Information on EC 6.1.1.9 - valine-tRNA ligase

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

EC NUMBER
COMMENTARY hide
6.1.1.9
-
RECOMMENDED NAME
GeneOntology No.
valine-tRNA ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-valine + tRNAVal = AMP + diphosphate + L-valyl-tRNAVal
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Aminoacylation
esterification
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
-
-
tRNA charging
-
-
valine metabolism
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-
SYSTEMATIC NAME
IUBMB Comments
L-valine:tRNAVal ligase (AMP-forming)
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CAS REGISTRY NUMBER
COMMENTARY hide
9023-47-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
cytoplasmic valRS
SwissProt
Manually annotated by BRENDA team
K12
-
-
Manually annotated by BRENDA team
MRE600
-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
strain WFD11
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-
Manually annotated by BRENDA team
strain WFD11
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
putative valyl tRNA synthetase, ValRSI
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain alphaS288C
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-
Manually annotated by BRENDA team
strain D273
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-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
2-chloroadenosine 5'-triphosphate + L-valine + tRNAVal
2-chloroadenosine 5'-monophosphate + diphosphate + L-valyl-tRNAVal
show the reaction diagram
-
-
-
-
-
ATP + DL-2-amino-3-chlorobutyrate + tRNAVal
AMP + diphosphate + DL-2-amino-3-chlorobutyryl-tRNAVal
show the reaction diagram
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as effective as valine
-
-
-
ATP + DL-2-aminobutyrate + tRNAVal
AMP + diphosphate + DL-2-aminobutyryl-tRNAVal
show the reaction diagram
-
30% of the activity with valine
-
-
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ATP + DL-allo-2-amino-3-chlorobutyrate + tRNAVal
AMP + diphosphate + DL-allo-2-amino-3-chlorobutyryl-tRNAVal
show the reaction diagram
-
15% of the activity with valine
-
-
-
ATP + L-isoleucine + tRNAVal
AMP + diphosphate + L-isoleucyl-tRNAVal
show the reaction diagram
-
very low activity
-
?
ATP + L-threonine + tRNAVal
?
show the reaction diagram
-
1 step performance of an originally two-step reaction, the enzyme can hardly differentiate between the cognate amino acid valine and others, especially threonine, to minimize misaminoacetylation the enzyme performs a proofreading, socalled editing reaction at a second active site, which is dependent on the presence of cognate tRNAVal whose 3'-end is involved in the editing reaction, a majority of editing by the enzyme entails prior charging of the tRNA, misacylated tRNA is a transient intermediate in the editing reaction
-
?
ATP + L-threonine + tRNAVal
AMP + diphosphate + L-threonyl-tRNAVal
show the reaction diagram
ATP + L-valine + tRNAiMet,GAC mutant
AMP + diphosphate + L-valyl-tRNAiMet,GAC mutant
show the reaction diagram
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anticodon CAU to GAC initiator tRNA mutant, i.e. G34C36 mutant, is aminoacylated in vitro and in vivo, its anticodon sequence corresponds to the one of tRNAVal
-
?
ATP + L-valine + tRNAVal
AMP + diphosphate + L-valyl-tRNAVal
show the reaction diagram
ATP + L-valine + tRNAVal(GAC)
AMP + diphosphate + L-valyl-tRNAVal(GAC)
show the reaction diagram
ATP + L-valine + tRNAVal(UAC)
AMP + diphosphate + L-valyl-tRNAVal(UAC)
show the reaction diagram
ATP + L-valine + tRNAVal(UAC-2)
AMP + diphosphate + L-valyl-tRNAVal(UAC-2)
show the reaction diagram
ATP + L-valine + tRNAVal,3' 2-aminopurine
AMP + diphosphate + L-valyl-tRNAVal, 3' 2-aminopurine
show the reaction diagram
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mutant tRNAVal with 3'-terminal 2-aminopurine base analogue substitution
-
?
ATP + L-valine + tRNAVal,3' inosine
AMP + diphosphate + L-valyl-tRNAVal, 3' inosine
show the reaction diagram
-
mutant tRNAVal with 3'-terminal inosine base analogue substitution
-
?
