Information on EC 6.1.1.16 - cysteine-tRNA ligase

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

EC NUMBER
COMMENTARY hide
6.1.1.16
-
RECOMMENDED NAME
GeneOntology No.
cysteine-tRNA ligase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-cysteine + tRNACys = AMP + diphosphate + L-cysteinyl-tRNACys
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Aminoacylation
esterification
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Aminoacyl-tRNA biosynthesis
-
-
cysteine metabolism
-
-
tRNA charging
-
-
SYSTEMATIC NAME
IUBMB Comments
L-cysteine:tRNACys ligase (AMP-forming)
-
CAS REGISTRY NUMBER
COMMENTARY hide
37318-56-2
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain 168T, gene cysS
-
-
Manually annotated by BRENDA team
strain 168T, gene cysS
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain NRC-1
-
-
Manually annotated by BRENDA team
gene cysS, canonical enzyme form CysRS
SwissProt
Manually annotated by BRENDA team
no activity in Archaeoglobus fulgidus
-
-
-
Manually annotated by BRENDA team
no activity in Methanococcus jannaschii
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
-
although the nucleotides in tRNA required for aminoacylation are conserved in evolution, bacterial aminoacyl-transfer RNA synthetases are unable to acylate eukaryote-specific tRNA. Whereas Escherichia coli CysRS cannot acylate human tRNACys, the fusion of a eukaryote-specific domain of human CysRS overcomes the cross-species barrier in human tRNACys. In addition to enabling recognition of the sequence differences in the tertiary core of tRNACys, the fused eukaryotic domain redirects the specificity of Escherichia coli CysRS from the A37 present in bacterial tRNACys to the G37 in mammals. The accuracy of codon recognition on the ribosome is also highly sensitive to the A37G transition in tRNACys
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-cysteine + tRNA1Cys
AMP + diphosphate + L-cysteinyl-tRNA1Cys
show the reaction diagram
-
substrate tRNA1Cys isoacceptor 1
-
-
?
ATP + L-cysteine + tRNA1CysA33U
AMP + diphosphate + L-cysteinyl-tRNA1CysA33U
show the reaction diagram
-
substrate tRNA1CysA33U isoacceptor 1, containing a A33U mutation
-
-
?
ATP + L-cysteine + tRNA2Cys
AMP + diphosphate + L-cysteinyl-tRNA2Cys
show the reaction diagram
-
substrate tRNA2Cys isoacceptor 2
-
-
?
ATP + L-cysteine + tRNA3Cys
AMP + diphosphate + L-cysteinyl-tRNA3Cys
show the reaction diagram
-
substrate tRNA3Cys isoacceptor 3
-
-
?
ATP + L-cysteine + tRNA3CysC20U/U21C/A44U/C46A/A47G
AMP + diphosphate + L-cysteinyl-tRNA3CysC20U/U21C/A44U/C46A/A47G
show the reaction diagram
-
substrate tRNA3CysC20U/U21C/A44U/C46A/A47G isoacceptor 3, containing a C20U/U21C/A44U/C46A/A47G mutation
-
-
?
ATP + L-cysteine + tRNA3CysC20U/U21C/A44U/C46A/A47G/G57A
AMP + diphosphate + L-cysteinyl-tRNA3CysC20U/U21C/A44U/C46A/A47G/G57A
show the reaction diagram
-
substrate tRNA3CysC20U/U21C/A44U/C46A/A47G/G57A isoacceptor 3, containing a C20U/U21C/A44U/C46A/A47G/G57A mutation
-
-
?
ATP + L-cysteine + tRNA3CysG57A
AMP + diphosphate + L-cysteinyl-tRNA3CysG57A
show the reaction diagram
-
substrate tRNA3CysG57A isoacceptor 3, containing a G57A mutation
-
-
?
ATP + L-cysteine + tRNA3CysU33A
AMP + diphosphate + L-cysteinyl-tRNA3CysU33A
show the reaction diagram
-
substrate tRNA3CysU33A isoacceptor 3, containing a U33A mutation
-
-
?
ATP + L-cysteine + tRNACys
AMP + diphosphate + L-cysteinyl-tRNACys
show the reaction diagram
ATP + L-cysteine + tRNACys mutant
AMP + diphosphate + L-cysteinyl-tRNACys mutant
show the reaction diagram
-
tRNA substrate is a tRNACys with mutation at the core tertiary Levitt pair from wild-type G15.C48 to mutant G15.G48, leading to a higher activity
-
?
ATP + L-cysteine + tRNACysA36G
AMP + diphosphate + L-cysteinyl-tRNACysA36G
show the reaction diagram
-
relative activity compared to wild-type tRNACys as a substrate: 0.01
-
-
?
