Information on EC 6.1.1.22 - asparagine-tRNA ligase

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

EC NUMBER
COMMENTARY hide
6.1.1.22
-
RECOMMENDED NAME
GeneOntology No.
asparagine-tRNA ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-asparagine + tRNAAsn = AMP + diphosphate + L-asparaginyl-tRNAAsn
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
-
-
aspartate and asparagine metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
L-asparagine:tRNAAsn ligase (AMP-forming)
-
CAS REGISTRY NUMBER
COMMENTARY hide
37211-76-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
wild-type and temperature-sensitive mutant strain lys65a
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
-
asparaginyl-tRNA synthetase is a member of the class-II aminoacyl-tRNA synthetases, and is responsible for catalyzing the specific aminoacylation of tRNAAsn with asparagine
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 + L-asparagine + tRNAAsn
AMP + diphosphate + L-asparaginyl-tRNAAsn
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-asparagine + tRNAAsn
AMP + diphosphate + L-asparaginyl-tRNAAsn
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
stimulates tRNA esterification with 5% of the efficiency compared to Mg2+
Co2+
-
stimulates ATP-diphosphate exchange with 17% the efficiency compared to Mg2+ and tRNA esterification with 45%
Mn2+
-
stimulates ATP-diphosphate exchange with 30% the efficiency compared to Mg2+ and tRNA esterification with 72% efficiency
Ni2+
-
stimulates tRNA esterification with 15% of the efficiency compared to Mg2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-cyanoalanine
-
aminoacylation of Asn, competitive
3D6
monoclonal antibody
-
5'-O-[N-(L-asparaginyl)sulfamoyl]adenosine
a non-hydrolysable analogue of asparaginyl adenylate, i.e. Asn-SA
Asn-S-adenylate
-
asparaginyl sulfamoyl adenylate
Aspartate 3-hydroxamate
-
Asn-dependent ATP-diphosphate exchange, competitive
Br-
-
1.2 M, 50% inhibition of ATP-diphosphate exchange
CH3COO-
-
1.3 M, 50% inhibition of ATP-diphosphate exchange
Cl-
-
2.5 M, 50% inhibition of ATP-diphosphate exchange
CNS-
-
0.4 M, 50% inhibition of ATP-diphosphate exchange
Cs+
-
1.3 M, 50% inhibition of ATP-diphosphate exchange
cycloadenosine
-
-
I-
-
0.5 M, 50% inhibition of ATP-diphosphate exchange
isoasparagine
-
aminoacylation of Asn, competitive
K+
-
2.5 M, 50% inhibition of ATP-diphosphate exchange
L-aspartate-beta-hydroxamate adenylate
-
i.e. LBHAMP
LCM01
LCM02
Li+
-
0.7 M, 50% inhibition of ATP-diphosphate exchange
Na+
-
2.0 M, 50% inhibition of ATP-diphosphate exchange
NH4+
-
0.9 M, 50% inhibition of ATP-diphosphate exchange
NO3-
-
1.0 M, 50% inhibition of ATP-diphosphate exchange
NSC114691
NSC12156
NSC35467
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i.e. 2-(3-methyl-1 lambda5-pyridin-1-yl)-1-(2-phenanthryl)ethanone, 53% inhibition at 0.2 mM
NSC363624
p-chloromercuribenzoate
-
-
rishirilide B
S-methyl-deoxyvariolin B
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i.e. SMEVAR
threo-3-Hydroxyasparagine
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aminoacylation of Asn, competitive
tirandamycin A
-
selective inhibitor
tirandamycin B
-
selective inhibitor
tirandamycin E
-
selective inhibitor
tirandamycin F
-
selective inhibitor
tirandamycin G
-
selective inhibitor
Tris
-
above 0.3 M
variolin B
-
-
additional information
-
no inhibition by NSC35467 at 0.2 mM
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ethanol
-
slight stimulation at low tRNA concentrations
additional information
-
the larger component of MW 80000-90000 requires the addition of an activator for enzyme transfer activity
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.8
3-cyanoalanine
-
ATP-diphosphate exchange
0.015 - 0.77
Asn
0.78
aspartate-3-hydroxamate
-
ATP-diphosphate exchange
0.0306 - 7
ATP
0.26
isoasparagine
-
ATP-diphosphate exchange
0.04
L-asparagine
-
in 50 mM HEPES-NaOH, pH 7.2, 10 mM MgCl2, at 37°C
0.00006 - 0.002
tRNAAsn
additional information
additional information
-
binding kinetics, affinities, and thermidynamics, molecular dynamics simulations, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.7 - 3
Asn
0.3 - 10.1
ATP
4.2
L-asparagine
Plasmodium falciparum
-
in 50 mM HEPES-NaOH, pH 7.2, 10 mM MgCl2, at 37°C
0.5
tRNAAsn
Plasmodium falciparum
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in 50 mM HEPES-NaOH, pH 7.2, 10 mM MgCl2, at 37°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
13.6
ATP
Plasmodium falciparum
-
in 50 mM HEPES-NaOH, pH 7.2, 10 mM MgCl2, at 37°C
4
105
L-asparagine
Plasmodium falciparum
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in 50 mM HEPES-NaOH, pH 7.2, 10 mM MgCl2, at 37°C
