Information on EC 6.1.1.12 - aspartate-tRNA ligase

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

EC NUMBER
COMMENTARY hide
6.1.1.12
-
RECOMMENDED NAME
GeneOntology No.
aspartate-tRNA ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-aspartate + tRNAAsp = AMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
aminoacyl group transfer
Aminoacylation
esterification
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
-
-
aspartate and asparagine metabolism
-
-
tRNA charging
-
-
SYSTEMATIC NAME
IUBMB Comments
L-aspartate:tRNAAsp ligase (AMP-forming)
-
CAS REGISTRY NUMBER
COMMENTARY hide
9027-32-1
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain TG1
-
-
Manually annotated by BRENDA team
gene aspS
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Methanothermobacter thermautotrophicum
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain KOD1, overexpressed in Escherichia coli
-
-
Manually annotated by BRENDA team
strain KOD1, overexpressed in Escherichia coli
-
-
Manually annotated by BRENDA team
strain C836
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain VK-1
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
LBSL, i.e. leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation, is a monogenic disease associated with a large variety of mutations affecting the human nuclear gene DARS2, encoding mt-AspRS, overview
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + Asp + tRNAAsn
AMP + diphosphate + aspartyl-tRNAAsn
show the reaction diagram
-
discriminating AspRS gains the ability to form Asp-tRNAAsn in vitro when the W26H or K85P changes are introduced independently or in combination
-
-
?
ATP + Asp + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-
-
-
-
?
ATP + D-aspartate + tRNAAsp
AMP + diphosphate + D-aspartyl-tRNAAsp
show the reaction diagram
-
aspartyl-tRNA synthetase can misacylate tRNAAsp with D-aspartate instead of its usual substrate, L-Asp, substrate specificity and molecular dynamics simulations, overview
-
-
?
ATP + L-Asp + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
-
low reaction with mutant tRNAAsp with A instead of G at position 73 (tRNAAspA73)
-
-
?
ATP + L-asparagine + tRNAAsp
AMP + diphosphate + L-asparaginyl-tRNAAsp
show the reaction diagram
-
-
-
?
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
ATP + L-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
GTP + L-aspartate + tRNAAsp
GMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
UTP + L-aspartate + tRNAAsp
UMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
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-aspartate + tRNAAsn
AMP + diphosphate + L-aspartyl-tRNAAsn
show the reaction diagram
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + aspartyl-tRNAAsp
show the reaction diagram
ATP + L-aspartate + tRNAAsp
AMP + diphosphate + L-aspartyl-tRNAAsp
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
can replace Mg2+, with 12% efficiency in activation of ATP-diphosphate exchange, with 34% efficiency in aminoacylation
K2HPO4
-
enzyme form PC-3 is stimulated, optimal concentration 75 mM, enzyme form PC-1 and enzyme form PC-2 are inhibited
KCl
-
enzyme form PC-3 is stimulated, optimal concentration 75 mM, enzyme form PC-1 and enzyme form PC-2 are inhibited
Mn2+
-
can replace Mg2+, with 35% efficiency in activation of ATP-diphosphate exchange, with 56% efficiency in aminoacylation
PO43-
-
appears to act synergistically with K+ in stimulation of enzyme form PC-3, no effect on either enzyme form PC-1 or enzyme form PC-2
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(3S)-3-amino-5-((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)(hydroxy)phosphoryl)-4-oxopentanoic acid
-
-
2-aminomalonic acid
-
inhibits transfer of Asp to tRNA
5'-O-[N-(L-aspartyl)sulfamoyl]adenosine
CaCl2
-
1 mM, more than 50% inhibition
CH3COO-
-
2.0 M, 50% inhibition of ATP-diphosphate exchange
ClO3-
-
0.1 M, 50% inhibition of ATP-diphosphate exchange
CNS-
-
0.1 M, 50% inhibition of ATP-diphosphate exchange
Cs+
-
1.1 M, 50% inhibition of ATP-diphosphate exchange, 0.16 M, 50% inhibition of aminoacylation
diethyldicarbonate
-
reversed by hydroxylamine
erythro-3-hydroxyaspartic acid
-
inhibits transfer of Asp to tRNA
F-
-
-
heptapeptide-nucleotide microcin C
-
i.e. McC, upon its entry into a susceptible cell, McC is processed to release a nonhydrolyzable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase, leading to the cessation of translation and cell growth. The rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC
IO3-
-
0.04 M, 50% inhibition of ATP-diphosphate exchange
K+
-
2.3 M, 50% inhibition of ATP-diphosphate exchange, 0.22 M, 50% inhibition of aminoacylation
K2HPO4
-
inhibits enzyme forms PC-1 and PC-2, PC-3 is stimulated (optimal concentration 75 mM)
KCl
-
inhibits enzyme forms PC-1 and PC-2, PC-3 is stimulated (optimal concentration 75 mM)