ATP + L-cysteine + tRNACysC35U
AMP + diphosphate + L-cysteinyl-tRNACysC35U
show the reaction diagram
-
relative activity compared to wild-type tRNACys as a substrate: 0.005
-
-
?
ATP + L-cysteine + tRNACysG15C/C48G
AMP + diphosphate + L-cysteinyl-tRNACysG15C/C48G
show the reaction diagram
-
relative activity compared to wild-type tRNACys as a substrate: 0.03
-
-
?
ATP + L-cysteine + tRNACysG34C
AMP + diphosphate + L-cysteinyl-tRNACysG34C
show the reaction diagram
-
relative activity compared to wild-type tRNACys as a substrate: 0.001
-
-
?
ATP + L-cysteine + tRNACysU73G
AMP + diphosphate + L-cysteinyl-tRNACysU73G
show the reaction diagram
-
relative activity compared to wild-type tRNACys as a substrate: 0.00002
-
-
?
ATP + L-cysteine + tRNAGln duoble-mutant
AMP + diphosphate + L-cysteinyl-tRNAGln double-mutant
show the reaction diagram
-
tRNA substrate is a tRNAGln with introduced tRNACys indentitiy nucleotides at the acceptor and anticodon ends and a core tertiary Levitt pair equivalent to tRNAGln of G15.G48, poor activity
-
?
ATP + L-cysteine + tRNAGln mutant
AMP + diphosphate + L-cysteinyl-tRNAGln mutant
show the reaction diagram
-
tRNA substrate is a tRNAGln with introduced tRNACys indentitiy nucleotides at the acceptor and anticodon ends and a core tertiary Levitt pair equivalent to tRNACys of G15.C48
-
?
L-Cysteinyl-tRNACys
Cysteine thiolactone + ?
show the reaction diagram
-
deacylation in which nucleophilic sulfur of the side chain of cysteine in Cys-tRNACys attacks its carboxyl carbon, synthesis of Cys-tRNACys and cyclization of cysteine to thiolactone occur in a single active site
-
-
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-cysteine + tRNACys
AMP + diphosphate + L-cysteinyl-tRNACys
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
15% of the efficiency of Mg2+ in activation of ATP-diphosphate exchange
KCl
-
required, optimal cocentration is 150 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-deoxy-ATP
-
-
2-L-aminobutyric acid
-
-
5'-O-[N-(L-cysteinyl)sulfamoyl] adenosine
-
i.e. Cys-AMS, a cysteinyl adenylate analogue
alpha,beta-CH2-ATP
-
competitive
AMP
-
noncompetitive
Ba2+
-
10 mM, 43% inhibition in presence of 10 mM Mg2+, no activation in absence of Mg2+
Ca2+
-
10 mM, 28% inhibition in presence of 10 mM Mg2+, no activation in absence of Mg2+
Co2+
-
10 mM, complete inhibition in presence of 10 mM Mg2+, no activation in absence of Mg2+
Cu2+
-
10 mM, complete inhibition in presence of 10 mM Mg2+, no activation in absence of Mg2+
Cys-AMP
-
inhibits deacylation of Cys-tRNACys
-
cysteamine
cysteine
D-Cysteine
diphosphate
-
at high concentrations
glutathione
GTP
-
noncompetitive
Hg2+
-
10 mM, complete inhibition in presence of 10 mM Mg2+, no activation in absence of Mg2+
iodoacetamide
L-cysteic acid
-
-
L-homocysteine
L-selenocysteine
-
-
Mg2+
-
free Mg2+ inhibits
O-acetylserine
p-chloromercuribenzoate
phenylhydrazine
S-methyl-L-cysteine
-
competitive
Sodium tripolyphosphate
-
-
Sulfhydryl group reagents
-
-
-
Zn2+
-
10 mM, complete inhibition in presence of 10 mM Mg2+, no activation in absence of Mg2+
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
methionine
-
stimulates
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
10
2-aminobutyric acid
0.057 - 1.8
ATP
0.03 - 0.17
Cys
0.16 - 1
diphosphate
0.0125
L-Cys
-
ATP-diphosphate exchange
0.00047 - 3.61
L-cysteine
0.00159
L-selenocysteine
-
ATP-diphosphate exchange
0.05 - 0.063
selenocysteine
0.08
tRNA1Cys
-
value above, substrate tRNA1Cys isoacceptor 1
-
0.09
tRNA1CysA33U
-
value above, substrate tRNA1Cys isoacceptor 1, containing a A33U mutation
-
0.16
tRNA2Cys
-
value above, substrate tRNA2Cys isoacceptor 2
-
0.14
tRNA3Cys
-
value above, substrate tRNA3Cys isoacceptor 3
-
0.11
tRNA3CysC20U/U21C/A44U/C46A/A47G
-
value above, substrate tRNA3Cys isoacceptor 3, containing a C20U/U21C/A44U/C46A/A47G mutation
-
0.09
tRNA3CysC20U/U21C/A44U/C46A/A47G/G57A
-
value above, substrate tRNA3Cys isoacceptor 3, containing a C20U/U21C/A44U/C46A/A47G/G57A mutation
-
0.12
tRNA3CysG57A
-
value above, substrate tRNA3Cys isoacceptor 3, containing a G57A mutation
-
0.13
tRNA3CysU33A
-
value above, substrate tRNA3Cys isoacceptor 3, containing a U33A mutation
-
0.00035 - 0.095
tRNACys
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.3 - 142
ATP
0.001 - 100
L-cysteine
0.017
thiolactone formation
Escherichia coli
-
-
-
0.17
tRNA1Cys
Methanosarcina mazei
-
value above, substrate tRNA1Cys isoacceptor 1
-
0.16
tRNA1CysA33U
Methanosarcina mazei
-
