Information on EC 1.1.1.82 - malate dehydrogenase (NADP+)

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

EC NUMBER
COMMENTARY hide
1.1.1.82
-
RECOMMENDED NAME
GeneOntology No.
malate dehydrogenase (NADP+)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(S)-malate + NADP+ = oxaloacetate + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
C4 photosynthetic carbon assimilation cycle, NADP-ME type
-
-
C4 photosynthetic carbon assimilation cycle, PEPCK type
-
-
Carbon fixation in photosynthetic organisms
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
Pyruvate metabolism
-
-
C4 and CAM-carbon fixation
-
-
photosynthesis
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SYSTEMATIC NAME
IUBMB Comments
(S)-malate:NADP+ oxidoreductase
Activated by light.
CAS REGISTRY NUMBER
COMMENTARY hide
37250-19-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
a strict aerobic hyperthermophilic archaeon isolated from a coastal thermal vent in Japan
Uniprot
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
weak activity
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
fragment
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
weak activity
-
-
Manually annotated by BRENDA team
(Poir.) C. Hubb.
SwissProt
Manually annotated by BRENDA team
L. Beauv., population Quebec and population Mississippi
-
-
Manually annotated by BRENDA team
SM-ZK
-
-
Manually annotated by BRENDA team
SM-ZK
-
-
Manually annotated by BRENDA team
A.M. Powell, clone B6
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
(L.) P. Beauv.
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
(Nees) Stapf
SwissProt
Manually annotated by BRENDA team
(Steud.) Hack.
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
no activity in Yarrowia lipolytica
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
(Lam.) Hack.
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
Sorghum sp.
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2S,3R)-tartrate + NAD(P)+
? + NAD(P)H + H+
show the reaction diagram
-
-
-
?
(2S,3S)-tartrate + NAD(P)+
? + NAD(P)H + H+
show the reaction diagram
-
-
-
?
(S)-malate + NAD(P)+
oxaloacetate + NAD(P)H + H+
show the reaction diagram
-
-
-
?
(S)-malate + NAD+
oxaloacetate + NADH + H+
show the reaction diagram
-
-
-
r
(S)-malate + NADP+
oxaloacetate + NADPH + H+
show the reaction diagram
oxaloacetate + NADH
(S)-malate + NAD+
show the reaction diagram
oxaloacetate + NADPH
(S)-malate + NADP+
show the reaction diagram
oxaloacetate + NADPH + H+
(S)-malate + NADP+
show the reaction diagram
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
(S)-malate + NADP+
oxaloacetate + NADPH + H+
show the reaction diagram
Q8H1E2
-
-
-
?
oxaloacetate + NADPH
(S)-malate + NADP+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD+
NADP+ is the preferred cofactor compared to NAD+ with substrate (S)-malate, while NAD+ is preferred with substrate tartrate. Ala at position 53 is responsible for coenzyme specificity, and the next residue, Arg, is important for NADP+ binding, structural analysis and comparison
NADP+
NADPH
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mn2+
-
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-oxoglutarate
-
5 mM
ATP
-
5 mM
citrate
-
5 mM
diethyldicarbonate
-
inactivation follows pseudo-first-order reaction kinetics, partial protection by NADP+, complete protection in presence of NADP+, 0.25 M hydroxylamine causes total reversal of inhibition
HgCl2
-
activity of the oxidized enzyme in presence of guanidine is unaffected by 0.1 mM HgCl2 in presence of 1 mM EDTA and by 0.005 mM HgCl2 in absence of EDTA, activity of the reduced form of enzyme is completely abolished
iodoacetamide
-
-
isocitrate
-
5 mM
KCl
-
oxidized truncated enzyme
malate
NADP+
NADPH
-
substrate inhibition
oxalate
-
5 mM
oxaloacetate
Urea
-
oxidized truncated enzyme
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acetone
-
increases the activity of the oxidized truncated enzyme form, optimal concentration 15%
carboxypeptidase Y
-
incubation of the native oxidized enzyme at pH 6.0, 25°C, results in slow activation. A small carboxy-terminal peptide of the native enzyme is accessible to proteolytic degradation followed by activation of the inactive oxidized enzyme. This peptide is involved in the regulation of activity, tetramer formation and thioredoxin binding