L-aspartol adenylate
-
-
L-aspartol-adenylate
L-aspartyl adenylate
-
-
Li+
-
0.6 M, 50% inhibition of ATP-diphosphate exchange, 0.11 M, 50% inhibition of aminoacylation
Microcin C
-
a ribosome-synthesized heptapeptide that contains a modified adenosine monophosphate covalently attached to the C-terminal aspartate, a potent inhibitor of bacterial growth, that targets the translation process via its degradation product, a modified aspartyl-adenylate containing an N-acylphosphoramidate linkage, overview, Microcystin is recombinantly expressed in Escherichia coli strain TG1
microcin C7-C51
-
an antimicrobial nucleotide peptide that targets aspartyl-tRNA synthetase and inhibits translation. Fragmentation and analysis of fragment structure and inhibitory potency, overview
-
Na+
-
1.4 M, 50% inhibition of ATP-diphosphate exchange, 0.18 M , 50% inhibition of aminoacylation
NaCl
-
inhibits enzyme forms PC-1 and PC-2 much more than PC-3
NH4+
-
0.9 M, 50% inhibition of ATP-diphosphate exchange, 0.14 M, 50% inhibition of aminoacylation
NiCl2
-
0.1 mM more than 50% inhibition
NO3-
-
0.7 M, 50% inhibition of ATP-diphosphate exchange
p-chloromercuribenzoate
-
-
succinate
-
50% inhibition at 210 mM
threo-3-hydroxyaspartic acid
-
inhibits transfer of Asp to tRNA
threo-3-Methylaspartic acid
-
inhibits transfer of Asp to tRNA
tRNA
-
above 0.4 mg/ml
additional information
-
the yeast AspRS initiates retro-inhibition of its expression, overview
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
putrescine
spermidine
spermine
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8
2-aminomalonic acid
-
ATP-diphosphate exchange
0.003 - 4.6
Asp
1.2 - 1.4
aspartate
0.03
aspartic acid
pH 7.2, 70C
0.023 - 1.11
ATP
0.32
L-Asp
-
-
0.0015 - 0.21
L-aspartate
0.005
L-aspartic acid
pH 7.2, 70C
24
threo-3-hydroxyaspartic acid
-
ATP-diphosphate exchange
0.000063 - 0.0091
tRNAAsn
0.000013 - 0.08
tRNAAsp
1.3
tRNAAspA73
-
pH 7.8, 28C
-
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2 - 18
Asp
16.9
aspartyl-tRNA
Saccharomyces cerevisiae
-
-
5.9 - 8.5
ATP
0.0008 - 0.12
tRNAAsn
0.00016 - 79
tRNAAsp
0.0029
tRNAAspA73
Escherichia coli
-
pH 7.8, 28C
-
additional information
additional information
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.9 - 6
tRNAAsn
3.7 - 293
tRNAAsp
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000123
(3S)-3-amino-5-((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)(hydroxy)phosphoryl)-4-oxopentanoic acid
-
-
0.0000098 - 0.00039
5'-O-[N-(L-aspartyl)sulfamoyl]adenosine
1
CaCl2
-
-
0.045
L-aspartol adenylate
-
pH 7.5, 37C
0.00033 - 0.045
L-aspartol-adenylate
6000
Li+
-
ATP-diphosphate exchange
0.1
NiCl2
-
-
additional information
additional information
-
Microcystin C in vivo inhibition kinetics
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00002
native enzyme, cell extract, 37C
0.000083
native enzyme, cell extract, 37C
0.017
-
wild-type enzyme
0.025
-
recombinant wild-type enzyme
0.0336
purified recombinant enzyme, 37C
0.034
-
truncated enzyme
0.0405
-
enzyme form AspRS2
0.043
recombinant enzyme, Escherichia coli DH5alpha cell extract, 37C
0.0452
-
enzyme form AspRS1
0.055
recombinant enzyme, Escherichia coli BL21 cell extract, 37C
0.063
-
recombinant mutant D560V
0.097
-
recombinant mutant R263Q
0.11
-
65C, pH is not specified in the publication; 65C, pH not specified in the publication
0.12
-
recombinant mutant L626V
0.124
recombinant enzyme, Escherichia coli BL21 cell extract, 37C
0.15
-
recombinant mutant Q184K
0.155
recombinant enzyme, Escherichia coli DH5alpha cell extract, 37C
0.321
purified recombinant enzyme, 37C
1.75
-
recombinant mutant C152F
3.7
-
purified recombinant His6-tagged DRS enzyme
10.9
-
purified recombinant His6-tagged DRS-ubiquitin fusion enzyme
11.5
-
purified recombinant biotin-tagged DRS-ubiquitin fusion enzyme
13.5
-
purified recombinant His6-tagged DRS-small ubiquitin-like modifier fusion enzyme
17
-
purified recombinant GST-tagged DRS enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
Methanothermobacter thermautotrophicum
-
assay at
7.3
assay at
7.8 - 8
-
potassium Hepes buffer
8.5
-
Tris/cacodylate buffer
additional information
-
very low kinetic effiency at 17C
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9
-
pH 5.5: about 60% of maximal activity, pH 9.0: about 30% of maximal activity
5.5 - 8
-
pH 5.5: about 60% of maximal activity, pH 8.0: about 65% of maximal activity
7.8 - 9.1
-
7.8: sharp loss of activity below, 9.1: plateau of high activity up to pH 9.1
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37 - 50
-
assay at 37C or 50C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35 - 75
-
35C: about 60% of maximal activity, 75C: about 50% of maximal activity
37 - 60
Methanothermobacter thermautotrophicum
-
-
45 - 85
-
45C: about 80% of maximal activity, 85C: about 25% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.23