266
4.2
tRNAAsn
Plasmodium falciparum
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in 50 mM HEPES-NaOH, pH 7.2, 10 mM MgCl2, at 37°C
1690
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0017 - 0.0045
asparaginyl sulfamoyl adenylate
0.004
L-aspartate-beta-hydroxamate adenylate
Brugia malayi
-
-
0.173 - 0.5
LCM01
0.123 - 1
LCM02
0.05 - 0.065
NSC114691
0.025
NSC363624
Brugia malayi
-
-
0.24
rishirilide B
Brugia malayi
-
-
0.03
tirandamycin G
Brugia malayi
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pH and temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.287
-
-
0.472
-
-
additional information
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.8
-
ATP-diphosphate exchange, tRNA esterification, Tris-chloride or Tris-acetate buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8
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25 C, the aminoacylation rate increases as the pH is raised from 6.0 to 8.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
wild-type
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8
-
isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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female and male adult worms
Manually annotated by BRENDA team
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intrauterine
Manually annotated by BRENDA team
-
primary
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50000
-
x * 50875, calculation from nucleotide sequence, x * 50000, SDS-PAGE
50875
-
x * 50875, calculation from nucleotide sequence, x * 50000, SDS-PAGE
51000
-
2 * 51000, sedimentation equilibrium measurement under denaturing conditions
65000
-
x * 65000, SDS-PAGE
120000
-
calculation from gel filtration and sedimentation velocity
127000
152000
-
wild-type and mutant enzyme, sucrose density gradient centrifugation
155000
-
gel filtration
175000
-
cytoplasmic enzyme, dissociated activity arises from high molecular weight aminoacyl-tRNA synthetase complex, gel filtration
189000
-
mitochondrial enzyme, dissociated activity arises from high molecular weight aminoacyl-tRNA synthetase complex, gel filtration
295000
-
dynamic light scattering
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
identification of peptidyl regions that are surface-accessible and available for antibody binding
purified apo-AsnRS, AsnRS in complex with AsnAMS, and in complex with Mg2+, ATP, and l-Asp-beta-NOH, X-ray diffraction structure determination and analysis at 1.9-2.4 A resolution
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purified dimeric enzyme with bound Mg2+ and a non-hydrolyzable analogue of asparaginyl adenylate, ASNAMS, X-ray diffraction structure determination and analysis at 1.9 A resolution, modeling
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sitting drop vapor diffusion method, using 100 mM Tris-HCl (pH 7.0), 0.2 M NaCl, and 32% (w/v) polyethylene glycol 3350
AsnRS complexed with asparaginyl-adenylate Asn-AMP, or free AsnRS and AsnRS complexed with an Asn-AMP analog Asn-SA, sitting-drop vapor diffusion method at 20°C, 0.001 ml protein solution is mixed with an equal volume of reservoir solution containing 25 mM sodium cacodylate buffer, pH 6.5, 50 mM sodium acetate trihydrate, and 7.5% w/v PEG 8000, equilibration against 0.5 ml reservoir solution, for ligand complex formation the crystals are soaked in a reservoir solution, containing 1.5 mM AMP-PNP, 1.5 mM asparagine, and 2 mM MgCl2, for approximately 10 h, followed by a reservoir solution containing 25% 2-methyl-2,4-pentanediol for a few seconds, X-ray diffraction strucure determination and analysis at 1.45 A, and 1.98 A and 1.8 A resolution, respectively
AsnRS with bound asparaginyladenylate, AsnAMP, X-ray diffraction structure anaylsis at 2.6 A resolution
-
recombinant enzyme, native or complexed withATP and asparaginyl-adenylate, 3 different crystal forms, X-ray diffraction structure determination at 2.6 A resolution and analysis
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
wild-type enzyme is stable, mutant enzyme is irreversibly inactivated, half-life: 4.8 min
42
-
very unstable at
50
-
50% loss of activity after 5 min (without addition of stabilizer), after 14 min (with addition of 0.004 mM ATP), after 27 min (with addition of 0.05 mM Asn and ATP)
60
-
3 min, stable, in presence of all substrates except amino acid
70
-
0.5 min, stable, in presence of all substrates except amino acid
85
-
21 h, retains 50% of its activity
90
-
1 h, retains 50% of its activity
additional information
-
ATP and Mg2+ stabilize against thermal inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
ATP and Mg2+ stabilize against thermal inactivation