value above, substrate tRNA1Cys isoacceptor 1, containing a A33U mutation
-
0.06
tRNA2Cys
Methanosarcina mazei
-
value above, substrate tRNA2Cys isoacceptor 2
-
0.03
tRNA3Cys
Methanosarcina mazei
-
value above, substrate tRNA3Cys isoacceptor 3
-
0.14
tRNA3CysC20U/U21C/A44U/C46A/A47G
Methanosarcina mazei
-
value above, substrate tRNA3Cys isoacceptor 3, containing a C20U/U21C/A44U/C46A/A47G mutation
-
0.18
tRNA3CysC20U/U21C/A44U/C46A/A47G/G57A
Methanosarcina mazei
-
value above, substrate tRNA3Cys isoacceptor 3, containing a C20U/U21C/A44U/C46A/A47G/G57A mutation
-
0.09
tRNA3CysG57A
Methanosarcina mazei
-
value above, substrate tRNA3Cys isoacceptor 3, containing a G57A mutation
-
0.05
tRNA3CysU33A
Methanosarcina mazei
-
value above, substrate tRNA3Cys isoacceptor 3, containing a U33A mutation
-
0.005 - 6
tRNACys
additional information
additional information
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3 - 1000
L-cysteine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.22
-
purified enzyme
1.2
-
enzyme form CRS-1
3.29
-
enzyme form CRS-2
6.25
-
enzyme form CRS-3
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
-
-
8
-
ATP-diphosphate exchange
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9
-
pH 7.0: about 30% of maximal activity, pH 9.0: about 50% of maximal activity, ATP-diphosphate exchange
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
cultured; from patients with cystinosis
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Borrelia burgdorferi (strain ATCC 35210 / B31 / CIP 102532 / DSM 4680)
Coxiella burnetii (strain RSA 493 / Nine Mile phase I)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42320
-
predicted from cDNA
54000
-
gel filtration
61000
-
gel filtration
82000
-
gel filtration, mitochondrial enzyme
122000
-
gel filtration, enzyme form, CRS-1
127000
-
sucrose density gradient centrifugation, enzyme form CRS-2
230000 - 240000
-
gel filtration, PAGE on gels of various porosities, enzyme form CRS-2
262000
-
gel filtration, cytoplasmic enzyme
270000 - 300000
-
gel filtration, enzyme form CRS-3
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, crystals of enzyme bound to tRNACys at a resolution of 2.3 A
-
purified enzyme complexed with ATP and cysteine, hanging-drop vapour diffusion from ammonium sulfate precipitant containing solution, X-ray diffraction structure analysis at 2.7 A resolution
-
purified recombinant free enzyme or cysteine and ATP complexed enzyme, hanging-drop vapour diffusion method, 17C, 0.001 ml protein solution: 6 mg/ml enzyme, 10 mM HEPES, pH 7.4, 50 mM NaCl, 1 mM DTT, 5 mM MgCl2, 5 mM ATP, 10 mM cysteine, plus equal volume of reservoir solution: 0.1 M sodium cacodylate, pH 6.5, 15-17% PEG 8000, 0.2 M magnesium acetate, 2% tert-butanol, 1-3 weeks, cysteine but not ATP is required for crystal growth, X-ray diffraction structure determination at 2.3-3.0 A resolution, and analysis
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
-
37C, in presence of 10% glycerol, stable for at least 15 min
184
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
pH 6-9, in presence of 10% glycerol, stable for at least 15 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
instability of the enzyme without the halophilic-specific peptide
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 50% glycerol
-
-20C, 50% glycerol, 1 mM DTE, at least 6 months, no loss of activity
-
4C, 50% glycerol, 0.5 mM DTT, 3 mM L-cysteine
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
8300fold from liver, 2 different forms, to homogeneity
-
multiple molecular forms: CRS-1, CRS-2, CRS-3
-
partial
-
recombinant C-terminally His-tagged wild-type and mutant enzymes from Escherichia coli to homogeneity
-
recombinant from overexpressing strain JM109
-
recombinant His-tagged wild-type enzyme and halophilic-specific peptide mutant enzyme variants from Escherichia coli strain BL21(DE3)
-
Talon resin column chromatography
-
using Ni-NTA chromatographay
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed as a His-tagged fusion protein in Escherichia coli
-
expressed in Escherichia coli BL21(DE3) cells
-
expression of His-tagged wild-type enzyme and halophilic-specific peptide mutant enzyme variants in Escherichia coli strain BL21(DE3)
-
gene cysS, stable expression of C-terminally His-tagged wild-type and mutant enzymes in Escherichia coli
-
overexpression in strain JM109
-
overexpression of the wild-type enzyme in Escherichia coli BL21(DE3) as N-terminally HIs-tagged protein
