2, at 37°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4.88
L-tryptophan
wild type enzyme, in 0.1 M Tris-HCl (pH 7.5), 0.01 M potassium fluoride, 5 mM MnCl2, at 37°C
5.45
L-tryptophan
wild type enzyme, in 0.1 M Tris-HCl (pH 7.5), 0.01 M potassium fluoride, 5 mM MgCl2, at 37°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
164
L-tryptophan
wild type enzyme, in 0.1 M Tris-HCl (pH 7.5), 0.01 M potassium fluoride, 5 mM MnCl2, at 37°C
1090
L-tryptophan
wild type enzyme, in 0.1 M Tris-HCl (pH 7.5), 0.01 M potassium fluoride, 5 mM MgCl2, at 37°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
indolmycin Michaelis-Menten inhibition kinetics
-
additional information
additional information
-
indolmycin Michaelis-Menten inhibition kinetics
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
aminoacyl-tRNA synthetases maintain the fidelity of the genetic code by ensuring the charging of tRNA with its cognate amino acid
evolution
-
an ancestral tryptophanyl-tRNA synthetase precursor achieves high catalytic rate enhancement without ordered ground-state tertiary structures. The TrpRS Urzyme catalytic activity arises neither from tiny amounts of wild-type enzyme, nor from a separate population of folded and highly active Urzyme molecules not in equilibrium with the general population. AaRS Urzymes lack much of the mass of modern aaRS, retaining only a small portion of the hydrophobic cores of the full-length enzymes. AaRS Urzymes contain 120-130 amino acids, and consist of little more than is required to form intact active sites. They retain over 60% of the transition-state stabilization free energy for amino acid activation and the ability to aminoacylate tRNA. Further, they preserve about 20% of the Gibbs energies necessary to discriminate between competing amino acid substrates and preferentially activate amino acids from within, rather than outside, their own class. A major fraction of TrpRS Urzyme molecules contribute to the rate acceleration by transiently forming tight transition-state complexes
additional information
additional information
both the ATP configuration and Mg2+ coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg2+
additional information
-
both the ATP configuration and Mg2+ coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg2+
additional information
-
15N tryptophanyl-tRNA synthetase Urzyme structure analysis by heteronuclear single quantum coherence (HSQC) NMR spectroscopy supplemented by circular dichroism, thermal melting, and induced fluorescence of bound dye. TrpRS Urzyme is not a typical protein domain. Transition state stabilization and catalytic activity from molten globules, overview
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
sequence homology of E. coli and Bacillus stearothermophilus enzyme
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
schematic model of subunit and subsite organization of the homodimeric enzyme
additional information
open and closed enzyme conformations, overview
additional information
-
open and closed enzyme conformations, overview
additional information
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primary sequence analysis and comparison to other class I and to class II aminoacyl-tRNA transferases, aaRS domain organization and sequence conservation and the sense/antisense coding hypothesis, overview
additional information
-
15N tryptophanyl-tRNA synthetase Urzyme structure analysis by heteronuclear single quantum coherence (HSQC) NMR spectroscopy supplemented by circular dichroism, thermal melting, and induced fluorescence of bound dye. Circular dichroism analysis demonstrates reversible folding of the alpha-helix formed by 61 of 130 residues
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystallization of native enzyme and selenomethionyl enzyme complexed with ATP, L-tryptophan and analogue indolmycin by microdialysis, formation of high-affinity or low-affinity complexes with ATP at 1 mM and 10 mM, respectively, for microclinic crystals: 0.002 ml enzyme solution, containing 4 mg/ml protein and 50% v/v glycerol, plus 2 ml well solution, 42°C, 2 M potassium phosphate, pH 6.6, 20 mM MgCl2, and 2 mM tryptophan or 1 mM ATP, for tetragonal crystals: 35°C, precipitant solution containing 1 M sodium citrate, 10 mM ATP, 10 mM MgCl2, 0.05 mM tryptophanamide, crystal growth within 1 week, X-ray diffraction structure determination at 2.3 A for the selenomethionyl-enzyme and 2.2 A for the native enzyme, structural data and analysis
purified recombinant selenomethionine-substituted BsTrpRS in complex with ATP, Mg2+, and indolmycin are grown by vapor diffusion against a reservoir of 1.4 M potassium citrate and 0.1 M Hepes, pH 7.4, X-ray diffraction structure determination and analysis at 1.9 A resolution, PDB ID 5DK4
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D146A
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site-directed mutagensis, the mutant shows highly reduced activity compared to the wild-type enzyme
additional information
additional information
multiple ligand binding conformation simulations with a virtual polyA mobile loop mutant and a virtual K111A mutant, interaction analysis, overview
additional information
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multiple ligand binding conformation simulations with a virtual polyA mobile loop mutant and a virtual K111A mutant, interaction analysis, overview
