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ATP + hydroxynorvaline + tRNAThr
AMP + diphosphate + hydroxynorvalyl-tRNAThr
10-70% less active than with L-threonine
-
?
ATP + L-serine + tRNAThr
AMP + diphosphate + L-seryl-tRNAThr
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
ATP + 3-hydroxynorvaline + tRNAThr
AMP + diphosphate + 3-hydroxynorvalyl-tRNAThr
-
the specificity constant kcat/KM for beta-hydroxynorvaline is only 20-30fold less than that of cognate threonine, amino acid activation is the potential rate-limiting step of b3-hydroxynorvaline aminoacylation
-
-
?
ATP + L-serine + tRNASer
AMP + diphosphate + L-seryl-tRNASer
-
-
-
-
?
ATP + L-serine + tRNAThr
AMP + diphosphate + L-seryl-tRNAThr
-
very low activity with the wild-type enzyme
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
additional information
?
-
ATP + L-serine + tRNAThr
AMP + diphosphate + L-seryl-tRNAThr
low activity
-
-
r
ATP + L-serine + tRNAThr
AMP + diphosphate + L-seryl-tRNAThr
1000fold less active than with L-threonine
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
r
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
regulatory mechanism
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
via AMP-L-threonine-enzyme intermediate in a two-step process
-
-
r
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
Escherichia coli ectRNAThr
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
-
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
tRNA aminoacylation
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
for cognate threonine, amino acid activation is likely to be the rate-limiting step. The inability of wild-type ThrRS to prevent utilization of beta-hydroxynorvaline as a substrate illustrates that the naturally occurring enzyme lacks the capability to effectively discriminate against nonproteogenic amino acids that are not encountered under normal physiological conditions
-
-
?
ATP + L-threonine + tRNAThr
AMP + diphosphate + L-threonyl-tRNAThr
-
tRNAThr3 of Escherichia coli
-
-
?
additional information
?
-
no activity with L-valine, determination of amino acid activation and discriminating editing mechanism
-
?
additional information
?
-
the enzyme represses the translation of its own mRNA by binding to an operator located upstream of the initiation codon thereby using the recognition mode of the tRNA anticodon loop to initiate binding
-
?
additional information
?
-
-
the enzyme represses the translation of its own mRNA by binding to an operator located upstream of the initiation codon thereby using the recognition mode of the tRNA anticodon loop to initiate binding
-
?
additional information
?
-
the enzyme needs to discriminate between threonine, serine, and valine in vivo, mechanism of proofreading of threonyl-tRNA synthetase at atomic resolution, overview
-
-
?
additional information
?
-
the interaction of ecRNAThr with the enzyme, interactions between the catalytically important loops and tRNA contribute to the change in dynamics of tRNA in free and bound states, respectively. Presence of bound Mg2+ ions around tRNA and dynamically slow bound water are other common features of the enzyme
-
-
?
additional information
?
-
-
enzyme also has a regulatory function by binding the so-called operator site located in the leader of its own mRNA and thereby inhibits translational initiation by competing with ribosome binding
-
?
additional information
?
-
-
regulation mechanism, 2 essential steps of regulation are operator recognition and inhibition of ribosome binding performed by different domains of the enzyme
-
?
additional information
?
-
-
in the pre-steady state, asymmetric activation of cognate threonine and noncognate serine is observed in the active sites of dimeric ThrRS, with similar rates of activation. In the absence of tRNA, seryl-adenylate is hydrolyzed 29old faster by the ThrRS catalytic domain than threonyl-adenylate. The rate of seryl transfer to cognate tRNA is only 2fold slower than threonine
-
-
?