the gene encoding the enzyme is expressed as a polycistronic gltX-cysE-cysS transcript, which is processed to a cysE-cysS transcript, regulatory implications
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C209S
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 0.0031% of the wild-type ratio
C28S
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 0.012% of the wild-type ratio
C28S/C209S
C28S/C209S/H234N/E238Q
C36S/C214S/C244S
-
site-directed mutagenesis, activity is similar to the wild-type enzyme, but the affinity for cysteine binding is increased
DELTA288-461
-
the ratio of turnover number to Km-value for ATP in ATP-diphosphate exchange is 7% of the wild-type ratio
DELTA328-461
-
the ratio of turnover number to Km-value for ATP in ATP-diphosphate exchange is 0.32% of the wild-type ratio, aminoacylation of tRNACys is not detectable
E354Q/R427A
-
the mutant has a decreased kcat and an increased Km leading to an overall decrease in kcat/Km by 22fold relative to the wild type enzyme
H206S
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 60% of the wild-type ratio
H224N/H235N
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 0.042% of the wild-type ratio
H224S
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 5.7% of the wild-type ratio
H234N/E238Q
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 0.0059% of the wild-type ratio
H234N/E238Q/H224N/H235N
-
aminoacylation of tRNACys is not detectable
H234S
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 0.015% of the wild-type ratio
H235S
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 0.38% of the wild-type ratio
H238S
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 30% of the wild-type ratio
H256S
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 10% of the wild-type ratio
H297A
-
the mutant has a decreased kcat and Km values leading to an overall decrease in kcat/Km by 3.4fold relative to the wild type enzyme
H404A/R42A
-
the mutant has a decreased kcat and an increased Km leading to an overall decrease in kcat/Km by 209fold relative to the wild type enzyme
H40A
-
the mutant has a decreased kcat and an increased Km leading to an overall decrease in kcat/Km by 204fold relative to the wild type enzyme
M294A
-
the mutant has decreased kcat and Km values leading to an overall decrease in kcat/Km by 3.7fold relative to the wild type enzyme
M294A/H297A
-
the mutant has a decreased kcat and Km values leading to an overall decrease in kcat/Km by 8.1fold relative to the wild type enzyme
M294A/R427A
-
the mutant has a decreased kcat and an increased Km leading to an overall decrease in kcat/Km by 370fold relative to the wild type enzyme
N351D
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 0.25% of the wild-type ratio
R427A
-
the mutant has a decreased kcat and an increased Km leading to an overall decrease in kcat/Km by 291fold relative to the wild type enzyme
R42A
-
the mutant has a decreased kcat and an increased Km leading to an overall decrease in kcat/Km by 1.5fold relative to the wild type enzyme
V27E
-
mutation does not affect the discrimination of the enzyme for serine. 4fold increase in Km-value for cysteine and 9fold reduction of turnover number for ATP
W205F
-
the ratio of turnover number to Km-value of aminoacylation of tRNACys is 0.55% of the wild-type ratio
W205Y
-
site-directed mutagenesis, highly reduced activity, highly increased Km for cysteine
D239A/D240A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
D417A/E420A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
D435A/D436A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
in order to evaluate genetic associations between candidate genes involved in oxidative stress and multiple system atrophy (MSA) 119 Japanese patients with MSA and 123 controls are examined and single-nucleotide polymorphisms are genotyped. Results revealed genetic associations of cysteinyl-tRNA synthetase, solute carrier family 1A4, sequestosome 1, and eukaryotic translation initiation factor 4E-binding protein 1 with MSA
molecular biology
-
Mycoplasmas hyopneumoniae are collected from bronchial alveolar lavage samples of infected pigs 28 days postinfection and compared to broth-grown cells using microarrays. During lung infection, the analysis indicates that 79 genes are differentially expressed. Of the down-regulated genes, 28 of 46 (61%) lacked an assigned function, in comparison to 21 of 33 (63%) of up-regulated genes. Cysteinyl-tRNA synthetase is down-regulated in vivo
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