Information on EC 1.13.11.20 - cysteine dioxygenase

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

EC NUMBER
COMMENTARY hide
1.13.11.20
-
RECOMMENDED NAME
GeneOntology No.
cysteine dioxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-cysteine + O2 = 3-sulfinoalanine
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dioxygenation
-
-
oxidation
-
-
-
-
redox reaction
-
-
-
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reduction
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
L-cysteine degradation I
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taurine biosynthesis I
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cysteine metabolism
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Cysteine and methionine metabolism
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Taurine and hypotaurine metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
L-cysteine:oxygen oxidoreductase
Requires Fe2+ and NAD(P)H.
CAS REGISTRY NUMBER
COMMENTARY hide
37256-59-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
BC2617 has 13.0% sequence identity with rat CDO, calculated by a BLASTP alignment using VEctor NTI; BC2617
Swissprot
Manually annotated by BRENDA team
BC2617 has 13.0% sequence identity with rat CDO, calculated by a BLASTP alignment using VEctor NTI; BC2617
Swissprot
Manually annotated by BRENDA team
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Swissprot
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
Cupriavidus necator H16 / ATCC 23440 / NCIB 10442 / S-10-1
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UniProt
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
no activity in Nostoc sp.
possesses no detectable CDO activity
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-
Manually annotated by BRENDA team
possesses no detectable CDO activity
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-
Manually annotated by BRENDA team
SCO3035 has 20.6% sequence identity with rat CDO, calculated by a BLASTP alignment using VEctor NTI; A3(2) SCO3035
Swissprot
Manually annotated by BRENDA team
SCO5772 has 16.4% sequence identity with rat CDO, calculated by a BLASTP alignment using Vector NTI; A3(2) SCO5772
Swissprot
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
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in mammals, excess cysteine is generally degraded by oxygenation to 3-sulfino-L-alanine. The majority of cysteine sulphinic acid is then deaminated to sulphinylpyruvate, which decomposes spontaneously byreleasing inorganic sulphite. The latter compound is then further oxidized to sulphate, which is excreted for the most part from the cell. In parallel, a variable proportion of cysteine sulphinic acid is decarboxylated to hypotaurine, then further oxidized to taurine. Although cysteine can be catabolized by some non-oxidative pathways, they are of minor importance. CDO activity is regulated by concentration of cysteine, and in mammals, both have been demonstrated to be important vital factors
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-mercaptopropionate + O2
3-sulfinopropanoate
show the reaction diagram
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-
-
-
?
3-mercaptopropionate + O2
3-sulfinopropionate
show the reaction diagram
beta-mercaptoethanol + O2
2-hydroxyethanesulfinate
show the reaction diagram
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slight activity
-
?
cysteamine + O2
?
show the reaction diagram
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-
-
-
?
L-cysteine + O2
3-sulfino-L-alanine
show the reaction diagram
L-cysteine + O2
3-sulfinoalanine
show the reaction diagram
additional information
?
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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
L-cysteine + O2
3-sulfino-L-alanine
show the reaction diagram
L-cysteine + O2
3-sulfinoalanine
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cys-Tyr cofactor
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FAD
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1 mol of enzyme contains about 0.1 mol of flavin
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe3+
0.048 mol Fe(III) per mol enzyme
Mg
-
purified human cystein dioxygenase yields trace amounts of magnesium about 0.41%
Mn
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purified human cystein dioxygenase yields trace amounts of manganese about 0.25%
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,2'-dipyridyl
2-amino-ethanethiol
2-mercaptoethanol
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2-oxoglutarate
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2-sulfanyl-ethanol
3,3'-thiodipropionic acid
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-
3-Mercaptopropionate
complete inhibition at 0.05 mM
3-Mercaptopropionic acid
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3-sulfanyl-propionic acid
3-sulfinopropionic acid
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8-hydroxyquinoline
alpha-ketoglutarate
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alpha-ketoglutarate inhibits cysteine dioxygenase with 50% inhibition at 6.8 mM
aspartic acid
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aspartic acid decreases enzyme activity to 50% at a concentration of 1.5 mM. Replacing the sulfydryl by the uncharged hydroxyl group of serine does not affect enzyme activity.