-
calculated from sequence
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)
Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)
Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)
Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15500
-
gel filtration
50000
-
determined by SDS-PAGE
64000
-
determined by SDS-PAGE
66000
determined by SDS-PAGE
83000 - 100000
-
sucrose density gradient centrifugation
89000 - 106000
-
sucrose density gradient centrifugation
96000
-
crystal structure determination
116000
-
sedimentation equilibrium determination, neutron scattering
117000
-
gel filtration, also a smaller peak of MW 57000 detected
119000
-
sedimentation equilibrium determination
122000
124000
-
gel filtration
132000
-
gel filtration
150000
-
gel filtration, PC-1 and PC-2
156000
-
nondenaturing PAGE
205000
-
gel filtration
260000
-
gel filtration
500000
-
gel filtration, PC-3, possibly PC-3 represents a large complex of aminoacyl-tRNA synthetases
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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
no glycoprotein
-
-
additional information
-
the enzyme does not perform autoaspartylation in vivo
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the crystal structure of aspartyl-tRNA synthetase is determined at 2.8 A resolution
analysis of the 2.6-A resolution crystal structure of Escherichia coli AspRS with bound aspartyl-adenylate, AspAMP, molecular dynamics simulations
-
of enzyme complexed with its cognate tRNA
-
purified recombinant enzyme, in ammonium sulfate, NaCl, bis Tris propane, isopropanol, X-ray diffraction structure determination at 2.7 A resolution and structure analysis
-
hanging drop vapor diffusion method, in 30% (w/v) PEG 4000, 100 mM ammonium sulfate, 100 mM sodium citrate at pH 5.6
-
vapor diffusion method, using 10% (m/v) PEG-3350 and 0.5M of ammonium sulfate in 25 mM Bis-Tris pH 5.5
-
binary complex formed by the enzyme and tRNAAsp
-
purified recombinant truncated enzyme, tri- and tetragonal crystals, X-ry structure determination at 3 and 2.3 A resolution, respectively, molecular replacement, structure analysis and modeling
7 different mutants, modification of crystal surfaces, investigation of crystallizability to determine the influence of surface residues and protein structure on crystal growth, packing arrangement, and quality
-
AspRS2, hanging drop vapour diffusion method, 0.002 ml of 14 mg/ml protein with 10% m/v PEG 8000, 200 mM NaCl, and 100 mM Na-CHES buffer, pH 9.5, X-ray diffraction structure determination and analysis at 2.3-3.2 A resolution
-
at 2.5 A resolution
-
enzyme complexed with a non-hydrolysable analogue of asparaginyl-adenylate and with ATP, X-ray diffraction structure determination at 2.6 A resolution
-
hanging-drop vapour-diffusion method, enzyme crystallizes either in a monoclinic or an orthorhombic habit. Minute amounts of protein impurities alter to a different extent the growth of each crystal form. The best synthetase crystals are obtained when the crystallizating solution is either enclosed in capillaries or immobilized in agarose gels
-
isozyme AspRS2, hanging-drop vapour diffusion method, pH 9.5, in presence of PEG 8000 and NaCl, structure determination
-
purified enzyme complexed with tRNAAsp from Thermus thermophilus or Escherichia coli, potential intermediate of the recognition process, protein solution: 15 mg/ml of enzyme, 7.35 mg/ml of tRNA, plus equal volume of reservoir solution: 10 mM MgCl2, 50 mM HEPES, pH 7.5, 0.7 M sodium citrate, 17C, 2 weeks, X-ray diffraction structure determination at 3.5 A resolution, structure analysis
-
purified recombinant enzyme, hanging-drop vapour diffusion method, in presence of PEG 8000, at 293K, growth kinetics and solubility measurements, X-ray diffraction structure determination at 3.1 A resolution
-
purified wild-type and seleno-Met isozymes AspRS2, vapour phase diffusion from mother liquid: 100 mM CHES buffer, pH 9.5, 200 mM NaCl, 10% w/v PEG 8000, X-ray diffraction structure determination at 2.3 A resolution, structure analysis
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 42
-
the enzyme is stable up to 42C
37
-
native enzyme is stable for 24 h, half-life of truncated enzyme hDRSDELTA32 is 7 h, half-life of the fusion protein of glutathione-S-transferase and hDRSDELTA32 is 3 h
42
-
half-life: 22 min for tRNA aminoacylation, 68 min for ATP/diphosphate exchange
50
-
half-life: 7 min without stabilizing reagents, 12 min in presence of 0.004 mM ATP, 23 min in presence of 0.004 mM ATP and 0.1 mM Asp
55
-
10 min stable, 95% loss of activity after 2 h
70
-
no denaturation below
85
-
the thermal melting temperature seems higher than 85C in the presence of Mg2+. In the absence of Mg 2+, melting temperature decreases to 85C
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