-
dithiothreitol
DTT
-
activates enzyme in free extracts
guanidine
-
can activate oxidized enzyme
methanol
-
increases the activity of the oxidized truncated enzyme form, optimal concentration 8%
reduced thioredoxin
-
thioredoxin
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.2 - 5.8
(2S,3R)-tartrate
1.2 - 3
(2S,3S)-tartrate
0.0145 - 32
(S)-malate
0.8
NAD+
-
cosubstrate malate
0.83
NADH
-
cosubstrate oxaloacetate
0.029 - 0.073
NADP+
0.011 - 0.472
NADPH
0.014 - 3
oxaloacetate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.16 - 0.2
(2S,3R)-tartrate
0.37 - 4.1
(2S,3S)-tartrate
0.5 - 233
(S)-malate
110 - 1520
oxaloacetate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
160
-
-
181
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enzyme from population Mississippi
211
-
enzyme from population Quebec
945
-
-
additional information
-
activity of NADP-MDH,PEPcase and PPDK are increased with leaf age after full expansion, reach the peak at around 32 days after emergence, and then fall down significantly
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 7.5
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reduction of oxaloacetate, oxidized truncated enzyme
6.5 - 8
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recombinant enzyme
7 - 8.5
-
activated enzyme
7.5 - 8.3
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-
8
-
assay at
8 - 8.5
-
active enzyme
8
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guanidine-activated oxidized enzyme and reduced enzyme
8 - 8.5
Sorghum sp.
-
-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8
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pH 5.5: about 90% of maximal activity, pH 8.0: about 40% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 55
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activity increases up to 3fold by increasing the temperature from 25°C to 55°C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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wall
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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in matrix and intermembrane space and slightly in inner membrane
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28400
-
2 * 28400, equilibrium sedimentation of the enzyme denatured by 6 M guanidinium chloride, pH 8, and 30 mM 2-mercaptoethanol
33000
Sorghum sp.
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x * 33000, DELTAN, a truncation mutant corresponding to the deletion of the 5 end of the mdh cDNA open reading frame until the 73rd codon, SDS-PAGE
33489
4 * 34000, recombinant enzyme, SDS-PAGE, 4 * 33489, sequence calculation
34000
4 * 34000, recombinant enzyme, SDS-PAGE, 4 * 33489, sequence calculation
36000
-
2 * 36000, mutant DELTAN, lacking the N-terminal 45 residues, SDS-PAGE
38000
-
2 * 38000, SDS-PAGE
38500
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1 * 38500, enzyme exists as monomer, dimer and tetramer, SDS-PAGE; 2 * 38500, enzyme exists as monomer, dimer and tetramer, SDS-PAGE; 4 * 38500, enzyme exists as monomer, dimer and tetramer, SDS-PAGE
38900
-
4 * 38900, native enzyme, SDS-PAGE
39000
-
2 * 39000, SDS-PAGE
40306
-
2 * 40306, enzyme from population Quebec, SDS-PAGE
41177
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2 * 41177, enzyme from population Mississippi, SDS-PAGE
42000
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2 * 42000, wild-type enzyme and mutant enzymes C23A, C28A, C23A/C28A, C23A/C28A/C206A, C23A/C28A/C364A, C23A/C28A/C376A, C23A/C28A/C206A/C364A, C23A/C28A/C206A/C376A and C23A/C28A/C364A/C376A, SDS-PAGE
42207
-
x * 42207, calculation from nucleotide sequence
50000 - 60000
-
unactivated enzyme, gel filtration
55000
-
equilibrium sedimentation
55680
-
equilibrium sedimentation
56000
-
gel filtration
57000
-
equilibrium sedimentation
61000
-
equilibrium sedimentation
72000
-
gel filtration
81000
-
gel filtration
83500
-
enzyme from population Quebec, non-denaturing PAGE
85450
-
isoenzyme 1 and 2 from Mississippi population, non-denaturing PAGE
86700
-
isoenzyme 3 from Mississippi population, non-denaturing PAGE
87000
-
dimer, gel filtration
88000
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gradient pore-PAGE