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H2O2
oxidation of ThrRS by H2O2 causes editing defects and Ser misincorporation at Thr codons due to oxidation of Cys182, zinc or nickel ions inhibit C182 oxidation by hydrogen peroxide. Reducing the oxidized ThrRS with DTT or sodium arsenite (NaAsO2) recovers the editing activity, cysteine residue C182 is reversibly oxidized
operator mRNA domain 2
-
-
Zn2+
inhibits the editing reaction
(2S,3R)-2,3-diamino-N-(((E)-3-(6-aminopyrimidin-4-yl)-styryl)sulfonyl)butanamide
-
-
(2S,3R)-2-amino-3-hydroxy-N-((3-(1-oxoisoindolin-5-yl)-phenyl)sulfonyl)butanamide
-
-
(2S,3R)-2-amino-3-hydroxy-N-((3-(3-methyl-1H-indazol-5-yl)phenyl)sulfonyl)butanamide
-
-
(2S,3R)-2-amino-3-hydroxy-N-((4-phenoxyphenyl)sulfonyl)-butanamide
-
-
(2S,3R)-2-amino-3-hydroxy-N-methyl-N-((3-(1-oxoisoindolin-5-yl)phenyl)sulfonyl)butanamide
-
-
(2S,3R)-2-amino-N'-(3-(4-amino-2-chloroquinazolin-7-yl)-phenyl)-3-hydroxybutanehydrazide
-
-
(2S,3R)-2-amino-N-(((E)-3-(6-aminopyrimidin-4-yl)styryl)-sulfonyl)-3-hydroxy-4-methylpentanamide
-
-
(2S,3R)-2-amino-N-(((E)-3-(6-aminopyrimidin-4-yl)styryl)-sulfonyl)-3-hydroxybutanamide
-
-
(2S,3R)-2-amino-N-(((E)-3-(6-aminopyrimidin-4-yl)styryl)-sulfonyl)-3-hydroxypentanamide
-
-
(2S,3R)-2-amino-N-((3-(1-amino-3-chloroisoquinolin-6-yl)-phenyl)sulfonyl)-3-hydroxybutanamide
-
-
(2S,3R)-2-amino-N-((3-(1-aminoisoquinolin-6-yl)phenyl)-sulfonyl)-3-hydroxybutanamide
-
-
(2S,3R)-2-amino-N-((3-(2,4-diaminoquinazolin-7-yl)phenyl)-sulfonyl)-3-hydroxybutanamide
-
-
(2S,3R)-2-amino-N-((3-(3-chloro-1H-indazol-5-yl)phenyl)-sulfonyl)-3-hydroxybutanamide
-
-
(2S,3R)-2-amino-N-((3-(4-amino-2-chloroquinazolin-7-yl)-phenyl)sulfonyl)-3-hydroxybutanamide
-
-
(2S,3R)-2-amino-N-((3-(4-amino-2-methylquinazolin-7-yl)-phenyl)sulfonyl)-3-hydroxybutanamide
-
-
(2S,3R)-2-amino-N-((3-(4-aminoquinazolin-7-yl)phenyl)-sulfonyl)-3-hydroxybutanamide
-
-
(2S,3R)-2-amino-N-((7-(6-aminopyrimidin-4-yl)naphthalen-2-yl)sulfonyl)-3-hydroxybutanamide
-
-
(2S,3R)-2-amino-N-(3-(4-amino-2-chloroquinazolin-7-yl)-benzyl)-3-hydroxybutanamide
-
-
(2S,3R)-N-(((E)-3-(6-aminopyrimidin-4-yl)styryl)sulfonyl)-2,3-dihydroxybutanamide
-
-
(2S,3R)-N-((7-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)-2-amino-3-hydroxybutanamide
-
-
(2S,3R)-N-((7-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)naphthalen-2-yl)sulfonyl)-2-amino-3-hydroxybutanamide
-
-
5'-O-[N-(threonyl)-sulfamoyl] adenosine
-
-
hydrogen peroxide
-
oxidizes cysteine182 residue critical for editing, which leads to Ser-tRNAThr formation and protein mistranslation that impaired growth of Escherichia coli. Presence of major heat shock proteases is required to allow cell growth in medium containing serine and hydrogen peroxide, which suggests that the mistranslated proteins are misfolded
tert-butyl((2S,3R)-1-(3-(1H-indazol-5-yl)-benzenesulfonamido)-3-(tert-butoxy)-1-oxobutan-2-yl)-carbamate
-
-
borrelidin
slowly but tight binding, noncompetitive with respect to threonine and ATP, inhibition mechanism via conformational change abolishing the activation of threonine, a unique hydrophobic cluster near the active site contributes to differences in borrelidin inhibition among threonyl-tRNA synthetases of different origin, comparison, overview
borrelidin
is an 18-membered macrolide polyketide produced by several actinomycete bacteria of the Streptomyces spp.. Identification of borrelidin binding site on threonyl-tRNA synthetase, molecular docking, overview. Borrelidin binds the pocket outside but adjacent to the active site of ThrRS, consisting of residues Y313, R363, R375, P424, E458, G459, and K465. Borrelidin may induce the cleft closure, which blocks the release of Thr-AMP and phosphate, to inhibit activity of ThrRS rather than inhibit the binding of ATP and threonine
additional information
development of novel borrelidin derivatives and rational design of structure-based ThrRS inhibitors, overview
-
additional information
-