azide
Bathocuproine sulfonate
bathophenanthroline sulfonate
Carboxyethyl-L-cysteine
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53% inhibition at 1 mM
carboxymethyl-L-cysteine
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37% inhibition at 1 mM
CdCl2
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cells transfected with wild-type enzyme show an enhanced sensitivity to CdCl2 that is limited to cells cultured in medium with cysteine levels of 0.1 and 0.3mM
Cu2+
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90% inhibition at 0.01 mM, 100% inhibition at 0.1 mM
cyanide
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inhibits with a 50% actvity reduction at 2.7 mM
cysteamine
cystine
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42% inhibition at 5 mM
cytokine tumor necrosis factor-alpha
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also TNF-alpha, down-regulation observed in hepatic and brain cells
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D-Cys
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competitive inhibitor, binding structure, overview
D-cysteine
D-cysteinesulfinate
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34.4% inhibition in hepatocytes from rats fed a low casein diet, 71.8% inhibition in hepatocytes from rats fed a moderate casein diet, 64.4% inhibition in hepatocytes from rats fed a high casein diet
diethyldithiocarbamate
DL-homocysteine
DL-homocystine
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47% inhibition at 5 mM
DL-propargylglycine
ethyl xanthate
44% inhibition at 0.004 mM
Glutarate
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homocysteine
L-cysteine
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concentrations of cysteine of 2 mM and above are inhibitory in assays of purified cysteine dioxygenase
Mercaptopropionic acid
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mercaptopropionic acid at a concentration of 1.2 mM inhibits cysteine dioxygenase activity by 50%
N-acetyl-L-cysteine
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35% inhibition at 10 mM
Neocuproine
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with protein-A: 18% inhibition at 0.1 mM, without protein-A: slight activation at 0.1 mM
o-phenanthroline
S-carboxymethylcysteine
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S-carboxymethylcysteine exhibits 50% inhibition at a concentration of 2.3 mM
S-methyl-L-cysteine
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34% inhibition at 1 mM
sodium ethylxanthate
11% inhibition at 0.004 mM
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thiosulfate
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competitive inhibitor, binding structure, overview
transforming growth factor-beta
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also TGF-beta, down-regulation observed in hepatic and brain cells
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additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,2'-dipyridyl
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slight activation at 0.01 mM
8-hydroxyquinoline
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slight activation at 0.01 mM
Carboxyethyl-L-cysteine
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at 10 mM, activation
carboxymethyl-L-cysteine
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at 10 mM, activation
cysteamine
cysteine
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protein expression of recombinant wild-type enzyme in HepG2/C3A cells increases by 160% when extracellular cysteine levels are increased from 0 to 1 mM cysteine
D-cysteine
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at 10 mM, activation
diethyldithiocarbamate
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without protein-A: 30% activation at 0.1 mM
DL-homocysteine
Fe2+
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stimulates
hydrocortisone
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induces
hydroxylamine
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activates, restores the inhibition by Fe2+
L-cysteine
methionine
N-acetyl-L-cysteine
NAD(P)H
NAD+
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stimulates
NADH
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stimulates
Neocuproine
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without protein-A: slight activation at 0.1 mM
S-methyl-L-cysteine
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.7
3-Mercaptopropionate
pH 6.5, 30C, recombinant His-tagged enzyme
16.6
beta-mercaptoethanol
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0.45 - 3
cysteine
0.002 - 6.7
L-cysteine
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.82
3-Mercaptopropionate
pH 6.5, 30C, recombinant His-tagged enzyme
0.01 - 10.35
L-cysteine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.144
3-Mercaptopropionate
pH 6.5, 30C, recombinant His-tagged enzyme
0.002 - 0.095
L-cysteine
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.5
cysteamine
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0.24
D-cysteine
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0.75
S-methyl-L-cysteine
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000068
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in isolated hepatocytes from rats fed the diet containing a low casein level
0.00016
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in hepatocytes isolated from rats fed diets containing 100 g casein/kg without sulfur amino acid supplementation
0.00017
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in hepatocytes isolated from rats fed diets containing 100 g casein/kg with 2.4 g L-cystine per kg supplementation
0.0003
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in isolated hepatocytes from rats fed the diet containing a moderate casein level
0.00046
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in hepatocytes isolated from rats fed diets containing 100 g casein/kg with 3 g L-methionine per kg supplementation
0.00047
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in isolated hepatocytes from rats fed the diet containing a high casein level
0.0012