polyamines stabilize the conformation of the tRNA-AspRS complex
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Glycerol
-
25% v/v, required throughout purification procedure to maintain stability
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 10 mM potassium phosphate, pH 7.6, 10 mM 2-mercaptoethanol, 50% glycerol, stable
-
-20C, 40% w/v glycerol, SH-group stabilizing reagent, 50% loss of activity after 72 h
-
-80C, 0.1 M potassium phosphate buffer, about 50% loss of activity after 6 months
-
in a dry form, e.g. as acetone precipitate, stable for 1 month
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 distinct enzyme forms: AspRS1 and AspRS2
-
2 enzyme forms: PC-1, PC-2 and PC-3
-
; recombinant His-tagged enzyme from Escherichia coli
affinity chromatography
-
by Ni2+-nitrilotriacetic acid chromatography, cytosolic and mitochondrial fractions of Trypanosoma brucei are prepared
enzyme form PC-1 (in addition 3 low MW enzyme forms exist: PC-2a, PC-2b and PC-2c)
-
HiTrap chelating HP column chromatography, HiTrap Q column chromatography, and Superdex 200 gel filtration
-
magnesium sulfate precipitation, column chromatography, nickel affinity chromatography, and anion-exchange chromatography
-
Ni-NTA column chromatography and gel filtration
-
Ni-NTA column chromatography and Hiprep 16/60 Sephacryl S-200 gel filtration
-
on a glutathione-Sepharose resin
on a Ni-NTA affinity column followed by cleavage of the N-terminal affinity tag
recombinant AspRS2 from Escherichia coli by two different steps of anion exchange chromatography and hydroxy apatite chromatography
-
recombinant from overexpressing Escherichia coli
-
recombinant from overexpressing Escherichia coli strain JM103, to homogeneity
-
recombinant from overexpressing Escherichia coli, 34.3fold; recombinant from overexpressing Escherichia coli, 7.4fold
recombinant GST-tagged enzyme from Escherichia coli strain Top10 by glutathione affinity chromatography, the tag is then cleaved of by thrombin, and the enzyme is further purified by adsorption chromatography yielding an AspRS with a short additional amino acid stretch at its N-terminus
-
recombinant His-tagged enzyme from Escherichia coli
Methanothermobacter thermautotrophicum
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
-
recombinant His-tagged enzyme from strain BL21 by nickel affinity chromatography
-
recombinant His6-tagged DRS-SUMO and DRS-ubiquitin fusion proteins by nickel affinity chromatography from Escherichia coli strain BL21(DE3), recombinant biotin-tagged DRS-ubiquitin fusion protein by avidin affinity chromatography from Escherichia coli strain Bl21(DE3)
-
recombinant truncated enzyme from Escherichia coli
recombinant wild-type and mutants
-
recombinant wild-type and seleno-Met isozyme 2 from overexpressing Escherichia coli
-
wild-type and mutant enzymes
-
wild-type and two truncated forms
-
wild-type enzyme and N-terminal 32-residue truncated form (hDRS delta 32), expressed in Escherichia coli as fusion proteins linked through a thrombin cleavage site with glutathione-S-transferase
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
aspS1 gene, DNA and amino acid sequence determination and analysis, expression in Escherichia coli strains DH5alpha and BL21; aspS2 gene, DNA and amino acid sequence determination and analysis, expression in Escherichia coli strains DH5alpha and BL21
cloning into pQE70 vectors and expression in Escherichia coli TOP 10
-
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli BL21(DE3) cells and in HEK-293T cells
-
expressed in Escherichia coli BL21(DE3) Rosetta 2 cells
-
expressed in Escherichia coli JM83 cells
-
expressed in Escherichia coli Rosetta2(DE3)pLysS cells
-
expression as His-tagged protein in Escherichia coli
Methanothermobacter thermautotrophicum
-
expression as His-tagged protein in Escherichia coli; expression in Escherichia coli
expression in Chinese hamster ovary cells
-
expression in CHO cells
-
expression in Escherichia coli
-
expression in Saccharomyces cerevisiae, and expression as His-tagged protein
-
expression in Saccharomyces cerevisiae, wild-type enzyme and two truncated forms lacking 20 or 36 amino acid residues from their amino-terminal polypeptide extension
-
expression of His6-tagged or GST-tagged enzyme in Escherichia coli strain BL21(DE3), and expression of biotin- or His6-tagged DRS-SUMO and DRS-ubiquitin fusion proteins in Escherichia coli strain BL21(DE3), since the free DRS is expressed as an inactive and insoluble protein in Escherichia coli, SUMO is a small ubiquitin-like modifier molecule
-
expression of wild-type and mutant enzymes in yeast YBC-603 cells, functional overexpression of GST- and His6-tagged enzyme in Escherichia coli strain Top10. Expression in yeast cells at low copy number, since overexpression is cytotoxic, while moderate AspRS accumulation in the cell is not deleterious
-