development of novel borrelidin derivatives and rational design of structure-based ThrRS inhibitors, overview
-
additional information
-
reactive oxygen species cause editing defect and misacylation by WT ThrRS
-
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1.95 - 7
hydroxynorvaline
0.00003 - 0.00043
tRNAThr
0.00003 - 0.897
L-threonine
additional information
additional information
-
1.95
hydroxynorvaline
pH 7.2, 37°C, wild-type enzyme
7
hydroxynorvaline
pH 7.2, 37°C, truncated enzyme DELTAN
81.5
L-serine
pH 7.2, 37°C, wild-type enzyme
142
L-serine
pH 7.2, 37°C, truncated enzyme DELTAN
0.101
L-threonine
pH 7.4, 30°C, recombinant mutant G459D
0.104
L-threonine
pH 7.4, 30°C, recombinant mutant E458D
0.105
L-threonine
pH 7.4, 30°C, recombinant wild-type enzyme
0.108
L-threonine
pH 7.4, 30°C, recombinant mutant P424K
0.11
L-threonine
pH 7.5, 37°C, wild-type enzyme
0.11
L-threonine
pH 7.2, 37°C, wild-type enzyme
0.18
L-threonine
pH 7.2, 37°C, truncated enzyme DELTAN
0.00003
tRNAThr
wild-type enzyme
0.00043
tRNAThr
truncated enzyme core DELTAN
0.267
ATP
-
wild-type enzyme, pH 7.2, 37°C
0.387
ATP
-
mutant enzyme W434Y, pH 7.2, 37°C
0.00003
L-threonine
-
wild-type enzyme complementing the null mutant
0.201
L-threonine
-
wild-type enzyme, pH 7.2, 37°C
0.897
L-threonine
-
mutant enzyme W434Y, pH 7.2, 37°C
0.00005
tRNAThr
-
of E. coli
0.0015
tRNAThr
-
pH 7.2, 37°C, ThrRS, aminoacylation with Thr
additional information
additional information
presteady-state and steady-state kinetic measurement
-
additional information
additional information
-
presteady-state and steady-state kinetic measurement
-
additional information
additional information
enzyme kinetics and stopped-flow fluorescence analysis, Michaelis-Menten steady-state kinetics of recombinant wild-type and mutant enzymes
-
additional information
additional information
-
enzyme kinetics and stopped-flow fluorescence analysis, Michaelis-Menten steady-state kinetics of recombinant wild-type and mutant enzymes
-
additional information
additional information
-
kinetics and kinetic mechanism
-
additional information
additional information
-
mutant enzymes complementing the null mutant
-
additional information
additional information
-
kinetics of diphosphate exchange activities and threonylation of tRNAThr of ThrRS with or without H2O2 treatment, overview
-
additional information
additional information
-
pre-steady-state kinetics, kinetics of ATPase activity in presence of 3-hydroxynorvaline, overview
-
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additional information
additional information
-
21
hydroxynorvaline
pH 7.2, 37°C, truncated enzyme DELTAN
22
hydroxynorvaline
pH 7.2, 37°C, wild-type enzyme
26
L-serine
pH 7.2, 37°C, wild-type enzyme
30
L-serine
pH 7.2, 37°C, truncated enzyme DELTAN
2 - 8
L-threonine
pH 7.4, 30°C, recombinant mutant G459D
3 - 6
L-threonine
pH 7.2, 37°C, wild-type enzyme
30
L-threonine
pH 7.4, 30°C, recombinant mutant P424K
33
L-threonine
pH 7.5, 37°C, wild-type enzyme
34
L-threonine
pH 7.4, 30°C, recombinant mutant E458D
35
L-threonine
pH 7.4, 30°C, recombinant wild-type enzyme
37
L-threonine
pH 7.2, 37°C, truncated enzyme DELTAN
42
ATP
-
mutant enzyme W434Y, pH 7.2, 37°C
90
ATP
-
wild-type enzyme, pH 7.2, 37°C
0.64
L-threonine
-
wild-type enzyme complementing the null mutant
6.08
L-threonine
-
wild-type enzyme complementing the null mutant
30
L-threonine
-
mutant enzyme W434Y, pH 7.2, 37°C
90
L-threonine
-
wild-type enzyme, pH 7.2, 37°C
0.05
tRNAThr
-
pH 7.2, 37°C, ThrRS, aminoacylation with Thr
0.52 - 0.7
tRNAThr
-
tRNAThr of Thermus thermophilus
0.53
tRNAThr
-
tRNAThr of E. coli
additional information
additional information
-
mutant enzymes complementing the null mutant
-
additional information
additional information
-
rate constants for adenylate synthesis by ThrRS in the absence of tRNA, overview
-
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0.0000034 - 0.006
borrelidin
0.05
(2S,3R)-2,3-diamino-N-(((E)-3-(6-aminopyrimidin-4-yl)-styryl)sulfonyl)butanamide