additional information
-
construction of a catalytically active wild-type and mutant TrpRS minimal catalytic domains, structures, overview
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
TrpRS Urzyme has substantially reduced hydrophobic stabilization with non-cooperative melting and cold-denaturation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
ligand-linked conformational stability changes associated with this catalytic cycle, overview
TrpRS Urzyme has substantially reduced hydrophobic stabilization with non-cooperative melting and cold-denaturation
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)pLysS by nickel affinity chromatography, followed by cleavage fo the tag by TEV protease, and dialysis
recombinant FLAG-tagged wild-type and mutant enzymes and isolated catalytic domains from Escherichia coli strain BL21(DE3) soluble fraction by anti-FALG immunoaffinity chromatography and dialysis
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recombinant solubilized FLAG- and His-tagged enzyme from Escherichia coli strains BL21 and HSM174 by nickel affinity chromatography, gel filtration, and ultrafiltration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)pLysS by autoinduction
DNA and amino acid sequence determination and anaylsis, genetic structure, aaRS domain organization and sequence conservation and the sense/antisense coding hypothesis, overview, expression of FLAG-tagged wild-type and mutant enzymes and isolated catalytic domains in Escherichia coli strain BL21(DE3) inclusion bodies
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recombinant expression of FLAG- and His-tagged enzyme in Escherichia coli strains BL21 and HSM174 in inclusion bodies, HSM174 cells produce about 5fold more enzyme protein
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RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
recombinant FLAG-tagged wild-type and mutant enzymes and isolated catalytic domains from Escherichia coli strain BL21(DE3) inclusion bodies by dilution in solubilization buffer containing 20 mM HEPES, pH 7.8, 6 M urea, 50 mM or 250 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM PMSF, 1 mM 2-mercaptoethanol, 5% glycerol, and dialysis
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solubilization and refolding of recombinant FLAG- and His-tagged enzyme from Escherichia coli strains BL21 and HSM174 by homogenization in 100 mM sodium phosphate, and 6 M guanidinium hydrochloride, pH 7.2 before purification
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Carter Jr., C.W.; Doublie, S.; Coleman, D.E.
Quantitative analysis of crystal growth. Tryptophanyl-tRNA synthetase crystal polymorphism and its relationship to catalysis
J. Mol. Biol.
238
346-365
1994
Geobacillus stearothermophilus
Winter, G.P.; Hartley, B.S.; McLachlan, A.D.; Lee, M.; Muench, K.H.
Sequence homologies between the tryptophanyl tRNA synthetases of Bacillus stearothermophilus and Escherichia coli
FEBS Lett.
82
348-350
1977
Geobacillus stearothermophilus, Escherichia coli
Carter, C.W.; Green, D.C.
Use of chromatofocusing in the purification of tryptophanyl-tRNA synthetase from Bacillus stearothermophilus
Anal. Biochem.
124
327-332
1982
Geobacillus stearothermophilus
Coleman, D.E.; Carter, C.W.
Crystals of Bacillus stearothermophilus tryptophanyl-tRNA synthetase containing enzymatically formed acyl transfer product. Tryptophanyl-ATP, an active site marker for the 3' CCA terminus of tryptophanyl-tRNATrp
Biochemistry
23
381-385
1984
Geobacillus stearothermophilus
Retailleau, P.; Huang, X.; Yin, Y.; Hu, M.; Weinreb, V.; Vachette, P.; Vonrhein, C.; Bricogne, G.; Roversi, P.; Ilyin, V.; Carter, C.W., Jr.
Interconversion of ATP binding and conformational free energies by tryptophanyl-tRNA synthetase: structures of ATP bound to open and closed, pre-transition-state conformations
J. Mol. Biol.
325
39-63
2003
Geobacillus stearothermophilus (P00953)
Kapustina, M.; Carter, C.W.
Computational studies of tryptophanyl-tRNA synthetase: activation of ATP by induced-fit
J. Mol. Biol.
362
1159-1180
2006
Geobacillus stearothermophilus (P00953), Geobacillus stearothermophilus
Pham, Y.; Li, L.; Kim, A.; Erdogan, O.; Weinreb, V.; Butterfoss, G.L.; Kuhlman, B.; Carter, C.W.
A minimal TrpRS catalytic domain supports sense/antisense ancestry of class I and II aminoacyl-tRNA synthetases
Mol. Cell
25
851-862
2007
Geobacillus stearothermophilus
Weinreb, V.; Carter, C.W.
Mg2+-free Bacillus stearothermophilus tryptophanyl-tRNA synthetase retains a major fraction of the overall rate enhancement for tryptophan activation
J. Am. Chem. Soc.
130
1488-1494
2008
Geobacillus stearothermophilus
Weinreb, V.; Li, L.; Chandrasekaran, S.N.; Koehl, P.; Delarue, M.; Carter, C.W.
Enhanced amino acid selection in fully evolved tryptophanyl-tRNA synthetase, relative to its urzyme, requires domain motion sensed by the D1 switch, a remote dynamic packing motif
J. Biol. Chem.
289
4367-4376
2014
Geobacillus stearothermophilus (P00953), Geobacillus stearothermophilus
Sapienza, P.J.; Li, L.; Williams, T.; Lee, A.L.; Carter, C.W.
An ancestral tryptophanyl-tRNA synthetase precursor achieves high catalytic rate enhancement without ordered ground-state tertiary structures
ACS Chem. Biol.
11
1661-1668
2016
Geobacillus stearothermophilus
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