expression of wild-type enzyme, amino acid residues 1-557, and a truncated mutant enzyme, amino acid resdiues 71-557, as His-tagged proteins in Escherichia coli
-
gene aspS, expression of His-tagged enzyme in Escherichia coli strain Bl21(DE3)
-
gene aspS, expression of the His-tagged enzyme in strain BL21
-
gene aspS2, expression in Escherichia coli strain JM103
-
gene DARS2, DNA and amino acid sequence determination and analysis
-
gene DARS2, located on chromosome 1, DNA and amino acid sequence determination and analysis, expression analysis, genotyping
-
into the vector pGEX-2T for expression in Escherichia coli ER2566 cells
mutants with substituted His residues expressed in Escherichia coli
-
native recombinant AspRS from rat and the N-terminal truncated derivatives AspRS-DELTA20 and AspRS-DELTA36 expressed in yeast
-
overexpression in Escherichia coli
overexpression in Escherichia coli strain JM103
-
overexpression of wild-type and mutants
-
overexpression of wild-type isozyme AspRS2 and seleno-Met isozyme AspRS2 in Escherichia coli
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the full length sequence is cloned into the AVA0421 vector
the sequence corresponding to the carboxyl-terminal Ty1-tagged Tb-AspRS1 is cloned into a derivative of pLew-100 to allow tetracycline-inducible expression of the tagged protein in transgenic cell lines; the sequence corresponding to the carboxyl-terminal Ty1-tagged Tb-AspRS1 is cloned into a derivative of pLew-100 to allow tetracycline-inducible expression of the tagged protein in transgenic cell lines, into the vector pET15b for expression in Escherichia coli BL21-CodonPlus DE3-RIL cells
truncated enzyme lacking 70 amino acid residues in the N-terminus, overexpression in Escherichia coli
wild-type enzyme and N-terminal 32-residues truncated form, expressed in Escherichia coli as fusion proteins linked through a thrombin cleavage site with glutathione-S-transferase
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A471T
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mutant enzyme is still able to aminoacylate the native tRNAAsp in vivo at a level sufficient to complement the defective strain CS143
D29N
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mutant enzyme is still able to aminoacylate the native tRNAAsp in vivo at a level sufficient to complement the defective strain CS143
E118K
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mutant enzyme is still able to aminoacylate the native tRNAAsp in vivo at a level sufficient to complement the defective strain CS143. 6.5fold increase in aminoacylation rate and 3fold decrease in amino acid activation reaction
E93K
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mutant enzyme is still able to aminoacylate the native tRNAAsp in vivo at a level sufficient to complement the defective strain CS143
G90S
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mutant enzyme is still able to aminoacylate the native tRNAAsp in vivo at a level sufficient to complement the defective strain CS143
G90V
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mutant enzyme is still able to aminoacylate the native tRNAAsp in vivo at a level sufficient to complement the defective strain CS143
K198L/D233E
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slightly increased binding of Asp, binding of ASn is strongly increased to the level of Asp, no ability to adenylate Asn, molecular dynamic simulation, binding mechansim and free energy
K198L/Q199E/D233E
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binding of Asp is strongly reduced, no ability to adenylate Asn, molecular dynamic simulation, binding mechansim and free energy
L30F
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mutant enzyme is still able to aminoacylate the native tRNAAsp in vivo at a level sufficient to complement the defective strain CS143
P555S
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thermosensitive mutant, resulting in substitution of Pro 555 by Ser. Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding
R383C
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mutant enzyme is still able to aminoacylate the native tRNAAsp in vivo at a level sufficient to complement the defective strain CS143
T89I
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mutant enzyme is still able to aminoacylate the native tRNAAsp in vivo at a level sufficient to complement the defective strain CS143
hDRSDELTA32
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N-terminal 32-residue truncated form, hDRSDELTA32, with lower thermal stability and ATP-diphosphate exchange activity, but higher aminoacylation activity. Fusion protein of glutathione-S-transferase and hDRSDELTA32 with lower thermal stability
L613F
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the mutant shows reduced specific activity compared to the wild type enzyme
