Information on EC 1.20.4.4 - arsenate reductase (thioredoxin)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.20.4.4
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RECOMMENDED NAME
GeneOntology No.
arsenate reductase (thioredoxin)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
arsenate + thioredoxin = arsenite + thioredoxin disulfide + H2O
show the reaction diagram
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SYSTEMATIC NAME
IUBMB Comments
thioredoxin:arsenate oxidoreductase
The enzyme, characterized in bacteria of the Firmicutes phylum, is specific for thioredoxin [1]. It has no activity with glutaredoxin [cf. EC 1.20.4.1, arsenate reductase (glutaredoxin)]. Although the arsenite formed is more toxic than arsenate, it can be extruded from some bacteria by EC 3.6.3.16, arsenite-transporting ATPase; in other organisms, arsenite can be methylated by EC 2.1.1.137, arsenite methyltransferase, in a pathway that produces non-toxic organoarsenical compounds. The enzyme also has the activity of EC 3.1.3.48, protein-tyrosine-phosphatase [3].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
enzyme additionally shows low phosphates activity
UniProt
Manually annotated by BRENDA team
enzyme additionally shows low phosphates activity
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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
arsenate + thioredoxin
arsenite + thioredoxin disulfide + H2O
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
arsenate + thioredoxin
arsenite + thioredoxin disulfide + H2O
show the reaction diagram
additional information
?
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assays are performed with different arsenate concentrations and arsenate reductase concentrations in the presence of 0.42 microM Escherichia coli thioredoxin, 0.14 microM Escherichia coli thioredoxin reductase and 125 microM NADPH
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
thioredoxin
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K+
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nonspecific binding
Na+
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nonspecific binding
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
antimonite
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SbO2-, pH 7.5, 37C
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arsenite
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pH 7.5, 37C
iodoacetate
sulfhydryl inhibitors N-ethylmaleimide -and iodoacetate inhibit arsenate reductase activity by 80% in crude cell-free preparations and by 90% with purified ArsC protein
N-ethylmaleimide
sulfhydryl inhibitors N-ethylmaleimide -and iodoacetate inhibit arsenate reductase activity by 80% in crude cell-free preparations and by 90% with purified ArsC protein
tellurite
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pH 7.5, 37C
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additional information
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arsenite, tellurite, and antimonite [Sb(III)] are inhibitors for NADPH oxidation; the substrate, arsenate, is not inhibitory at concentrations up to 40 mM
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dithiothreitol
stimulates ArsC-dependent arsenate reduction, but not 2-mercaptoethanol or reduced glutathione
thioredoxin
and dithiothreitol are needed for arsenate reduction
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000066 - 75
arsenate
0.033
thioredoxin
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additional information
arsenate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.061 - 3.65
arsenate
1.9
thioredoxin
Staphylococcus aureus
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.045 - 1170
arsenate
255
57.6
thioredoxin
Staphylococcus aureus
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-
121
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01
antimonite
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pH 7.5, 37C
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0.5
arsenite
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pH 7.5, 37C
0.0005
tellurite
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pH 7.5, 37C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.2
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pH 7.5, 37C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9.5
ArsC arsenate reductase activity is decreased >80% at pH 6.3 but is stable in the pH range 7.0-9.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.9
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calculated
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
14440
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calculated from amino acid sequence
14500
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electrospray mass spectrometry shows two molecular masses of 14810.5 and 14436.0 Da, suggesting that 70% of the purified protein lacks the N-terminal three amino acids
14800
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reduced C82S mutant
14820
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reduced C89L mutant
15030
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calculated, SDS-PAGE
17000
gel filtration
20000
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gel filtration
21000
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gel filtration
47000
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Western blot
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
x-ray structures of the mutants
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seleno-methionine derivative, to 2.2 A resolution
triple mutant, hanging-drop vapour diffusion method
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x-ray structures of the mutants
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9.5
ArsC arsenate reductase activity is decreased >80% at pH 6.3 but is stable in the pH range 7.0-9.5
706565
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80
heat-stable, surviving 2 min heating at 80C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; gel filtration, SDS-PAGE
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; wild-type ArsC and the Cys mutants, gel filtration more than 97% pure
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by fast protein liquid chromatography
C10S/C15A/C89L mutant and C10S/C15A/C82S mutant purified by Ni2+-NTA Superflow
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ion-exchange chromatography
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mutant is purified on Ni2+-NTA Superflow
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recombinant protein
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
C10S/C15A/C89L mutant and C10S/C15A/C82S mutant expressed in Escherichia coli
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expressed in Escherichia coli
expressed in Escherichia coli and yeast
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expressed in Escherichia coli; expression in Escherichia coli. Wild-type enzyme and the Cys mutants (C15A, C10A, C82A, C82S, C89A, C10SC15S, C10SC15A) are expressed in Escherichia coli. Wild-type enzyme, mutant enzyme C15A, mutant enzyme C10A, mutant enzyme C82S, mutant enzyme C89A, and mutant enzyme C10SC15A are expressed soluble and with high yields. Mutant enzyme C82A is found in inclusion bodies, and the double mutant C10S/C15S is not expressed
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expression in Escherichia coli
mutant expressed in Escherichia coli
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mutant is expressed in Escherichia coli
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overproduced in Escherichia coli
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overproduced in Escherichia coli K38(pGP1-2)
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
arsenate up-regulates expression
expression is 4fold increased in presence of arsenate in the growth medium
isooform ArsC1' is constitutively expressed at low levels using its own promoter site. It reduces arsenate to arsenite that can then induce the expression of isoforms ArsC1 and ArsC2
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C10S/C15A/C82S
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mutant in complex with the thioredoxin C32S mutant. Solve the structure of the Trx-ArsC complex by NMR spectroscopy
K33D
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site directed mutagenesis
K33N
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site directed mutagenesis
C10S/C15A
C10S/C15A/C82S
C10S/C15A/C89L
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mutant, determination of the redox potential of the Cys82-Cys89 redox couple
C10S/C15S
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double mutation, no enzymatic activity
C10SC15A
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inactive mutant enzyme
C82S
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inactive mutant enzyme; site-directed mutagenesis, mutation of Cys 10, 82, and 89 leads to redox-inactive enzymes
H62Q
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change of the P-loop geometry
H62Q/N33K
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site directed mutagenesis
H62Q/N33K/E30D/G31E
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site directed mutagenesis
C7S
complete loss of activity
additional information