Information on EC 1.6.5.4 - monodehydroascorbate reductase (NADH)

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

EC NUMBER
COMMENTARY hide
1.6.5.4
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RECOMMENDED NAME
GeneOntology No.
monodehydroascorbate reductase (NADH)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
NADH + H+ + 2 monodehydroascorbate = NAD+ + 2 ascorbate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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redox reaction
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
ascorbate recycling (cytosolic)
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Ascorbate and aldarate metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
NADH:monodehydroascorbate oxidoreductase
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CAS REGISTRY NUMBER
COMMENTARY hide
9029-26-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
var. columbia
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
acerola
UniProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
interspecific cross of Nicotiana lansdorfii x Nicotiana sanderae
SwissProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
Pterocladia sp.
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
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
2 ferricyanide + NADH
2 ferrocyanide + NAD+ + H+
show the reaction diagram
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enzyme also shows diaphorase activity, i.e. ferricyanide reductase
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?
2 ferricyanide + NADPH
2 ferrocyanide + NADP+ + H+
show the reaction diagram
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?
2 ferricytochrome b5 + NADH
2 ferrocytochrome b5 + NAD+ + H+
show the reaction diagram
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?
2 ferricytochrome c + NADH
2 ferrocytochrome c + NAD+ + H+
show the reaction diagram
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?
CO2 + H2O
HCO3- + H+
show the reaction diagram
bifunctional enzyme showing monodehydroascorbate reductase and carbonic anhydrase, EC 4.2.1.1, activities
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?
ferricyanide + reduced 2,6-dichlorophenolindophenol
ferrocyanide + 2,6-dichlorophenolindophenol
show the reaction diagram
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enzyme also shows diaphorase activity, i.e. ferricyanide reductase
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?
monodehydroascorbate + NADH
ascorbate + NAD+
show the reaction diagram
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?
NAD(P)H + monodehydroascorbate
NAD(P)+ + ascorbate
show the reaction diagram
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reduction of the radical monodehydroascorbate, via FAD as intermediary electron acceptor, FAD reduction by NADH producing a charge-transfer complex
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?
NADH + 1,4-benzoquinone
NAD+ + ?
show the reaction diagram
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?
NADH + 2,6-dichlorophenol indophenol
NAD+ + ?
show the reaction diagram
NADH + H+ + 2 monodehydroascorbate
NAD+ + 2 ascorbate
show the reaction diagram
NADH + monodehydroascorbate
NAD+ + ascorbate
show the reaction diagram
NADH + monodehydroisoascorbate
NAD+ + ?
show the reaction diagram
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?
NADH + monodehydroisoascorbate
NAD+ + isoascorbate
show the reaction diagram
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?
NADH + p-benzoquinone
NAD+ + ?
show the reaction diagram
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?
NADPH + H+ + 2 monodehydroascorbate
NADP+ + 2 ascorbate
show the reaction diagram
NADPH + monodehydroascorbate
NADP+ + ascorbate
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
CO2 + H2O
HCO3- + H+
show the reaction diagram
Q84UV8
bifunctional enzyme showing monodehydroascorbate reductase and carbonic anhydrase, EC 4.2.1.1, activities
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?
NADH + H+ + 2 monodehydroascorbate
NAD+ + 2 ascorbate
show the reaction diagram
NADH + monodehydroascorbate
NAD+ + ascorbate
show the reaction diagram
NADPH + H+ + 2 monodehydroascorbate
NADP+ + 2 ascorbate
show the reaction diagram
additional information
?
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Q84UV8
reaction scheme, the enzyme is involved in nectary production in floral organs, multiple molecular mechanism responsible for expression in the nectary gland
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD(P)H
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NADPH
additional information
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MDHAR catalyses the reduction of the MDHA to AsA, using NADH or NADPH as an electron donor
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Zn2+
coordinated by residues His122, His124, and His141
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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10 mM, 16% inhibition
1-(2-thenoyl)-3,3,3-trifluoracetone
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0.3 mM, about 10% inhibition
2,2'-dipyridyl
2,4,6-Trinitrobenzenesulfonic acid
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1 mM, 54% inhibition
2-Iodoacetamide
2-[[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-amino]ethanesulfonic acid
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5,5'-dithiobis(2-nitrobenzoic acid)
8-hydroxyquinoline
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10 mM, 15% inhibition
beta-chloromercuribenzoate
PpMDHAR1 is almost completely inactivated after a 20 min preincubation with 0.25 mM beta-chloromercuribenzoate. At 0.05 mM the enzyme retains 11% of the initial activity.