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in hepatocytes isolated from rats fed diets containing 100 g casein/kg with 8 g L-cystine per kg supplementation
0.00202
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in hepatocytes isolated from rats fed diets containing 100 g casein/kg with 10 g L-methionine per kg supplementation
0.034
purified recombinant enzyme, in ammonium acetate buffer, pH 4.5, at 37C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8 - 6.2
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highest activity
6
SCO3035, pH optimum is determined by using 2-morpholinoethanesulfonic acid or Tris buffers at a final concentration of 62.5 mM
6.2
YubC, pH optimum is determined by using 2-morpholinoethanesulfonic acid or Tris buffers at a final concentration of 62.5 mM
6.8 - 9.5
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for the anaerobic activation of the purified enzyme by L-cysteine
6.8
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assay at
7.7
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60% cross-linked wild-type enzyme
8.5 - 9
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purified enzyme, of enzyme reaction
9
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assay at
additional information
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pH-dependence of wild-type and mutant enzyme kinetics, detailed overview
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 9.5
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
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assay at
22
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assay at room temperature
30
assay at; assay at
38 - 40
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for the anaerobic activation of the purified enzyme by L-cysteine
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 37
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activity at 37C 2-fold higher than at 25C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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hepatoblastoma cells, enzyme expression is up-regulated under hypertonic conditions
Manually annotated by BRENDA team
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CDO is found specifically in the mucus-secreting goblet cells
Manually annotated by BRENDA team
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intense staining for CDO in ductal cells of pregnant rats but not in other mammary epithelial cells or in ductal cells of non-pregnant rats
Manually annotated by BRENDA team
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in Streptomyces spp. CDO is expressed in the vegetative state, and an increase in its activity is detected after the initiation of conidia production
Manually annotated by BRENDA team
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exocrine cell, but not in islet endocrine cells
Manually annotated by BRENDA team
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-
Manually annotated by BRENDA team
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detectable levels
Manually annotated by BRENDA team
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barely detectable levels
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10500
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gel filtration, SDS-PAGE, the estimate of molecular weight may be inaccurate because it is based on use of the iodinated protein and the assumption that iodination does not affect molecular weight
23060
MALDI-TOF mass spectrometry
23830
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calculated from deduced amino acid sequence, His-tag fusion protein
24950
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mass spectrometry, thrombin cleaved enzyme
25000
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SDS-PAGE, native protein
25700
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SDS-PAGE, His-tag fusion protein
26800
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His-tag fusion protein
63900
recombinant His-tagged enzyme CdoB, gel filtration
68000
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detection in liver whole homogenate, immunoabsorption of anti-enzyme antibodies
70000
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detected by antibody for the enzyme
additional information
-
no 68000 Da species detected as reported in different publications
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
trimer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme free or with bound substrate L-Cys, hanging drop vapour diffusion method, mixing of 0.004 ml of 10 mg/ml protein solution with 0.004 ml of reservoir solution containing 18% w/v PEG 4000, 0.1 M potassium acetate, 0.05 M 2-(N-morpholino)ethanesulfonic acid, pH 6.0, 25C, 2-7 days, X-ray diffraction structure determination and analysis at 2.38-2.82 A resolution
co-crystallization of L-cysteine and purified wild-type cysteine dioxygenase by the hanging drop, vapor-diffusion method at 16C, the crystals grow as rods up to 0.2 x 0.2 x 0.8 mm in 1 week. Refinement of the crystal stucture leads to a final model with a crystallographic R-factor of 18.1% and a free R-factor of 21.5% at 2.7 A resolution.
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enzyme with or without bound cysteine and formation of persulfenate, usage of crushed CDO crystals at 8 mg/mL in 10 mM Tris, pH 7.4, recrystallized by hanging drop vapour diffusion method, mixing of 0.0005 ml of crystal seed stock solution with 0.001 ml protein solution and 0.001 ml of reservoir solution containing 0.1 M trisodium citrate, pH 5.6, 24% PEG 4000, and 0.15 M ammonium acetate, final pH of 6.2, at room temperature, X-ray diffraction structure determination and analysis at pH 4.0-9.0 and 1.25-1.60 A resolution, overview
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crystals are grown at 25C by hanging-drop vapor-diffusion. The reservoir contains 20% methosylpolyethylene glycol 5000, 160 mM CaCl2, and 100 mM 2-morpholinjoethane-sulfonic acid (pH 6.5). Hanging drops consist of 2 microl of protein solution mixed with 2 microl of reservoir solution. Structure is solved to a nominal 1.75 A resolution.