-
Ki above 0.05 mM with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0000077
(2S,3R)-2-amino-3-hydroxy-N-((3-(1-oxoisoindolin-5-yl)-phenyl)sulfonyl)butanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.000127
(2S,3R)-2-amino-3-hydroxy-N-((3-(3-methyl-1H-indazol-5-yl)phenyl)sulfonyl)butanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0365
(2S,3R)-2-amino-3-hydroxy-N-((4-phenoxyphenyl)sulfonyl)-butanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.05
(2S,3R)-2-amino-3-hydroxy-N-methyl-N-((3-(1-oxoisoindolin-5-yl)phenyl)sulfonyl)butanamide
-
Ki above 0.05 mM, with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.000136
(2S,3R)-2-amino-N'-(3-(4-amino-2-chloroquinazolin-7-yl)-phenyl)-3-hydroxybutanehydrazide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0000324
(2S,3R)-2-amino-N-(((E)-3-(6-aminopyrimidin-4-yl)styryl)-sulfonyl)-3-hydroxy-4-methylpentanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0000027
(2S,3R)-2-amino-N-(((E)-3-(6-aminopyrimidin-4-yl)styryl)-sulfonyl)-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0000043
(2S,3R)-2-amino-N-(((E)-3-(6-aminopyrimidin-4-yl)styryl)-sulfonyl)-3-hydroxypentanamide
-
Ki above 0.05 mM, with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.00002
(2S,3R)-2-amino-N-((3-(1-amino-3-chloroisoquinolin-6-yl)-phenyl)sulfonyl)-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.00009
(2S,3R)-2-amino-N-((3-(1-aminoisoquinolin-6-yl)phenyl)-sulfonyl)-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0000107
(2S,3R)-2-amino-N-((3-(2,4-diaminoquinazolin-7-yl)phenyl)-sulfonyl)-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.000052
(2S,3R)-2-amino-N-((3-(3-chloro-1H-indazol-5-yl)phenyl)-sulfonyl)-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0000008 - 0.05
(2S,3R)-2-amino-N-((3-(4-amino-2-chloroquinazolin-7-yl)-phenyl)sulfonyl)-3-hydroxybutanamide
0.0000172
(2S,3R)-2-amino-N-((3-(4-amino-2-methylquinazolin-7-yl)-phenyl)sulfonyl)-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0000029
(2S,3R)-2-amino-N-((3-(4-aminoquinazolin-7-yl)phenyl)-sulfonyl)-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0000027
(2S,3R)-2-amino-N-((7-(6-aminopyrimidin-4-yl)naphthalen-2-yl)sulfonyl)-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.000329
(2S,3R)-2-amino-N-(3-(4-amino-2-chloroquinazolin-7-yl)-benzyl)-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.05
(2S,3R)-N-(((E)-3-(6-aminopyrimidin-4-yl)styryl)sulfonyl)-2,3-dihydroxybutanamide
-
Ki above 0.05 mM, with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0000095
(2S,3R)-N-((7-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,4-dihydroisoquinolin-2(1H)-yl)sulfonyl)-2-amino-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0000024
(2S,3R)-N-((7-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)naphthalen-2-yl)sulfonyl)-2-amino-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.0000131
5'-O-[N-(threonyl)-sulfamoyl] adenosine
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.000182
tert-butyl((2S,3R)-1-(3-(1H-indazol-5-yl)-benzenesulfonamido)-3-(tert-butoxy)-1-oxobutan-2-yl)-carbamate
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
additional information
additional information
-
0.0000034
borrelidin
pH 7.4, 30°C, recombinant wild-type enzyme
0.000004
borrelidin
pH 7.5, 37°C, wild-type enzyme
0.0000191
borrelidin
pH 7.4, 30°C, recombinant mutant E458D
0.0000651
borrelidin
pH 7.4, 30°C, recombinant mutant P424K
0.0001114
borrelidin
pH 7.4, 30°C, recombinant mutant G459D
0.006
borrelidin
pH 7.5, 37°C, mutant L489W
0.0000008
(2S,3R)-2-amino-N-((3-(4-amino-2-chloroquinazolin-7-yl)-phenyl)sulfonyl)-3-hydroxybutanamide
-
with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
0.05
(2S,3R)-2-amino-N-((3-(4-amino-2-chloroquinazolin-7-yl)-phenyl)sulfonyl)-3-hydroxybutanamide
-
Ki above 0.05 mM, with L-serine as substrate, in 60 mM Tris, pH 7.6, 10 mM MgCl2, 20 mM KCl, temperature not specified in the publication