chloromercuribenzoate
at 0.05 mM PpMDHAR3 retains 80% of the initial activity; PpMDHAR2 is almost completely inactivated after a 20 min preincubation with 0.25 mM chloromercuribenzoate. At 0.05 mM the enzyme retains 14% of the initial activity. The C69A mutant is more resistant to the inhibitory effects of chloromercuribenzoate than the wild type PpMDHAR2 protein
citrate
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1 mM, about 10% inhibition
Cu2+
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0.5 mM, complete inhibition
diacetyl
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1 mM, 55% inhibition
dicoumarol
diethyl dicarbonate
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0.1 mM, 62% inhibition
EDTA
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10 mM, 11% inhibition
Hg2+
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0.5 mM, complete inhibition
Imidazole buffer
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Insulin
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inhibition of the enzyme in plasma membrane
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iodoacetamide
iodoacetate
lectin
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ConA, WGA and LFA
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Mersalyl
NaN3
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16 mM, 16% inhibition
p-chloromercuribenzoate
phenyl mercuric acetate
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0.1 mM, 85% inhibition
Phenylglyoxal
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100 mM, 46% inhibition
phosphate
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pyridoxal 5'-phosphate
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1 mM, 46% inhibition
sodium diethyldithiocarbaminate
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1 mM, about 10% inhibition
Thenoyltrifluoroacetone
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0.15 mM, complete inhibition
thiol-modifiying reagents
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inhibition at the step of FAD reduction by NADH producing a charge-transfer complex
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Trinitromethane
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10 mM, 11% inhibition
additional information
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not inhibited by 5 mM N-ethylmaleimide
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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1 mM, stimulates
diphosphate
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1 mM, stimulates
NADH
NADH is the preferred electron donor
NADPH
NADH is the preferred electron donor
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.03
2,6-dichloroindophenol
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0.052
2,6-dichlorophenol indophenol
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maltose-binding protein-monodehydroascorbate reductase fusion protein
0.0013 - 0.012
ferricyanide
0.00144 - 0.6
monodehydroascorbate
0.0006 - 0.05
NADH
0.0215 - 0.99
NADPH
0.0012 - 0.005
semidehydroascorbic acid
additional information
additional information
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kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
62.67 - 200
NADH
150
NADPH
Cucumis sativus
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4290 - 20420
NADH
8
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.095
dicoumarol
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.056
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cell extract, wild type enzyme
13.1
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cell extract, recombinant enzyme
24
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after 429fold purification, wild type enzyme
100
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after 7.6fold purification, recombinant enzyme
185
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using ascorbate free radical as substrate
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 7
activity at pH 6 is higher than at pH 7
7
stable pH of nectar in vivo
7.8
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9.5
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pH 5.5: about 60% of maximal activity, pH 9.5: about 75% of maximal activity
6.7 - 7.9
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about 50% of maximal activity at pH 6.7 and at pH 7.9
7.6 - 8.7
85% or greater activity between pH 7.6 and pH 8.7; 85% or greater activity between pH 7.6 and pH 8.7; 85% or greater activity between pH 7.6 and pH 8.7
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.9
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major enzyme form, native isoelectric focusing
7
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minor enzyme forms, native isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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normal and virus-transformed
Manually annotated by BRENDA team
lower expression level
Manually annotated by BRENDA team
early to post-fertilized
Manually annotated by BRENDA team
different developmental stages, high expression level
Manually annotated by BRENDA team
lower expression level
Manually annotated by BRENDA team
lower expression level
Manually annotated by BRENDA team
lower expression level
Manually annotated by BRENDA team
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etiolated hook
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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activity of the enzyme depends on the interaction of proteins and lipids and is therefore of necessity dependent upon the integrity of a certain micellar structure in the endoplasmic reticulum
Manually annotated by BRENDA team
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000
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SDS-PAGE
42000
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gel filtration
47350
calculated from amino acid sequence
54000
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MDAR4, SDS-PAGE
66000
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gel filtration
127000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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2 * 62000, SDS-PAGE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme, immediately after purification by hanging drop vapour diffusion method, 0.005 ml of protein solution containing 10 mg/ml protein, mixed with equal volume of reservoir solution containing 20-25% PEG 6000, 0.1 M CaCl2, 50 mM Tris-HCl, pH 8.0, equilibration against 1 ml reservoir solution, 20C, 1 week, cryoprotection by 30% glycerol, X-ray diffraction structure determination and analysis at 2.4 A resolution
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 11
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stable