91000
-
gel filtration
98000
-
gel filtration
110000 - 120000
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unactivated enzyme, gel filtration
110000
recombinant enzyme, gel filtration
135000
-
gel filtration
143000 - 150000
-
activated enzyme, gel filtration
150000
-
gel filtration
163000
-
tetramer, gel filtration
additional information
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-
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
-
1 * 38500, enzyme exists as monomer, dimer and tetramer, SDS-PAGE
tetramer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
-
a small carboxy-terminal peptide of the native enzyme is accessible to proteolytic degradation followed by activation of the inactive oxidized enzyme. This peptide is involved in the regulation of activity, tetramer formation and thioredoxin binding
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified refolded reconbinant enzyme, sitting drop vapor diffusion method, 0.002 ml of protein solution containing 8.9 mg/ml protein in 50 mM Tris-HCl, pH7.5, containing 0.2 M NaCl, are mixed with an equal volume of the reservoir solution, comprised of 0.1 M CHES, pH 9.5, and 40% PEG 600, and equilibrated against 0.15 ml of reservoir solution at 20°C, 2 weeks, X-ray diffraction structure determination and analysis at 2.9 A resolution and room temperature
structure of the oxidized, inactive enzyme form containing the coenzyme NADP+ at a resolution of 2.8 A
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10.5
purified recombinant enzyme, 30 min, 50°C, stable
696461
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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pH 8.0, 10 min, reduced enzyme without NADPH, about 50% loss of activity
55
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pH 8.0, 10 min, oxidized enzyme without NADPH, about 75% loss of activity
60
-
pH 8.0, 10 min, oxidized enzyme in presence of NADPH, about 50% loss of activity
90
purified recombinant enzyme, 10 min, stable. The hyperthermostability of the Aeropyrum pernix MDH is likely attributable to its smaller cavity volume and larger numbers of ion pairs and ion-pair networks, but the molecular strategy for thermostability may be specific for each enzyme
100
purified recombinant enzyme, 10 min, stable. The hyperthermostability of the Aeropyrum pernix MDH is likely attributable to its smaller cavity volume and larger numbers of ion pairs and ion-pair networks, but the molecular strategy for thermostability may be specific for each enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
a small carboxy-terminal peptide of the native enzyme is accessible to proteolytic degradation followed by activation of the inactive oxidized enzyme. This peptide is involved in the regulation of activity, tetramer formation and thioredoxin binding
-
denaturation by 2 M guanidine-HCl
-
no effect of guanidine/HCl up to 0.25 M on the quarternary structure of the enzyme in its oxidized and reduced form. In the oxidized state the enzyme undergoes guanidine-dependent dissociation to the monomer with a midpoint of transition at 0.5 M. The kinetic of unfolding is significantly faster for the reduced than for the oxidized enzyme
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme undergoes reversible oxidation/reduction during its photoregulation
-
287411
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15°C, 37.5 mM Na-acetate, pH 5.5, 0.37 mM EDTA, 25% glycerol, no significant loss of activity after 3 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
4 isoenzymes
-
one protein band from the Quebec population, three isozymes from Mississippi population
-
partial, enzyme exists in at least two molecular forms
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recombinant MDH from Escherichia coli inclusion bodies by solubilization, refolding, dialysis, and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli, expression vectors pJ2PCC1 and pJ2PCC2
-
expression of MDH in Escherichia coli strain BL21(DE3) in inclusion bodies
high-level expression in Escherichia coli, wild-type enzyme and mutant enzymes C64S, C69S, C64S/C69S and DELTAN, a truncation mutant corresponding to the deletion of the 5' end of the mdh cDNA open reading frame until the 73rd codon
Sorghum sp.