additional information
additional information
-
-
additional information
additional information
-
-
-
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Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
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malfunction
threonyl-tRNA synthetase (ThrRS) misactivates serine and utilizes an editing site cysteine (C182 in Escherichia coli) to hydrolyze Ser-tRNAThr. Hydrogen peroxide oxidizes C182, leading to SertRNAThr production and mistranslation of threonine codons as serine. C182 is oxidized to sulfenic acid by air, hydrogen peroxide, and hypochlorite. Air oxidation increases the Ser-tRNAThr level in the presence of elongation factor Tu. C182 forms a putative metal binding site with three conserved histidine residues (H73, H77, and H186). H73 and H186, but not H77, are critical for activating C182 for oxidation. Zinc or nickel ions inhibit C182 oxidation by hydrogen peroxide. Bacteria may use ThrRS editing to sense the oxidant levels in the environment. C182 oxidation modeling, overview. C182 is directly activated by H73 and H186 rather than by a metal ion. Chronic oxidative stress leads to ThrRS mistranslation in vivo
malfunction
-
reactive oxygen species cause editing defect and misacylation by WT ThrRS. H2O2-induced Ser-tRNAThr formation causes protein mistranslation
physiological function
-
amino acid discrimination does not occur at the aminoacyl transfer step. pre-Transfer hydrolysis contributes to proofreading only when the rate of transfer is slowed significantly. Thus, the relative contributions of pre- and posttransfer editing in ThrRS are subject to modulation by the rate of aminoacyl transfer
evolution
crucial importance of the only absolutely conserved residue within the N1 domain in regulating post-transfer editing activityand editing active sites by mediating an N1-N2 domain interaction in Escherichia coli ThrRS. Translational quality control of various ThrRSs and the role of the N1 domain in translational fidelity, overview
evolution
the enzyme belongs to the class II of aminoacyl-tRNA sythetases
physiological function
aminoacyl-tRNA synthetases maintain the fidelity during protein synthesis by selective activation of cognate amino acids at the aminoacylation site and hydrolysis of misformed aminoacyl-tRNAs at the editing site, crystal structure PDB ID 1TJE
physiological function
correct aminoacyl-tRNA generation is critical for the faithful transduction of genetic information, which is supported by the high levels of amino acid conservation in editing active sites of specific aaRSs across the three domains of life. Enzyme EcThrRS is an editing-capable enzyme, that can remove noncognate Ser. The N1 domain is essential for editing by EcThrRS. The enzyme shows strong tRNA-dependent editing, including the pre- and post-transfer editing of EcThrRS
additional information
molecular dynamics simulation study of the dynamics of the active site organization during charging step of dimeric ThrRS from Escherichia coli (ecThrRS) bound with ectRNAThr. The active site residues of the motif 2 loop approach the proximal bases of tRNA and adenylate by slow diffusive motion (in nanosecond time scale) and make conformational changes of the respective side chains via ultrafast librational motion to develop precise hydrogen bond geometry. Presence of bound Mg2+ ions around tRNA and dynamically slow bound water are other common features of the enzyme. The presence of dynamically rigid zinc ion coordination sphere and bipartite mode of recognition of ectRNAThr are observed. Molecular dynamic simulation, overview
additional information
structure analysis of EcThrRS, functional importance of the Asp46 in the N1 domain and the Tyr173 in the N2 domain
additional information
-
structure analysis of EcThrRS, functional importance of the Asp46 in the N1 domain and the Tyr173 in the N2 domain
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C182A