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 40% w/v glycerol, 2 months with less than 10% loss of activity
-
4°C, 15% w/v glycerol, 48 h, stable
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
as a fusion protein with maltose binding protein
-
Ni-NTA resin column chromatography
-
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange and hydroxyapatite chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA sequence determination and analysis, functional expression in Escherichia coli
DNA sequence determination and analysis, multicopy gene, present in 5 gene clusters, the most abundant of all aminoacyl-tRNA synthetases in Brugia malayi
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DNA sequence determination and phylogenetic analysis
expressed in Escherichia coli Top10 cells
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expression in Escherichia coli
NARS, transient transfection of MC3T3-E1 cells and primary mouse calvarial cells
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overexpression as a fusion protein with maltose binding protein
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overexpression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
overproduced in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
engineering of aspartyladenylate, AspAMP, specificity into asparaginyl-tRNA synthetase, AsnRS, whose substrate is asparaginyl-adenylate by CPD method, that uses a polar-hydrogen energy function for protein interactions and a coulomb/accessible surface area model for solvent effects, method development, overview. Application of an all-atom energy for protein interactions and a residue-pairwise generalized Born model for solvent effects, overview. The conformations and interactions are well maintained in molecular dynamics simulations and the sequences have an inverted specificity, favoring AspAMP over AsnAMP
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fibroblast growth factor 2, FGF2, that functions in intracellular signaling in bone development, induces the enzyme in MC3T3-E1 preosteoblast cells more than 900fold, the overexpression of NARS significantly increases the proliferation of both the MC3T3-E1 and the primary mouse calvarial cells, overview
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L65A
-
the mutant strain lys65a produces an enzyme with greater temperature lability in vitro, a higher temperature-independent Km for Asn, and a lower temperature-dependent catalytic capacity than the enzyme from the wild-type strain
P231L
-
temperature-sensitive mutant HO202, P231L replacement leads to a change in aminoacylation activity. 50fold increased Km for Asn compared to the wild-type, and 8fold increase for ATP in ATP-diphosphate exchange. Significantly increased Km-values for Asn and ATP in aminoacylation. Pro-231 does not seem to be implicated directly in substrate binding. Instead it seems to have a structural role on the positioning of the loop formed by the motif 2
Y426F
-
wild-type and mutant enzymes Y426F and Y426S. Y426S replacement leads to a 15fold increase in Km for ATP, while all the other kinetic parameters remain unchanged. Y426F replacement does not affect the kinetic behaviour of the enzyme
Y426S
-
wild-type and mutant enzymes Y426F and Y426S. Y426S replacement leads to a 15fold increase in Km for ATP, while all the other kinetic parameters remain unchanged. Y426F replacement does not affect the kinetic behaviour of the enzyme
P231L
-
temperature-sensitive mutant HO202, P231L replacement leads to a change in aminoacylation activity. 50fold increased Km for Asn compared to the wild-type, and 8fold increase for ATP in ATP-diphosphate exchange. Significantly increased Km-values for Asn and ATP in aminoacylation. Pro-231 does not seem to be implicated directly in substrate binding. Instead it seems to have a structural role on the positioning of the loop formed by the motif 2
-
R83A
site-directed mutagenesis, active site amino acid binding mutant, structure analysis in comparison to the wild-type enzyme
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
concentrations of urea above 4 M inactivate during incubation at 37°C for 5 min. 70% of the activity is recovered by dialysis
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
-
Brugia malayi asparaginyl-tRNA synthetase, a target for anti-parasitic drug design
medicine
filariasis caused by the nematode, Brugia malayi, is an important worldwide tropical disease in which parasites disappear from the bloodstream during daylight hours, thus hampering standard microscopic diagnostic methods
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