L626Q
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the mutant shows 43fold decreased specific activity compared to the wild type enzyme
L626V
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the mutant shows 210fold reduced activity compared to the wild-type enzyme
R58G
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the mutant shows an increase in specific activity compared to the wild type enzyme
S45G
a naturally occuring mutation identified in patients suffering leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation. The mutant enzyme is not processed due to nontranslocation of the protein
T136S
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the mutant shows an increase in specific activity compared to the wild type enzyme
Asp-DELTA20
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native recombinant AspRS from rat and the N-terminal truncated derivatives Asp-DELTA20 and AspRS-DELTA36 expressed in yeast. A moderate but significant drop in affinity towards the multisynthetase complex is inferred by the removal of the N-terminal domain. This domain is absolutely required in vivo for association within the multisynthetase structure
D210A
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central core binding mutant, increased acylation activity, increased dissociation constant
E188A
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anticodon loop binding mutant, slightly reduced acylation activity, increased dissociation constant
E188A/S239A
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anticodon-G73 binding mutant, reduced acylation activity, highly increased dissociation constant
E188A/T331A
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anticodon-G73 binding mutant, highly reduced acylation activity, highly increased dissociation constant
E202A
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central core binding mutant, slightly increased acylation activity, increased dissociation constant
E327A
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acceptor arm binding mutant, reduced acylation activity
F127A
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anticodon loop binding mutant, increased acylation activity, increased dissociation constant
F127A/D210A
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anticodon binding mutant, reduced acylation activity, highly increased dissociation constant
F127A/E188A
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anticodon binding mutant, reduced acylation activity, highly increased dissociation constant
F127A/S239A
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anticodon-G73 binding mutant, reduced acylation activity, highly increased dissociation constant
F127A/T331A
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anticodon-G73 binding mutant, highly reduced acylation activity, highly increased dissociation constant
F304A
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terminal A binding mutant, reduced acylation activity
H116G
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mutants with substituted His residues, His116Gly mutant with a slightly reduced rate of amino acid activation without affecting the other kinetic parameters, His271Gly mutant with completely destroyed activity, His332Gly mutant with 60% decrease in rate of tRNA aminoacylation and no significant changes in the other parameters, His334Gly mutant with 70% decrease in amino acid activation, complete loss of tRNA aspartylation and slightly increased Km for ATP, His271Ala mutant with 25% decrease in the rate of tRNA charging. His334 seems do be part of the active site, while His271 and His332 play an important structural role
H334A
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acceptor arm binding mutant, highly reduced acylation activity, increased dissociation constant
K142A
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anticodon loop binding mutant, increased acylation activity, increased dissociation constant
K142A/E188A
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anticodon binding mutant, reduced acylation activity, highly increased dissociation constant
K155A
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central core binding mutant, increased acylation activity, increased dissociation constant
K180A
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anticodon loop binding mutant, slightly increased acylation activity, increased dissociation constant
K293A
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acceptor arm binding mutant, highly increased acylation activity, increased dissociation constant
K428A
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acceptor arm binding mutant, increased acylation activity, increased dissociation constant
K553A
