Information on EC 1.8.4.9 - adenylyl-sulfate reductase (glutathione)

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

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EC NUMBER
COMMENTARY hide
1.8.4.9
-
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RECOMMENDED NAME
GeneOntology No.
adenylyl-sulfate reductase (glutathione)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
AMP + sulfite + glutathione disulfide = adenylyl sulfate + 2 glutathione
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
selenate reduction
-
-
sulfate reduction II (assimilatory)
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-
sulfate reduction
-
-
Sulfur metabolism
-
-
Microbial metabolism in diverse environments
-
-
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SYSTEMATIC NAME
IUBMB Comments
AMP,sulfite:glutathione-disulfide oxidoreductase (adenosine-5'-phosphosulfate-forming)
This enzyme differs from EC 1.8.99.2, adenylyl-sulfate reductase, in using glutathione as the reductant. Glutathione can be replaced by gamma-glutamylcysteine or dithiothreitol, but not by thioredoxin, glutaredoxin or mercaptoethanol. The enzyme from the mouseear cress, Arabidopsis thaliana, contains a glutaredoxin-like domain. The enzyme is also found in other photosynthetic eukaryotes, e.g., the Madagascar periwinkle, Catharanthus roseus and the hollow green seaweed, Enteromorpha intestinalis.
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CAS REGISTRY NUMBER
COMMENTARY hide
9027-75-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain DSM 4304T
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-
Manually annotated by BRENDA team
fragment; autotrophic sulphide-oxidizing bacterium, symbiont of the clam Lucinoma kazani
SwissProt
Manually annotated by BRENDA team
indian mustard
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
L. Merr., cv Williams 82
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-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Tetraselmis sp.
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3'-phosphoadenosine 5'-phosphosulfate + dithioerythritol
?
show the reaction diagram
-
-
-
-
?
3'-phosphoadenosine 5'-phosphosulfate + glutathione
adenosine 3',5'-diphosphate + sulfite + oxidized glutathione
show the reaction diagram
-
-
-
-
?
3'-phosphoadenosine-5'-phosphosulfate + glutathione
adenosine 3',5'-bisphosphate + sulfite + oxidized glutathione
show the reaction diagram
-
-
-
-
?
3'-phosphoadenosine-5'-phosphosulfate + glutathione
adenosine 3',5'-diphosphate + sulfite + oxidized glutathione
show the reaction diagram
5'-adenylyl sulfate + glutathione
AMP + sulfite + glutathione disulfide
show the reaction diagram
5'-adenylyl sulfate + thioredoxin
AMP + sulfite + thioredoxin disulfide
show the reaction diagram
-
recombinant enzyme in transgenic Arabidopsis thaliana plants
-
-
?
5'-adenylylsulfate + dithioerythritol
adenosine 5'-monophosphate + sulfite + oxidized dithioerythritol
show the reaction diagram
5'-adenylylsulfate + dithiothreitol
adenosine 5'-monophosphate + sulfite + oxidized dithiothreitol
show the reaction diagram
5'-adenylylsulfate + glutathione
adenosine monophosphate + sulfite + oxidized glutathione
show the reaction diagram
5'-adenylylsulfate + glutathione + glutathione
adenosine monophosphate + sulfite + oxidized glutathione
show the reaction diagram
adenosine 5'-phosphosulfate + glutathione
?
show the reaction diagram
-
PpAPR-B is much more active with adenosine 5'-phosphosulfate as a substrate than with 3'-phosphoadenosine 5'-phosphosulfate
-
-
?
adenylyl sulfate + DTT
?
show the reaction diagram
-
-
-
-
?
adenylyl sulfate + glutathione
AMP + sulfite + glutathione disulfide
show the reaction diagram
cystine + glutathione
cysteine + glutathione disulfide
show the reaction diagram
catalyzed by the holoenzyme APR1p and the C-domain
-
-
?
dehydroascorbate + glutathione
ascorbate + glutathione disulfide
show the reaction diagram
catalyzed by the holoenzyme APR1p and the C-domain
-
-
?
hydroxyethyldisulfide + glutathione
?
show the reaction diagram
catalyzed by the holoenzyme APR1p and the C-domain
-
-
?
insulin disulfide + glutathione
insulin + glutathione disulfide
show the reaction diagram
catalyzed by the C-domain, not by the holoenzyme APR1p or the R-domain
-
-
?
oxidized ribonucleotide reductase + glutathione
reduced ribonucleotide reductase + glutathione disulfide
show the reaction diagram
catalyzed by the C-domain, not by the holoenzyme APR1p or the R-domain
-
-
?