ATP + L-valine + tRNAVal,3' isoguanosine
AMP + diphosphate + L-valyl-tRNAVal, 3' isoguanosine
show the reaction diagram
-
mutant tRNAVal with 3'-terminal isoguanosine base analogue substitution
-
?
ATP + L-valine + tRNAVal,3' purine riboside
AMP + diphosphate + L-valyl-tRNAVal, 3' purine riboside
show the reaction diagram
-
mutant tRNAVal with 3'-terminal purine riboside base analogue substitution
-
?
formycin 5'-triphosphate + L-valine + tRNAVal
formycin 5'-monophosphate + diphosphate + L-valyl-tRNAVal
show the reaction diagram
-
-
-
-
-
L-threonyl-tRNAVal
L-threonine + tRNAVal
show the reaction diagram
-
substrate bound to the enzyme, intermediate in the posttransfer editing reaction
-
ir
L-threonyl-tRNAVal-AMP
L-threonine + tRNAVal + AMP
show the reaction diagram
-
substrate bound to the enzyme, intermediate in the pretransfer editing reaction
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ir
tubercidin 5'-triphosphate + L-valine + tRNAVal
tubercidin 5'-monophosphate + diphosphate + L-valyl-tRNAVal
show the reaction diagram
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-
-
-
-
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + L-valine + tRNAVal
AMP + diphosphate + L-valyl-tRNAVal
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
can partially replace Mg2+ in reaction with chloroplastic enzyme only
Zn2+
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2 Zn2+ tightly bound
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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D-allo-2-Amino-3-chlorobutyrate
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DL-2-amino-3-chlorobutyrate
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L-Penicillamine
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Mg2+
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50% inhibition at 7 mM, optimal activation at 4 mM
p-hydroxymercuribenzoate
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pyridoxal 5'-triphospho-5'-adenosine
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aminoacylation and ATP-diphosphate exchange abolished
S-adenosylhomocysteine
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competitive with respect to ATP and valine in the ATP-diphosphate exchange reaction, noncompetitive with respect to ATP and valine and uncompetitive with respect ro tRNA in the tRNA aminoacylation
Thr-tRNAValC76
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-
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tRNAVal(-ACCA)
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-
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tRNAVal(-CCA)
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-
-
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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stimulates
spermine
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chloroplastic enzyme: unable to promote aminoacylation in absence of Mg2+. In presence of suboptimal concentrations of Mg2+, spermine can restore maximal activity (not in cytoplasmic enzyme)
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.029 - 13.4
ATP
0.33
DL-2-amino-3-chlorobutyrate
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-
3.7
DL-2-aminobutyrate
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-
1
DL-allo-2-amino-3-chlorobutyrate
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-
12
DL-threonine
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-
1.9
L-isoleucine
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pH 7.5, 37C
0.3
L-threonine
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ATP-diphosphate exchange reaction, native wild-type enzyme, pH 7.5, 25C
0.0019 - 0.1926
L-valine
0.0002 - 0.001
L-valyl-tRNAVal
0.00005 - 0.042
tRNAVal
0.00061
tRNAVal(GAC)
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in 100 mM HEPES (pH 7.2), 20 mM KCl, 30 mM MgCl2, at 30C
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0.00208
tRNAVal(UAC)
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in 100 mM HEPES (pH 7.2), 20 mM KCl, 30 mM MgCl2, at 30C
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0.00108
tRNAVal(UAC-2)
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in 100 mM HEPES (pH 7.2), 20 mM KCl, 30 mM MgCl2, at 30C
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0.001 - 0.1
valine
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.28 - 57
ATP
0.064 - 14.4
L-valine
0.06 - 0.2
L-valyl-tRNAVal
0.36 - 51
tRNAVal
2.9 - 5.65
valine
additional information
additional information
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.03 - 1.37
ATP
4
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7
Mg2+
-
-
0.0085
Thr-tRNAValC76
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-
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0.02
tRNAVal(-ACCA)
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pH 7.5, 37C
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0.029
tRNAVal(-CCA)
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pH 7.5, 37C
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additional information
additional information
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inhibition kinetics
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 7.5
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7.5 - 8
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Tris-HCl or cacodylate buffer
8
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52C, aminoacylation
