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Information on EC 1.8.3.2 - thiol oxidase and Organism(s) Saccharomyces cerevisiae and UniProt Accession P27882
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1.8.3.2 thiol oxidaseIUBMB Comments
R may be =S or =O, or a variety of other groups. The enzyme is not specific for R'.
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Word Map
- 1.8.3.2
- hepatocytes
-
intermembrane
- oxidases
-
relay
-
qsoxs
- thioredoxin
-
redox-active
-
hepatectomy
-
hepatotrophic
-
reoxidized
-
hepatectomized
-
flavoenzyme
-
fad-dependent
-
fad-binding
-
oxidoreductins
-
flavin-linked
-
redox-regulated
- isoalloxazine
-
3h-tdr
- diagnostics
- medicine
- nutrition
- analysis
The taxonomic range for the selected organisms is: Saccharomyces cerevisiae
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
qsox1, sulfhydryl oxidase, augmenter of liver regeneration, ero1p, thiol oxidase, hepatopoietin, erv2p, erv1p, sulphydryl oxidase, sfalr, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
DTT-oxidase
-
-
-
-
Erv1p
ERv2p
oxidase, thiol
-
-
-
-
sulfhydryl oxidase
sulfhydryl oxidase SOx-3
-
-
-
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thiooxidase
-
-
-
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ERv2p
-
-
-
-
sulfhydryl oxidase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 R'C(R)SH + O2 = R'C(R)S-S(R)CR' + H2O2
enzyme contains a functionally involved redox-active motif CXXC
-
2 R'C(R)SH + O2 = R'C(R)S-S(R)CR' + H2O2
the CXXC motif in the active site sequence of Erv2p is catalytically essential, reaction mechanism involving reactive cysteine residues C121 and C124 of the A subunit, and C176 and C178 of the B subunit
-
2 R'C(R)SH + O2 = R'C(R)S-S(R)CR' + H2O2
the N-terminal cysteine pair of the enzyme is essential for in vivo activity and interacts with the primary redox centre
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
redox reaction
-
-
-
-
oxidation
-
-
-
-
reduction
-
-
-
-
PATHWAY SOURCE
PATHWAYS
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-
SYSTEMATIC NAME
IUBMB Comments
thiol:oxygen oxidoreductase
R may be =S or =O, or a variety of other groups. The enzyme is not specific for R'.
CAS REGISTRY NUMBER
COMMENTARY
9029-39-4
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
glutathione + O2
glutathione disulfide + H2O2
-
Evr2p, not Evr1p
-
-
?
protein disulfide isomerase + O2
protein disulfide isomerase disulfide + H2O2
-
-
-
?
dithiothreitol + O2
dithiothreitol disulfide + H2O2
-
Evr2p, not Evr1p
-
-
?
dithiothreitol + O2
dithiothreitol disulfide + H2O2
-
low concentrations of dithiothreitol stimulate the import efficiency of Erv1, whereas higher concentrations of dithiothreitol decrease it
-
-
?
protein Mia40 + O2
protein Mia40 disulfide + H2O
-
recombinantly expressed substrate amino acids 284-403, which is the C-terminal domain of Mia40, electron transfer between the shuttle and active site disulfides of Erv1p. Both intersubunit and intermolecular electron transfer can occur, overview
-
-
?
R-SH + O2
R-S-S-R + H2O2
3 cysteine pairs are required for optimal enzyme function
-
-
?
additional information
?
-
-
essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic but not of mitochondrial Fe-S proteins
-
-
?
additional information
?
-
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Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential FAD-dependent oxidase of protein disulfide isomerase
-
-
?
additional information
?
-
-
Evr1p is involved in cellular iron homeostasis, physiological role of the ERV1/ALR family enzymes, overview
-
-
?
additional information
?
-
-
the enzyme is involved in mitochondrial biogenesis
-
-
?
additional information
?
-
does not oxidize reduced thioredoxin
-
-
?
additional information
?
-
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Erv2p is a modest catalyst of disulfide bond formation. None of the monothiols (at 10 mM), including beta-mercaptoethanol, N-acetylcysteamine, reduced glutathione and CoASH, prove detectable substrates of the yeast oxidase at pH 7.5. In contrast, dithiols are significant substrates
-
-
?
additional information
?
-
-
Erv1p contains three conserved disulfide bonds arranged in two CXXC motifs and one CX16C motif, the CX16C disulfide plays an important role in stabilizing the folding of Erv1p, both CXXC disulfides are required for Erv1 oxidase activity, but none of the disulfide is essential for FAD binding, overview
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
additional information
?
-
-
essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic but not of mitochondrial Fe-S proteins
-
-
?
additional information
?