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acceptor arm binding mutant, acylation activity similar to wild-type, increased dissociation constant
N117A
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anticodon loop binding mutant, increased acylation activity, increased dissociation constant
N227A
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central core binding mutant, slightly increased acylation activity, increased dissociation constant
N328A
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acceptor arm binding mutant, reduced acylation activity, increased dissociation constant
N328A/S329A/T331A
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G73 binding mutant, highly reduced acylation activity, increased dissociation constant
P273G
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Pro273Gly mutant. Catalytic properties of native and Pro273Gly homodimers or heterodimers of AspRS molecules, confirm the participation of Pro273 in subunit association
Q121A
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anticodon loop binding mutant, increased acylation activity, increased dissociation constant
Q138A
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anticodon loop binding mutant, slightly increased acylation activity, increased dissociation constant
Q138A/E188A
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anticodon binding mutant, reduced acylation activity, highly increased dissociation constant
Q138A/R119A
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anticodon binding mutant, reduced acylation activity, highly increased dissociation constant
Q300A
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terminal A binding mutant, reduced acylation activity
R119A
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anticodon loop binding mutant, increased acylation activity, increased dissociation constant
R119A/E188A
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anticodon binding mutant, highly reduced acylation activity, highly increased dissociation constant
R485K
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site-directed mutagenesis, the substitution in the catalytic site completely inhibits aspartylation by impairing ATP binding, this mutant still retains the capacity to be modified and shows the same pattern as wild-type AspRS on the two-dimensional gel, thus the modifications are not the result of autoaspartylation
S181A
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anticodon loop binding mutant, increased acylation activity, increased dissociation constant
S280A
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terminal A binding mutant, reduced acylation activity
S301A
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terminal A binding mutant, reduced acylation activity, slightly increased dissociation constant
S329A
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acceptor arm binding mutant, increased acylation activity, increased dissociation constant
S423A
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acceptor arm binding mutant, reduced acylation activity
T124A
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anticodon loop binding mutant, slightly reduced acylation activity, increased dissociation constant
T230A
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central core binding mutant, increased acylation activity, increased dissociation constant
T331A
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acceptor arm binding mutant, reduced acylation activity, increased dissociation constant
T424A
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acceptor arm binding mutant, acylation activity similar to the wild-type, increased dissociation constant
K85P
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discriminating AspRS gains the ability to form Asp-tRNAAsn. Mutation impairs the ability to synthesize Asp-trNAASp in vitr, 8fold increase in KM-value for tRNAAsp; the wild-type enzyme shows no activity with tRNAAsn, the mutant is active with tRNAAsn
W26H
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discriminating AspRS gains the ability to form Asp-tRNAAsn. Mutation causes a 1.5fold decrease in overall catalytic efficiency for Asp-tRNAASp synthesis; the wild-type enzyme shows no activity with tRNAAsn, the mutant is active with tRNAAsn
W26H/K85P
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discriminating AspRS gains the ability to form Asp-tRNAAsn; the wild-type enzyme shows no activity with tRNAAsn, the mutant is active with tRNAAsn
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
denaturation by 4 M urea, 30% recovery after dialysis
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
pharmacology
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the enzyme is the target of peptide nucleotide antibiotic Microcin C
Show AA Sequence (4610 entries)
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