8.5 - 9
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in Tris glycine or glycine buffer
8.5
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30E, aminoacylation
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
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pH 6-7.5: optimum, pH 8.5: 50% of maximal activity
6.5 - 10
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6.5: 30-40% of maximal activity, 10: 10% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
27.5
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aminoacylation
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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no activity detectable at 60C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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-
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
Schizosaccharomyces pombe contains two distinct nuclear ValRS genes, one encoding the mitochondrial form and the other its cytosolic counterpart. The cytosolic form closely resembles other yeast ValRS sequences. Gene is active and essential for the survival of the yeast
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30000
-
4 * 30000, SDS-PAGE
100000
108000
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1 * 108000, SDS-PAGE
108100
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calculated from nucleotide sequence
110000
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gel filtration
125000
126000
135000
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glycerol density gradient centrifugation
137000
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gel filtration
140000
145000
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sucrose density gradient centrifugation, monomeric enzyme form
585000
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gel filtration, tetrameric enzyme form
800000
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high-molecular-mass complex, gel filtration
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1.7 A resolution crystal structure of the ValRS editing domain and 1.7 A resolution crystal structure of the editing domain bound with [N-(L-threonyl)-sulfamoyl]adenosine, hanging drop vapor diffusion method
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enzyme complexed with tRNAVal and valyl-adenylate, hanging-drop vapour diffusion method, 20C, 1 month, equal volumes of protein and crystallization solution: 50 mM N-(2-acetoamide)iminodiacetic acid sodium salt buffer, pH 6.5, 2% 2-propanol, 0.1 M lithium sulfate, 12% PEG4000, equilibration against reservoir solution: 50 mM N-(2-acetoamide)iminodiacetic acid sodium salt buffer, pH 6.5, 2% propanol, 0.1 M lithium sulfate, and 14% PEG 4000, ligand-free crystals by macro-seeding, X-ray diffraction structure determination at 2.9 A resolution, and analysis
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enzyme in complex with tRNAValCAC isoacceptor and an analogue of the Val-adenylate intermediate, hanging-drop vapour diffusion method, 2 months, 4C, 7-10 mg/ml protein/tRNA/Val-AMS solution in a molar ration of 1:1.1:2, plus equal volume of crystallization solution containing 50 mM sodium cacodylate, pH 6.5, 1.0 ammonium sulfate, 10 mM MgSO4, 6% 1,8-diaminooctane, equilibration against a reservoir solution of 50 mM sodium cacodylate, pH 6.5, 2.8 M ammonium sulfate, 10 mM MgSO4, X-ray structure determination at 2.9 A resolution and analysis
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molecular dynamics simulation studies based on PDB structure ID 1wk9. Noncognate substrates Thr-AMP and Thr-A76, bind more strongly than the cognate substrates Val-AMP and Val-A76 in both pre- and post-transfer editing, respectivel
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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half-life: 90 min
20
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half-life: 30 min, when enzyme is stabilized by 1 mg/ml bovine serum albumin and 50% glycerol
40
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5 min, chloroplastic enzyme loses 30% of its activity, mitochondrial enzyme 75%
60
-
1 min, stable
70
-
1 min, complete inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
20-30% loss of activity after each cycle of freezing and thawing
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bovine serum albumin protects from thermal inactivation
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relatively unstable in the absence of a sulfhydryl-containing compound
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-180C, purified enzyme highly unstable, stored immediately after purification at -180C
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-20C, 50% glycerol, 0.1% detergent, stable for several months
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-20C, 50% glycerol, 5 months stable, heterotypic complex
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-20C, homogenate and ammonium sulfate fraction are stable for 6 months
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-20C, stable for at least 1 year
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-20C, without addition of tRNA about 80% loss of activity during 1 week
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0C, stable for several months
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4C, 1 month, less than 20% loss of activity