site-directed mutagenesis, the mutation leads to loss of editing activity and to Ser misacylation to tRNAThr. C182A and C182S mutations reduce the kcat value of editing over 500fold
C182S
site-directed mutagenesis, the mutation leads to loss of editing activity and to Ser misacylation to tRNAThr. C182A and C182S mutations reduce the kcat value of editing over 500fold
D435A
site-directed mutagenesis, mutant shows a similar Ki for inhibitor borrelidin compared to the wild-type enzyme
D46E
site-directed mutagenesis, the mutant has a modest reduction in its aminoacylation activity compared to wild-type, and its post-transfer editing activity is abolished
D46E/H186G
site-directed mutagenesis, the mutant EcThrRS strongly supports growth of a yeast thrS deletion strain (ScDELTAthrS)
D46E/Y173F
site-directed mutagenesis, the mutant EcThrRS supports growth of a yeast thrS deletion strain (ScDELTAthrS)
D46E/Y173H
site-directed mutagenesis, the mutant does not support growth of a yeast thrS deletion strain (ScDELTAthrS)
D46E/Y173K
site-directed mutagenesis, the mutant does not support growth of a yeast thrS deletion strain (ScDELTAthrS)
D46E/Y173R
site-directed mutagenesis, the mutant does not support growth of a yeast thrS deletion strain (ScDELTAthrS)
D46E/Y173S
site-directed mutagenesis, the mutant does not support growth of a yeast thrS deletion strain (ScDELTAthrS)
D46R
site-directed mutagenesis, the mutant has a modest reduction in its aminoacylation activity compared to wild-type, and its post-transfer editing activity is abolished
E458D
site-directed mutagenesis, the sensitivity of the mutant enzyme to borrelidin is reduced markedly compared to wild-type, mutant shows decreased apparent rate constants
G459D
site-directed mutagenesis, the sensitivity of the mutant enzyme to borrelidin is reduced markedly compared to wild-type, mutant shows decreased apparent rate constants
H186A
site-directed mutagenesis, the mutant does not show oxidation of Cys182 by H2O2 and only partially by NaOCl
H186G
site-directed mutagenesis, the mutant EcThrRS strongly supports growth of a yeast thrS deletion strain (ScDELTAthrS)
H309A
site-directed mutagenesis, mutant shows highly increased Ki for inhibitor borrelidin compared to the wild-type enzyme
H337A
site-directed mutagenesis, mutant shows increased Ki for inhibitor borrelidin compared to the wild-type enzyme
H73A
site-directed mutagenesis, the mutant does not show oxidation of Cys182 by H2O2 and NaOCl
H77A
site-directed mutagenesis, the mutant shows oxidation of Cys182 by H2O2 and NaOCl
K136A
site-directed mutagenesis, the mutant EcThrRS supports growth of a yeast thrS deletion strain (ScDELTAthrS)
K136E
site-directed mutagenesis, the mutant does not support growth of a yeast thrS deletion strain (ScDELTAthrS)
K136R
site-directed mutagenesis, the mutant EcThrRS supports growth of a yeast thrS deletion strain (ScDELTAthrS)
L489W
site-directed mutagenesis, mutant has a reduced space of the hydrophobic cluster near the active site resulting in a 1500fold increase in Ki for inhibitor borrelidin compared to the wild-type enzyme
P296A
site-directed mutagenesis, mutant shows a similar Ki for inhibitor borrelidin compared to the wild-type enzyme
P296S
site-directed mutagenesis, mutant shows slightly increased Ki for inhibitor borrelidin compared to the wild-type enzyme
P335A
site-directed mutagenesis, mutant shows increased Ki for inhibitor borrelidin compared to the wild-type enzyme
P424K
site-directed mutagenesis, the sensitivity of the mutant enzyme to borrelidin is reduced markedly compared to wild-type, the mutant shows decreased apparent rate constants
P464A
site-directed mutagenesis, mutant shows a similar Ki for inhibitor borrelidin compared to the wild-type enzyme
R282A
site-directed mutagenesis, mutant shows a similar Ki for inhibitor borrelidin compared to the wild-type enzyme
S429A
site-directed mutagenesis, mutant shows a similar Ki for inhibitor borrelidin compared to the wild-type enzyme