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electron paramagnetic study of substrate-O2 binding. Ordered binding of L-cysteine prior to NO and presumably O2. Upon addition of NO to cysteine dioxygenase in the presence of substrate L-cysteine, a low-spin(FeNO)7 signal with spin S of 1/2that accounts for 85% of the iron within the enzyme develops. Substitution of L-cysteine with isosteric substrate analogues cysteamine, 3-mercaptopropionic acid, and propane thiol does not produce any analogous signals.The unusual (FeNO)7, spin 1/2 electronic configuration adopted by the substrate-bound iron-nitrosyl cysteine dioxygenase is a result of the bidentate thiol/amine coordination of L-cysteine in the NO-bound active site
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crystallization in sitting drops at 25C using a reservoir of 0.1-0.25 M ammonium acetate, 0.1 M tri-sodium citrate, pH 5.6, with 22-26% (w/v) polyethylene glycol 4000. The co-crystals with 5 mM are grown using a reservoir of 0.15 M ammonium sulfate, 0.2 M sodium cacodylate, pH 6.5, with 26% (w/v) polyethylene glycol 8000. The crystal sturcture, solved by SAD phasing using selenomethionine-substituted protein, yields a final refined model with r = 18.0 and Rfree = 20.8 at 1.5-A resolution. Data from a co-crystallization experiment with 5 mM cysteine shows structural changes in the binding pocket, they are determined to 1.5 A resolution (final r = 19.8 and Rfree = 22.4).
generation of 21 CDO crystal structures at resolutions between 1.25 and 1.65 A. Of these, 16 are of C93A or Y157F CDO mutants either alone or bound to L-Cys, D-Cys, or the inhibitor homocysteine, the other five are of wild-type CDO bound to homocysteine, azide, or thiosulfate. Cys-soaked wild-type CDO crystals are analysed at pH 6.2 and pH 8.0, detailed overview
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purified enzyme, hanging drop vapour diffusion method, mixing of 0.0015 ml of 7 mg/mL CDO mutant C93G in 10 mM sodium phosphate and 20 mM NaCl, pH 7.5, and 0.0006 ml of crushed wild-type CDO crystal seeds in their own growth solution consisting of 25% w/v PEG 1500, 13 mM succinate, 44 mM sodium phosphate, and 44 mM glycine, pH 5.2, with 0.0015 ml of reservoir buffer containing 26% w/v PEG 4000, 200 mM ammonium acetate, and 100 mM sodium citrate, pH 6.3, equilibration against reservoir solution, X-ray diffraction structure determination and analysis at 1.82 A resolution
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purified recombinant enzyme complexed with cysteine persulfide or 3-mercaptopropionic acid persulfide, hanging drop vapor diffusion method, mixing of 0.0015 ml of 8 mg/ml protein with 0.0015 ml of reservoir solution containing 24-34% w/v PEG 4000, 100-250 mM ammonium acetate, 100 mM sodium citrate, pH 5.6, 0-4 mM dithionite, and 40 mM ligand, final pH is 6.1-6.2, 24C, one week, X-ray diffraction structure determination and analysis at 1.63-2.05 A resolution
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X-ray crystal structure
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
pronase destroys activity
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
rapid and irreversible inactivation under aerobic conditions, inactivation can be prevented by a distinct cytoplasmic protein, i.e. protein A
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439542, 439546, 439548
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 3 months
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-70C, crude enzyme, stable for up to 4 weeks
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0C, no significant loss of activity after 1 month
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1 ml HisTrap HP column, metal ion (nickel) affinity chromatography, gel filtration, MonoQ 4.6/100 PE ion exchange column. The thioredoxin/6x His cysteine dioxygenase fusion protein separates into two apparent isoforms that elute as two distinct peaks, one that elutes at 50 mM imidazole and one that elutes at 100 mM imidazole during metal ion affinity chromatograohy. The protein in the second peak has a specific activity, that is 50-60% less than that of the protein in the first peak. In contrast to peak 1 peak 2 elutes as two pronounced peaks (A and B) from the MonoQ column. The cysteine dioxygenase in peak A has no detectabel activity. In the the standard cysteine dioxygenase purification procedure, only the form from peak 1 is retained and further purified.