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4C, 50 mM potassium phosphate buffer, pH 7.0, 1 mM MgCl2, 0.002 mM L-valine, 100 mM NaCl, 15% v/v 1,2-propanediol, stable for several weeks
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
heterotypic complex in association with elongation factor
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His-Trap column chromatography, and PD-10 gel filtration
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recombinant from Escherichia coli strain JM109(DE3)
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recombinant His-tagged ValRS from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography
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recombinantly expressed His-tagged wild-type and mutant enzymes
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using Ni-NTA chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli C41(DE3) cells
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expressed in Saccharomyces cerevisiae as a His-tagged fusion protein
expression in Escherichia coli
gene valS, expression of His-tagged wild-type and mutant enzymes in strain JM101Tr
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gene VARS2
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gene VAS1, codes for both cytoplasmic and mitochondrial isozymes of ValRS through alternative transcription and translation, expression of wild-type and mutant enzymes in yeast
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overexpression of His-tagged ValRS in Escherichia coli strain BL21(DE3)
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overexpression of wild-type and mutant emnzymes in Escherichia coli strain JM109(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D286A
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site-directed mutagenesis, the ValRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations
K277A
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site-directed mutagenesis, mutant exhibits a reduced posttransfer editing activity compared to the wild-type, also the specificiy of the editing reaction is modulated, the mutant hydrolyzes the correctly formed Val-tRNAVal, increased sensitivity to Mg2+, high concentrations inactivate
K277P
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site-directed mutagenesis, the ValRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations
K277P/D286A
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site-directed mutagenesis, the ValRS CP1 domain mutant is unable to deacylate misacylated tRNA even at high enzyme concentrations
C25T
-
site-directed mutagenesis
DELTA1-97
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an N-domain-deleted yeast valyltRNA synthetase mutant (DELTA1-97) form Saccharomyces cerevisiae can be rescued by fusion of the equivalent domain from its human homologue
D750G
-
the mutation is associated with a temperature-sensitive phenotype. Although depicting slower growth at 26C, the mutant strain is unable to grow at 37C
DELTA1-97
-
deletion of N-terminal polypeptide extension of 97 residues, which is absent in bacteria, severely impairs tRNA binding, aminoacylation, and complementation activities of the enzyme. This N-domain-deleted yeast valyl-tRNA synthetase mutant can be rescued by fusion of the equivalent domain from its human homologue
DELTA32-71
-
deletion of a lysine rich insert impairs aminoacylation activity of the enzyme in vitro but aminoacylation activity is still significantly higher than in DELTA1-97 mutant. Km: 0.001 mM (L-valyl-tRNAVal), kcast: 0.06/sec (L-valyl-tRNAVal)
D276A
-
valylation activity is similar to that of wild-type enzyme
F264A
-
valylation activity is similar to that of wild-type enzyme, about 15% decrease in ATP consumption rate compared to wild-type enzyme, somewhat reduced deacylation activityith Thr-tRNAVal, mutation impairs editing activity of ValRS
R216A
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valylation activity is similar to that of wild-type enzyme, about 15% decrease in ATP consumption rate compared to wild-type enzyme, somewhat reduced deacylation activity with Thr-tRNAVal
R818A/R843A
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slightly reduced activity compared to the wild-type enzyme with tRNAValCAC mutant substrate
T272A
-
valylation activity is similar to that of wild-type enzyme, about 15% decrease in ATP consumption rate compared to wild-type enzyme, somewhat reduced deacylation activity with Thr-tRNAVal
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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a C25U point mutation in the MTTV gene, gene encoding tRNAVal, is associated with a metabolic disorder resulting in the neonatal deaths of numerous siblings. In primary myoblasts and transmitochondrial cybrids established from the proband and offspring, the steady-state levels of the mutated mt-tRNAVal are greater than in the biopsy material, but are still lower than in control myoblasts. Decrease in steady-state mt-tRNAVal observed cell lines is caused by a rapid degradation of the deacylated form of the abnormal mt-tRNAVal. By both establishing the identity of the human mitochondrial valyltRNA synthetase then inducing its overexpression in transmitochondrial cell lines, steady-state levels of the mutated mt-tRNAVal can be partially restored
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