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
5'-adenylyl sulfate + glutathione
AMP + sulfite + glutathione disulfide
show the reaction diagram
5'-adenylyl sulfate + thioredoxin
AMP + sulfite + thioredoxin disulfide
show the reaction diagram
-
recombinant enzyme in transgenic Arabidopsis thaliana plants
-
-
?
adenylyl sulfate + glutathione
AMP + sulfite + glutathione disulfide
show the reaction diagram
additional information
?
-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
glutathione
thioredoxin
additional information
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
(NH4)2SO4
-
500 mM, stimulates; highly activating
KNO3
-
500 mM, stimulates
MgCl2
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500 mM, stimulates
MgSO4
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500 mM, stimulates
NaCl
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all three APR isoforms increased 3fold in roots after 5 h of treatment with 150 mM NaCl, regulation of salt stress by plant hormone signalling, overview
additional information
-
no iron is associated with recombinant PpPAPR
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5'-adenylylsulfate
adenosine 5'-phosphosulfate
-
inhibits activity with 3'-phosphoadenosine 5'-phosphosulfate
adenosine-5'-phosphate
AMP
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competitive inhibitor with respect to 5'-adenylyl sulfate
glutathione
KNO3
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abolishes enzyme activation by other salts and inhibits the enzyme
additional information
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sulfite (up to 100 mM) has no inhibitory effect
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(NH4)2SO4
-
less effective than Na2SO4
DTT
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DTT can replace glutathione as reductant, 5 mM DTT increases activity 40fold
sulfate
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optimal concentration between 350 and 500 mM
additional information
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.02
3'-phosphoadenosine 5'-phosphosulfate
-
-
0.031
3'-phosphoadenosine-5'-phosphosulfate
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assay conditions: APS reductase (0.0009 mM), EST (0.10 unit/ml), thioredoxin (0.037 mM), MgCl2 (3.0 mM), DTT (2.0 mM), pNPS (10 mM), GSH (5.0 mM), and Hepes (50 mM, pH 8.0), at 25°C
0.011 - 0.0174
5'-adenylyl sulfate
0.00057 - 0.0122
5'-adenylylsulfate
0.05
adenosine 5'-phosphosulfate
-
-
0.595
dithiothreitol
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30°C, pH 8.5
0.4
DTT
-
with 5'-adenylylsulfate as substrate
0.0002 - 16.8
glutathione
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00075
3'-phosphoadenosine 5'-phosphosulfate
-
-
0.012
3'-phosphoadenosine-5'-phosphosulfate
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assay conditions: APS reductase (0.0009 mM), EST (0.10 unit/ml), thioredoxin (0.037 mM), MgCl2 (3.0 mM), DTT (2.0 mM), pNPS (10 mM), GSH (5.0 mM), and Hepes (50 mM, pH 8.0), at 25°C
8.4 - 11.3
5'-adenylyl sulfate
0.153 - 56
5'-adenylylsulfate
0.176
adenosine 5'-phosphosulfate
-
-
10.1
dithiothreitol
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30°C, pH 8.5
8.4
glutathione
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30°C, pH 8.5
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0074 - 0.081
5'-adenylylsulfate
0.176
adenosine 5'-phosphosulfate
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-
1
AMP
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-
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000002
-
with 3'-phosphoadenosine 5'-phosphosulfate as substrate and 5 mM DTT, recombinant protein expressed in Escherichia coli
0.000007
-
with 3'-phosphoadenosine 5'-phosphosulfate as substrate and 5 mM DTT, recombinant protein expressed in Escherichia coli
0.00022
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with 5'-adenylylsulfate as substrate and 5 mM DTT, recombinant protein expressed in Escherichia coli
0.0005 - 0.005
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crude cell lysate
0.00059
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with 5'-adenylylsulfate as substrate and 5 mM DTT, recombinant protein expressed in Escherichia coli
0.00077
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wild-type plants
0.001
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crude cell lysate from young leaves
0.0013
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crude cell lysate from young leaves
0.0023 - 0.006
0.004 - 0.02
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crude cell lysate
0.013
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after purification of His-tagged protein with Nickel-affinity chromatography
0.02 - 0.025
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crude extract, plants treated with 10 mM NH4Cl
0.04
-
purified recombinant enzyme
0.05 - 0.1
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crude cell lysate
0.08 - 0.13
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crude cell lysate
0.1 - 0.4
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crude cell lysate
0.1 - 0.25
-
-
0.1 - 0.5
Tetraselmis sp.