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all buffers are supplemented with 5 mM dithiothreitol to prevent oxidation of the selenomethionine
amylose column chromatography and Superdex 200 gel filtration
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HisTrap HP column, immobilized metal affinity chromatography, during purification two peaks of highly purified recombinant protein are found to elute: one at 10% (peak1) and the second at 20% (peak 2), kinetic data show that peak 1 has lower Km and higher Vmax values for cysteine than peak 2
HisTrap HP column, immobilized metal affinity chromatography, during purification two peaks of highly purified recombinant protein are found to elute: one at 10% (peak1) and the second at 20%(peak 2), kinetic datat show that peak 1 has lower Km and higher Vmax values for cysteine thant peak 2.
HisTrap HP column, immobilized metal affinity chromatography; HisTrap HP column, immobilized metal affinity chromatography, during purification of SCO30335 two peaks of highly purified recombinant protein are found to elute: one at 10% (peak1) and the second at 20% (peak 2), kinetic data show that peak 1 has lower Km and higher Vmax values for cysteine than peak 2
immobilized metal chelate affinity chromatography
-
immobilized nickel affinity chromatography
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Ni-NTA agarose column chromatography
recombinant enzyme
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recombinant enzyme from Escherichia coli strain BL21(DE3)
recombinant His-tagged enzyme CdoA from Escherichia coli by nickel affinity chromatography to homogeneity; recombinant His-tagged enzyme CdoB from Escherichia coli by nickel affinity chromatography to homogeneity
recombinant N-terminal maltose-binding protein fusion enzyme from Escherichia coli strain BL21(DE3) by anion exchange and amylose affinity chromatography
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recombinant N-terminal maltose-binding protein fusion enzyme from Escherichia coli strain BL21(DE3)RIPL by anion exchange and amylose affinity chromatography
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recombinant protein using His-tag
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recombinant Strep-tagged enzyme
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recombinant Strep-tagged wild-type and mutant enzyme by affinity chromatography to over 95% purity
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recombinant thioredoxin-His6-tagged enzyme by affinity chromatography
-
recombinnat enzyme from Escherichia coli strain BL21(DE3) with cleavage of the Thx-His6 tag
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standard glutathione S-transferase fusion protein purification protocol, gel-filtration chromatography after removing the glutathione S-transferase tag
-
the fusion protein is purified by immobilized metal (Ni2+) affinity chromatography. The liberated enzyme is separated from the His-8-maltose binding protein by immobilized metal affinity chromatography, further purified by gel filtration chromatography, and concentrated. EDTA is obmitted from the buffers used to purifiy enzyme for catalytic studies, and the gel filtration is not used.
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using acetone fractionation, column chromatography on DEAE-cellulose, Sephadex G-100, hydroxylapatite, DEAE-Sephadex A-25 and Sephadex G-75
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using acetone precipitation, first chromatography on DEAE-cellulose column, second chromatography on DEAE-cellulose column, chromatography on Sephadex G-100 column, hydroxyapatite column, DEAE-Sephadex A-25 column and Sephadex G-75 column
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using acid treatment, ammonium sulfate fractionation and column chromatography with DEAE-cellulose. The purified enzyme is composed of two distinct proteins, it appears that one of them is a catalytic protein named protein-B having tightly bound iron as a prosthetic group, while the other is either a modifier or activating protein named protein-A. Protein-B is found to exist in both an active and an inactive form
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using filtration, centrifugation, column chromatography on DEAE-cellulose, Sephadex G-50 and cysteine-Sepharose
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using heat treatment, ammonium sulfate fractionation and column chromatography on DEAE-cellulose, Sephadex G-200 and Sephadex G-100
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
amplification by PCR and cloning into TA cloning vector, sequencing
-
cloned in plasmid pVP16 to produce the enzyme in Escherichia coli B834 pRARE2, an N-terminal fusion to a His-8-maltose binding protein
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cloned into pGEX-6p-1 expression vector, expressed in Escherichia coli Bl21(DE3), glutathione S-transferase-fused CDO is purified and GTS tag is removed by cleavage with PreScission Protease
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cloning of the gene and comparison of the gene to the known rat and human genes, sequencing, profiling of the enzyme mRNA and protein levels in mouse tissues
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expressed as a His-tag fusion protein in Escherichia coli BL21(DE3)pLysS
-
expressed as His-tag fusion protein in Escherichia coli
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expressed in BL21(D3)pLysS cells containing the pET-14b/CDO-ORF plasmid
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expressed in BL21(DE3) cells
expressed in Escherichia coli
-
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli BL21(DE3)pLysS cells
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expressed in Escherichia coli strain BL21(DE3)
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expressed in Hep-G2 cells