T307A
site-directed mutagenesis, mutant shows increased Ki for inhibitor borrelidin compared to the wild-type enzyme
Y173D
site-directed mutagenesis, the mutant has a modest reduction in its aminoacylation activity compared to wild-type, and its post-transfer editing activity is abolished
Y173R
site-directed mutagenesis, the mutant has a modest reduction in its aminoacylation activity compared to wild-type, and its post-transfer editing activity is abolished
Y313A
site-directed mutagenesis, mutant shows a similar Ki for inhibitor borrelidin compared to the wild-type enzyme
D180A
-
charging of tRNAThr with serine, mutant is no longer able to rapidly deacetylate Ser-tRNAThr
D549A
-
modified interation of anticodon loop/C-ter domain, activity similar to the wild-type
E258K
-
modified interation with the superrepressor, no activity
E259K
-
modified interation with the superrepressor, unaltered activity
E600A
-
modified interation of anticodon loop/C-ter domain, 710fold increased activity
H73A
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, and a 2fold higher rate of ATP consumption relative to the rate of Ser-tRNAThr synthesis
H73A/H309A
-
site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme, and a 2fold higher rate of ATP consumption relative to the rate of Ser-tRNAThr synthesis
H73A/H77A
-
charging of tRNAThr with serine, mutant is no longer able to deacetylate Ser-tRNAThr
K246A
-
modified interation of acceptor stem and catalytic domain, 2.9fold increased activity
K249A
-
modified interation of acceptor stem and catalytic domain, 3.5fold increased activity
K577A
-
modified interation of anticodon loop/C-ter domain, 118fold increased activity
N324A
-
modified interation of cross-subunit contacts, 3.5fold increased activity
N502A
-
modified interation of cross-subunit contacts, 2.1fold increased activity
N575A
-
modified interation of anticodon loop/C-ter domain, 9.4fold increased activity
R349A
-
modified interation of cross-subunit contacts, 42fold increased activity
R583H
-
modified interation of anticodon loop/C-ter domain, no activity
R609A
-
modified interation of anticodon loop/C-ter domain, 35fold activity
S347A
-
modified interation of cross-subunit contacts, similar to the wild-type
S367A
-
modified interation of acceptor stem and catalytic domain, 11fold increased activity
W434Y
-
reduced activity, Trp434 is involved in conformational changes during substrate binding
Y205F
-
modified interation of acceptor stem and N-terminal domain, 7.7fold increased activity
Y219F
-
modified interation of acceptor stem and N-terminal domain, similar to the wild-type
Y348F
-
modified interation of cross-subunit contacts, 6.5fold increased activity
additional information
construction of a truncated enzyme: core domain DELTAN comprising residues 242-642
additional information
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construction of a truncated enzyme: core domain DELTAN comprising residues 242-642
additional information
truncated lamdaN-enzyme mutant, lacking the N-terminal domains N1 and N2, produces Ser-tRNAThr, reduced activity and altered substrate recognition compared to the wild-type which does nearly not incorporate serine
additional information
the mutant EcThrRS-DELTAN1 lacking domain N1 shows no post-transfer editing activity
additional information
-
the mutant EcThrRS-DELTAN1 lacking domain N1 shows no post-transfer editing activity
additional information
-
borrelidin-resistant mutant
additional information
-
construction of chromosomal disruption null mutant strain with no activity, construction of a truncated mutant lacking the N1 and N2 domains, 93.5fold increased activity
additional information
-
truncated lamdaN-enzyme mutant, lacking the N-terminal domains N1 and N2, produces Ser-tRNAThr, reduced activity and altered substrate recognition compared to the wild-type which does nearly not incorporate serine
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