-
-
Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential FAD-dependent oxidase of protein disulfide isomerase
-
-
?
additional information
?
-
-
Evr1p is involved in cellular iron homeostasis, physiological role of the ERV1/ALR family enzymes, overview
-
-
?
additional information
?
-
-
the enzyme is involved in mitochondrial biogenesis
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
enzyme contains a thioredoxin and an ERV1 domain
-
FAD
dependent on, N-terminal cysteine pair contributes to the correct arrangement of the FAD-binding fold
FAD
-
dithionite and photochemical reductions of Erv2p show full reduction of the flavin cofactor after the addition of 4 electrons with a midpoint potential of -200 mV at pH 7.5. No charge-transfer complex between a proximal thiolate and the oxidized flavin
FAD
-
dependent on. The three disulfides of the enzyme are not essential for FAD binding
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Zn2+
-
upon coordination with Zn2+, full reduction of Erv2p requires 6 electrons. Strongly inhibits Erv2p when assayed using tris(2-carboxyethyl)phosphine as the reducing substrate of the oxidase. Restoration of 1 mM EDTA effects rapid recovery of 80% of the original activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
oxygen consumption kinetic parameters for the WT and Erv1p mutants, overview
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0.0001
O2
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with 0.3 mM reduced thioredoxin as the substrate of the reductive half-reaction and an initial oxygen concentration of around 0.3 mM such that the reduced thioredoxin would be depleted by 10% during the course of the reaction
0.018
O2
-
25°C, recombinant mutant C159S/C176S, in presence of 10 3.5 mM tris(2-carboxyethyl)phosphine
0.027
O2
-
25°C, recombinant wild-type enzyme, in presence of 3.5 mM tris(2-carboxyethyl)phosphine
0.057
O2
-
25°C, recombinant wild-type enzyme, in presence of 10 mM DTT
0.062
O2
-
25°C, recombinant mutant C30S/C33S, in presence of 10 mM DTT
0.087
O2
-
25°C, recombinant mutant C159S/C176S, in presence of 10 mM DTT
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.7
O2
-
25°C, recombinant mutant C159S/C176S, in presence of 10 3.5 mM tris(2-carboxyethyl)phosphine
0.8
O2
-
25°C, recombinant mutant C159S/C176S, in presence of 10 mM DTT
1.1
O2
-
25°C, recombinant wild-type enzyme, in presence of 3.5 mM tris(2-carboxyethyl)phosphine
1.3
O2
-
25°C, recombinant wild-type enzyme, in presence of 10 mM DTT
1.5
O2
-
25°C, recombinant mutant C30S/C33S, in presence of 10 mM DTT
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0023
O2
-
25°C, recombinant wild-type enzyme, in presence of 10 mM DTT
0.0024
O2
-
25°C, recombinant mutant C30S/C33S, in presence of 10 mM DTT
0.0039
O2
-
25°C, recombinant mutant C159S/C176S, in presence of 10 3.5 mM tris(2-carboxyethyl)phosphine
0.0041
O2
-
25°C, recombinant wild-type enzyme, in presence of 3.5 mM tris(2-carboxyethyl)phosphine
0.0093
O2
-
25°C, recombinant mutant C159S/C176S, in presence of 10 mM DTT
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
-
enzyme is expressed in a large number of different cell types and tissues
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
high concentration of Evr1p
-
Erv1 requires the redox-regulated receptor Mia40 for its import into mitochondria. Interacts via disulfide bonds with Mia40. It does not need two CX2C motifs for import into mitochondria
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
-
Erv1p is a FAD-dependent sulfhydryl oxidase and is an essential component of the redox regulated Mia40/Erv1 import and assembly pathway used by many of the cysteine-containing intermembrane space proteins
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
15000
-
2 * 22000, long Erv1p form, SDS-PAGE, 2 * 15000, short Evrp1 form, SDS-PAGE
22000
-
2 * 22000, long Erv1p form, SDS-PAGE, 2 * 15000, short Evrp1 form, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
additional information
dimer
-
2 * 22000, long Erv1p form, SDS-PAGE, 2 * 15000, short Evrp1 form, SDS-PAGE
additional information
-
structure, active site structure containinbg a CXXC motif
additional information
-
Erv1p contains three conserved disulfide bonds arranged in two CXXC motifs and one CX16C motif in the highly conserved central catalytic core. The CX16C disulfide plays an important role in stabilizing the folding of Erv1p, both CXXC disulfides are required for Erv1 oxidase activity, but none of the disulfide is essential for FAD binding, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structures at 2.0 A resolution of the C-terminal domain and at 3.0 A resolution of a C30S/C133S double mutant. The C-terminal domain exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The N-terminal domain is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the N-terminal domain to the C-terminal domain of another subunit. The four-helix bundle of the C-terminal domain forms a wide platform for the electron donor N-terminal domain. Moreover,the amphipathic helix close to the shuttle redox enter may be critical for the recognition of Mia40, the upstream electron donor
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C130S