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crude cell lysate
0.1 - 0.25
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crude cell lysate
0.2
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crude cell lysate
0.207
-
-
additional information
-
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
substrate: 3'-phosphoadenosine 5'-phosphosulfate
8.5
-
recombinant protein, expressed in Escherichia coli
8.8
-
recombinant protein, expressed in Escherichia coli
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 25
-
purified, recombinant enzyme, Ea 10.1 kcal per mol between 0°C and 20°C
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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accumulation of enzyme mRNA at 25°C, 3fold increased enzyme activity at 12°C
Manually annotated by BRENDA team
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-
Manually annotated by BRENDA team
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enzyme mRNA appears after chilling
Manually annotated by BRENDA team
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sulfur and phosphorus deprivation increases expression level, while nitrogen starvation represses APS reductase mRNA transcript and protein levels
Manually annotated by BRENDA team
additional information
-
chilling at 12°C induces oxidative stress which increases oxidized glutathione concentration in mesophyll cells and bundle-sheath, increases enzyme activity in bundle-sheath cells, mesophyll cells, and root tips, chilling alters the intercellular distribution of the enzyme
Manually annotated by BRENDA team
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20000
-
2 * 75000 + 2 * 20000
29000
-
x * 29000, recombinant enzyme, SDS-PAGE
32000
-
x * 32000, SDS-PAGE
43640
-
calculated from cDNA sequence
45000
-
mature protein without chloroplast transit peptide, calculated from cDNA sequence
45600
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clone APR2, calculated from cDNA sequence
45700
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mature protein without chloroplast transit peptide, calculated from cDNA sequence
46000
-
2 * 46000, SDS-PAGE
47300
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clone APR3, calculated from cDNA sequence
50000
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x * 50000, recombinant His-tagged PpAPR, SDS-PAGE
50450
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clone Prh-43, calculated from cDNA sequence
50500
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clone Prh-26, calculated from cDNA sequence
50800
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clone APR1, calculated from cDNA sequence
51300
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clone Prh-19, calculated from cDNA sequence
51390
-
calculated from cDNA sequence
75000
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2 * 75000 + 2 * 20000
91300
-
estimation by gel filtration with Superose 12
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotetramer
-
2 * 75000 + 2 * 20000
additional information
amino acid residues 73-327 form the R-domain which is homologous to microbial 5'-phosphoadenylylsulfate reductase, and of residues 328-465 form the C-domain which is homologous to thioredoxin, the domains alone are inactive, but mixing of both can partially rstore activity
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour diffusion
-
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme is highly stable in vitro
-
enzyme unstable during purification and storage, addition of 5 mM 5'-adenosine monophosphate stabilizes activity
-
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 1 day, PpAPR is completely inactivated, plant APRs are very unstable enzymes because of the rapid loss of their iron-sulfur clusters
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-20°C, 1 day, PpAPR-B retains 30% of its activity
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4°C, PpAPR, completely stable for 48 h, inactivation after longer than 2 dayswith 15% remaining activity after 4-5 days
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4°C, PpAPR-B is stable over 5 days
-
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purification of recombinant 6xHis-tagged protein with Nickel-affinity chromatography
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purification to homogeneity
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recombinant holoenzyme APR1p, C-domain, and R-domain from Escherichia coli strain BL21(DE3)
recombinant N-terminally His-tagged PpAPR from Escherichia coli
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Sepharose column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a heterologous system is constructed in which the C domain of EiAPR was fused to the carboxyl terminus of the APS reductase from Pseudomonas aeruginosa (PaAPR), an enzyme that normally uses thioredoxin as an electron donor and is incapable of using glutathione for this function. The hybrid enzyme, which retains the [4Fe-4S] cluster from PaAPR can use both thioredoxin and glutathione as an electron donor for 5'-adenylylsulfate reduction. Expression in Escherichia coli
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APR, DNA and amino acid sequence determination and analysis, semiquantitative expression analysis, overexpression of His-tagged APR isozyme in Escherichia coli
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expressed in Escherichia coli strain BL21(DE3)-pLysS
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expression in Escherichia coli
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expression of N-terminally His-tagged PpAPR in Escherichia coli, expression of GFP-tagged APR in Physcomitrella patens plants using the mAV4 vector containing the chloroplast transit peptide from Physcomitrella patens FtsZ2-1 and transient transfection of protoplasts; overexpression in Escherichia coli
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gene APR1, expression of the holoenzyme APR1p in Escherichia coli strain BL21(DE3), separate expression in Escherichia coli strain BL21(DE3) of amino acid residues 73-327, forming the R-domain, and of residues 328-465, forming the C-domain, the domains alone are inactive, but mixing of both can partially restore activity
recombinant expression of the enzyme, fused to the transit peptide rbcS, in Arabidopsis thaliana under transcriptional control of the CaMV 35S promotor
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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LINKS TO OTHER DATABASES (specific for EC-Number 1.8.4.9)
ExplorEnz
ExPASy
KEGG
MetaCyc
SABIO-RK
NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
InterPro (database of protein families, domains and functional sites)