392751
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35 - 62
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the activity of wild type MDAR has no much change after heat treatment at temperatures below 35C, steep decrease of activity is observed between 35 and 55C, and a complete loss in activity occurs above 62C. The midpoint of thermal inactivation of wild type MDAR is about 52.1C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, maltose-binding protein-monodehydroascorbate reductase fusion protein, in presence of 2-mercaptoethanol, 1 month, stable
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freezing without glycerol, complete loss of activity
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in whole glyoxysomes the enzyme is insensitive to trypsin
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 10 mM Tris/HCl buffer, pH 7.4, 2 mM dithiothreitol, 25% glycerol, 1 month, stable
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-80C, buffer containing 10 mM potassium phosphate at pH 6.5 10 mM 2-mercaptoethanol and 0.1 mM EDTA, several months, no loss of activity
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4C, 4C, in absence of dithiothreitol, complete loss of activity within 1 day
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4C, pH 8, 30 d, almost complete loss of activity, residual activity up to 38% when stored in presence of 1-10 mM MgSO4
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Butyl-Toyopearl column chromatography, HiTrap Q column chromatography and Blue Sepharose 6FF column chromatography
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glutathione Sepharose column chromatography
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Ni-NTA agarose chromatography
Phenyl Superose gel filtration, Blue Sepharose column chromatography and DEAE cellulose column chromatography
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recombinant enzyme
recombinant from Escherichia coli
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several native enzyme forms from lens soluble fraction by gel filtration, affinity chromatography, native isoelectric focusing, and 2D-electrophoresis, about 1400fold
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis
DNA and amino acid sequence determination and analysis, construction of a cDNA library, expression in Escherichia coli
expressed as GST-tagged enzyme in Escherichia coli BL21 cells
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expressed in Escherichia coli
expressed in Escherichia coli BL21(DE3) cells and in Nicotiana tabacum cv. petit havana
expressed in Nicotiana tabacum
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expressed in Nicotiana tabacum; overexpression in Nicotiana tabacum. The transgenic plants exhibit up to 2.1fold higher monodehydroascorbate reductase activity and 2.2fold higher level of reduced ascorbate compared to non-transformed control plants. The transgenic plants show enhanced stress tolerance in term of significantly higher net photosynthesis rates under ozone, salt and polyethylene glycol stresses and greater PSII effective quantum yield under ozone and salt stresses
expression in Escherichia coli
expression in Escherichia coli fused to maltose-binding protein
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expression in Escherichia coli; expression in Escherichia coli; expression in Escherichia coli
expression of mitochondrial and chloroplastic isozymes as GFP-fusion proteins in transgenic Arabidopsis plants, targeting of recombinant fusion proteins to the organelles in vivo in transgenic plants, dual targeting is achieved by multiple transcription initiation sites, 2 transcripts, a small and a large one, both have an N-terminal extension
gene MDHAR3, functional overexpression in transgenic Solanum lycopersicum plant leaves, quantitative reverse-transcriptase PCR expression analysis
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high level expression in Escherichia coli, using the T7 RNA polymerase expression system
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overexpression in Escherichia coli
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overproduced in Escherichia coli
quantitative reverse-transcription PCR expression analysis, functional recombinant enzyme overexpression driven by a CaMV double 35S promoter in Oryza sativa in three stable transgenic lines, MT22, MT24 and MT25 as single or multiple copy gene, using the Agrobacterium tumefaciens strain LBA4404 transfection method. All transgenic lines show better yield attributes such as a higher tiller number and increased 1000-grain weight compared to non-transgenics. They also show tolerance to salt at germination and seedling stages
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
ascorbate, the MDAR2 (At3g09940) and the dehydroascorbate reductase 5 (DHAR5, At1g19570) mRNA levels are upregulated in Arabidopsis roots colonized by the beneficial endophytic fungus Piriformosporaindica
exposure to 0.4 mM aluminium decreases the activity of MDAR
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MDAR is inducible after exposure to 200 mM NaCl
MDHAR transcripts and enzyme activities are significantly up-regulated in the leaves of acerola under cold (4C for 24 h) and salt (treatment with 150 mM NaCl over 9 days) stress conditions. MDHAR activity increases gradually and significantly as ripening progresses,with overripe fruits having the highest activity
the MDHAR expression level is decreased firstly and then increased, the maximum level is observed after treatment for 12 h with 200 mM NaCl or 20% PEG6000, overview. The stressful treatment accelerates degradation of mRNA at the early stage, then with the duration of treatment the plants respond to the stress and the synthesis of MDHAR increases to protect the plant from stressful damage
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C69A
mutation has a negligible effect on enzyme activity under standard reaction conditions. The C69A mutant is more resistant to the inhibitory effects of beta-chloromercuribenzoate than the wild type PpMDHAR2 protein
C117A
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the binding affinity of NADH in the mutant MDAR is lower than that of wild type MDAR as indicated by about 1.5-3fold increase of the Km value for NADH, the mutant is less thermo-stable than the wild type enzyme
C117S
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the binding affinity of NADH in the mutant MDAR is lower than that of wild type MDAR as indicated by about 1.5-3fold increase of the Km value for NADH, the mutant is less thermo-stable than the wild type enzyme
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
using 0.8 M guanidine-HCl at 25C for 24 h
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