site-directed mutagenesis, inactive mutant, no complementation of an enzyme-defect mutant strain, no complementation of an enzyme-defect mutant strain
C130S/C133S
-
site-directed mutagenesis, the active site mutant shows no or very little activity, and the mutant shows a shifted protein-bound FAD spectrum compared to the wild-type enzyme Erv1p, the active site disulfide is located proximal to the isoalloxazine ring of FADa nd the mutation changes bound-FAD absorption slightly, the mutant is active in presence of DTT, but not with tris(2-carboxyethyl)phosphine
C133S
C159S
site-directed mutagenesis, about 70% reduced activity in vitro compared to the wild-type enzyme, complementation of an enzyme-defect mutant strain
C159S/C176S
-
site-directed mutagenesis, the mutant shows the same protein-bound FAD spectrum as the wild-type enzyme Erv1p
C176S
site-directed mutagenesis, about 60% reduced activity in vitro compared to the wild-type enzyme, complementation of an enzyme-defect mutant strain
C30S
C30S/C33S
C33S
site-directed mutagenesis, about 50% reduced activity in vitro compared to the wild-type enzyme, no complementation of an enzyme-defect mutant strain
D24A
-
the mutant enzyme oxidizes GSH and gamma-glutamylcysteine at much lower rates than the wild-type enzyme
N131A
-
the mutant enzyme oxidizes GSH and gamma-glutamylcysteine at much lower rates than the wild-type enzyme
N34A
-
enhancement of catalytic activity for GSH, whereas the catalytic activity for gamma-glutamylcysteine remains unchanged. The mutant enzyme shows slightly decreased maximum temperatures at 55°C (compared to 60°C for the wild-type enzyme)
N34Q
-
the mutant enzyme oxidizes GSH more efficiently (201%) than the wild-type enzyme
P129A
-
the mutant enzyme oxidizes GSH and gamma-glutamylcysteine at much lower rates than the wild-type enzyme
S32A
-
the mutant enzyme oxidizes GSH more efficiently (169%) than the wild-type enzyme. About 1.5fold increase in GSSG production compared to that of the parental ERV1 gene
S32A/N34A
-
the mutant enzyme oxidizes GSH more efficiently (240%) than the wild-type enzyme and shows comparable activity for gamma-glutamylcysteine (96%). The mutant enzyme shows slightly decreased maximum temperatures at 55°C (compared to 60°C for the wild-type enzyme)
S32T
-
the mutant enzyme oxidizes GSH more efficiently (178%) than the wild-type enzyme
S32T/N34A
-
the mutant enzyme shows almost the same activity for GSH (192%) compared to mutant enzyme S32A, S32T, and N34A, and high activity for gamma-glutamylcysteine (161%). The mutant enzyme shows slightly decreased maximum temperatures at 55°C (compared to 60°C for the wild-type enzyme)
W132A
-
the mutant enzyme oxidizes GSH and gamma-glutamylcysteine at much lower rates than the wild-type enzyme
additional information
C133S
site-directed mutagenesis, inactive mutant, no complementation of an enzyme-defect mutant strain, no complementation of an enzyme-defect mutant strain
C133S
-
imported into mitochondria with similar efficiencies as wild-type Erv1
C30S
site-directed mutagenesis, about 70% reduced activity in vitro compared to the wild-type enzyme, complementation of an enzyme-defect mutant strain
C30S
-
imported into mitochondria with similar efficiencies as wild-type Erv1
C30S/C33S
-
imported into mitochondria with similar efficiencies as wild-type Erv1
C30S/C33S
-
site-directed mutagenesis, the mutant shows the same protein-bound FAD spectrum as the wild-type enzyme Erv1p
additional information
construction of a His-tagged truncated enzyme form comprising the 15 kDa C-terminus, the mutant shows in vitro activity similar to the wild-type enzyme, dimerization behaviour of the mutant enzymes, overview
additional information
-
construction of a His-tagged truncated enzyme form comprising the 15 kDa C-terminus, the mutant shows in vitro activity similar to the wild-type enzyme, dimerization behaviour of the mutant enzymes, overview
additional information
-
the conserved C-terminal domain of the human Alrp can functionally replace the yeast domain in vivo, genetic system to study function of sulfhydryl oxidases, overview, enzyme-defective Erv1p mutant shows highly altered mitochondrial membrane morphology with loss of cristae, overview
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recobinant His6-tagged enzyme from Escherichia coli by nickel affinity chromatography and gel filtration
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli strains DH5alpha and BL21(DE3) of wild-type enzyme and a His-tagged truncated enzyme form comprising the 15 kDa C-terminus, expression of full-length point mutants
genes ERV1 and ERV2, DNA and amino acid sequence determination and analysis, gene structure, phylogenetic analysis
-
truncated version of Erv2p, without the N-terminal 34 residues, cloned into a pET24(a+) plasmid carrying kanamycin resistance. Amplified in DH5alpha Escherichia coli strain. Expressed from the Escherichia coli strain BL21(DE3) or BL21(DE3 star)
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
Erv1 represents a substrate of the Mia40-dependent translocation pathway
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Lange, H.; Lisowsky, T.; Gerber, J.; Muhlenhoff, U.; Kispal, G.; Lill, R.