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expression in Escherichia coli and HepG2/C3A cell
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expression in Escherichia coli strain BL21(DE3)
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expression in HepG2/C3A cell
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gene cdo, expression as N-terminal maltose-binding protein fusion protein in Escherichia coli strain BL21(DE3)
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gene cdo, expression as N-terminal maltose-binding protein fusion protein in Escherichia coli strain BL21(DE3)RIPL
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gene cdo, expression of Thx-His6 -tagged enzyme in Escherichia coli strain BL21(DE3)
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gene cdoA, recombinant enzyme expression in Escherichia coli strain BL21(DE3), subcloning in Escherichia cli strain DH5alpha
gene H16_A1614, located on chromosome 1, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of His-tagged enzyme CdoB in Escherichia coli; gene H16_B1863, located on chromosome 2, DNA and amino acid sequence determination and analysis, phylogenetic analysis, recombinant expression of His-tagged enzyme CdoA in Escherichia coli. Escherichia coli cells expressing CdoA can convert 3-mercaptopropionate to 3-sulfinopropionate
heterologously expressed in human HepG2/C3A cells
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primary structure of the cDNA for liver enzyme, sequence determination
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recombinant expression of Strep-tagged enzyme
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recombinant expression of the thioredoxin-His6-tagged enzyme
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recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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recombinant expression of wild-type and mutant Strep-tagged enzyme
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subcloned into an expression vector, pESC-TRP, for Saccharomyces cerevisiae; subcloned into an expression vector, pESC-TRP, for Saccharomyces cerevisiae
the open reading frame of putative cysteine dioxygenase NP_390992(yubC, Bacillus subtilis) is cloned and overexpressed in Escherichia coli.
the open reading frame of putative cysteine dioxygenase NP_627257 (SCO30335, Streptomyces coelicolor A3(2))is cloned and overexpressed in Escherichia coli; the open reading frame of putative cysteine dioxygenase NP_629897 (SCO5772, Streptomyces coelicolor A3(2)) is cloned and overexpressed in Escherichia coli
the open reading frame of putative cysteine dioxygenase NP_832375(BC2617, Bacillus cereus ATCC 14579) is cloned and overexpressed in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C164A
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mutation of C164 shows a about 20% abatement of enzymatic activity
C164S
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mutation of C164 shows a about 20% abatement of enzymatic activity
C93S
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C93 mutation reduces activity to 50%, zinc content of about 45%, specific activity of Cys-93 mutants is proportional to the measured iron content.
R60A
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similar results as R60Q, R60 mutation reduces activity to 30%
R60Q
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R60 mutation reduces activity to 30%
Y157F
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in the gel-filtration chromatography Y157F shows an additional peak with an estimated molecular weight equivalent to a cysteine dioxygenase dimer. The results for monomer and dimer are similar. Activity reduced to 5% of the wild type activity. Zinc content of about 45%
C164A
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mutations of nonessential residues has little effect
C93A
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site-directed mutagenesis, mutation of the active site residue, no crosslink formation resulting in reduced activity compared to the wild-type enzyme. The mutant variant structure has a new chloride ion coordinating the active site iron. Cys binding is also different from wild-type CDO, and no Cys-persulfenate forms in the C93A active site at pH 6.2 or pH 8.0
C93S
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mutant can not be converted to the mature form due to the loss of the cysteine residue involved in thioether crosslink formation
R60A
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mutant forms with low activity, which has a markedly decreased affinity for cysteine, probably due to the loss of the hydrogen bonding partner for the carboxylate of the substrate, forms the crosslink more slowly
S153A
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mutations of nonessential residues has little effect
Y157F
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site-directed mutagenesis, mutation of the active site residue, no crosslink formation resulting in reduced activity compared to the wild-type enzyme. The mutant variant structure has a new chloride ion coordinating the active site iron. Cys binding is also different from wild-type CDO, and no Cys-persulfenate forms in the Y157F active site at pH 6.2 or pH 8.0
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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lack of methionine sulfoxide reductase MsrA in liver of MsrA -/- mice leads to a significant drop in the cellular level of thiol groups and lowers the level of cysteine dioxygenase expression. Following selenium deficient diet applied to decrease the expression levels of selenoproteins like MsrB, the latter effect is maintained while the basal levels of thiol decreased in both wild-type strains and strains deficient for methionine dioxygenase
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