An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins
EMBO Rep.
2
715-720
2001
Saccharomyces cerevisiae
Gerber, J.; Muhlenhoff, U.; Hofhaus, G.; Lill, R.; Lisowsky, T.
Yeast Erv2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family
J. Biol. Chem.
276
23486-23491
2001
Saccharomyces cerevisiae
Lee, J.E.; Hofhaus, G.; Lisowsky, T.
Erv1p from Saccharomyces cerevisiae is FAD-linked sulfhydryl oxidase
FEBS Lett.
477
62-66
2000
Saccharomyces cerevisiae
Thorpe, C.; Hoober, K.L.; Raje, S.; Glynn, N.M.; Burnside, J.; Turi, G.K.; Coppock, D.L.
Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes
Arch. Biochem. Biophys.
405
1-12
2002
Aspergillus niger, Cavia porcellus, Gallus gallus, Homo sapiens (O00391), Homo sapiens, Rattus norvegicus (Q6IUU3), Saccharomyces cerevisiae, Trypanosoma brucei
Hofhaus, G.; Lee, J.E.; Tews, I.; Rosenberg, B.; Lisowsky, T.
The N-terminal cysteine pair of yeast sulfhydryl oxidase Erv1p is essential for in vivo activity and interacts with the primary redox centre
Eur. J. Biochem.
270
1528-1535
2003
Saccharomyces cerevisiae (Q12284), Saccharomyces cerevisiae
Hofhaus, G.; Lisowsky, T.
Sulfhydryl oxidases as factors for mitochondrial biogenesis
Methods Enzymol.
348
314-324
2002
Saccharomyces cerevisiae, Homo sapiens
Vitu, E.; Bentzur, M.; Lisowsky, T.; Kaiser, C.A.; Fass, D.
Gain of function in an ERV/ALR sulfhydryl oxidase by molecular engineering of the shuttle disulfide
J. Mol. Biol.
362
89-101
2006
Arabidopsis thaliana, Saccharomyces cerevisiae (Q12284)
Gross, E.; Sevier, C.S.; Heldman, N.; Vitu, E.; Bentzur, M.; Kaiser, C.A.; Thorpe, C.; Fass, D.
Generating disulfides enzymatically: reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p
Proc. Natl. Acad. Sci. USA
103
299-304
2006
Saccharomyces cerevisiae
Wang, W.; Winther, J.R.; Thorpe, C.
Erv2p: characterization of the redox behavior of a yeast sulfhydryl oxidase
Biochemistry
46
3246-3254
2007
Saccharomyces cerevisiae
Fass, D.
The Erv family of sulfhydryl oxidases
Biochim. Biophys. Acta
1783
557-566
2008
African swine fever virus, Oryza sativa, Vaccinia virus, Saccharomyces cerevisiae (Q12284), Rattus norvegicus (Q63042), Arabidopsis thaliana (Q8GXX0)
Terziyska, N.; Grumbt, B.; Bien, M.; Neupert, W.; Herrmann, J.M.; Hell, K.
The sulfhydryl oxidase Erv1 is a substrate of the Mia40-dependent protein translocation pathway
FEBS Lett.
581
1098-1102
2007
Saccharomyces cerevisiae
Ang, S.K.; Lu, H.
Deciphering structural and functional roles of individual disulfide bonds of the mitochondrial sulfhydryl oxidase Erv1p
J. Biol. Chem.
284
28754-28761
2009
Saccharomyces cerevisiae
Guo, P.C.; Ma, J.D.; Jiang, Y.L.; Wang, S.J.; Bao, Z.Z.; Yu, X.J.; Chen, Y.; Zhou, C.Z.
Structure of yeast sulfhydryl oxidase erv1 reveals electron transfer of the disulfide relay system in the mitochondrial intermembrane space
J. Biol. Chem.
287
34961-34969
2012
Saccharomyces cerevisiae (P27882), Saccharomyces cerevisiae
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