-
structure and characterization of the gene, the gene contains 13 introns and 14 exons which encode a 429 amino acid precursor. The gene transcript contains an untranslated leader sequence of 44 nucleotides
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
biotic stress applied on C3 plants leads to increase in malate concentration during the night and in consequence to induction of isoform NADP-ME3 of NADP-dependent malate dehydrogenase
-
CAM-performing exhibit a decrease in malate concentration and decay in its diurnal fluctuations as a reaction to Botrytis cinerea infection. This correlates with significant decrease in activities of NADP-dependent malate dehydrogenase isoforms on the 2nd post infection as well as no fluctuations in their activities on the 9th day post infection
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C23A
-
minute activity under oxidizing conditions, up to 1300fold activation under reducing conditions
C23A/C28A
-
minute activity under oxidizing conditions, up to 1300fold activation under reducing conditions
C23A/C28A/C206A
-
only marginal activity in the oxidized state, does not exhibit full activity in the crude extract
C28A
-
minute activity under oxidizing conditions, up to 1300fold activation under reducing conditions
DELTAN
-
mutant lacking the N-terminal 45 residues
C207A
Sorghum sp.
-
strictly thioredoxin-dependent, totally insensitive to diethyl dicarbonate
C207A/DETAEV
Sorghum sp.
-
very low spontaneous activity, high Km-value for oxaloacetate, fast activation kinetics in the presence of reduced thioredoxin, ability to be activated by dithiothreitol alone at a slow rate
C207A/E387Q
Sorghum sp.
-
very low spontaneous activity, high Km-value for oxaloacetate, fast activation kinetics in the presence of reduced thioredoxin, ability to be activated by dithiothreitol alone at a slow rate
C24S/C207A
Sorghum sp.
-
oxidation-reduction midpoint potential is -310 mV at pH 7.0, compared to -330 mV for the wild-type enzyme
C24S/C29S
Sorghum sp.
-
oxidation-reduction midpoint potential is -280 mV at pH 7.0, compared to -330 mV for the wild-type enzyme
C29S
Sorghum sp.
-
strictly thioredoxin-dependent, totally insensitive to diethyl dicarbonate
C29S/C207A
Sorghum sp.
-
no spontaneous activity, activated much faster than the wild-type protein, strictly dependent on reduced thioredoxin for activation
C29S/C207A/DELTAEV
Sorghum sp.
-
high spontaneous activity, activated by reduced thioredoxin almost instantaneously and also by dithiothreitol alone, although at a much slower rate. The Km-values for both the oxidized and reduced enzyme show no significant differences in the apparent affinity for NADPH, whereas the Km for oxaloacetate is dramatically increased in the oxidized form
C29S/C207A/E387Q
Sorghum sp.
-
high spontaneous activity, activated by reduced thioredoxin almost instantaneously and also by dithiothreitol alone, although at a much slower rate. The Km-values for both the oxidized and reduced enzyme show no significant differences in the apparent affinity for NADPH, whereas the Km for oxaloacetate is dramatically increased in the oxidized form
C29S/DELTAEV
Sorghum sp.
-
very low spontaneous activity, high Km-value for oxaloacetate, fast activation kinetics in the presence of reduced thioredoxin, ability to be activated by dithiothreitol alone at a slow rate
C39S/E387Q
Sorghum sp.
-
very low spontaneous activity, high Km-value for oxaloacetate, fast activation kinetics in the presence of reduced thioredoxin, ability to be activated by dithiothreitol alone at a slow rate
C64S
Sorghum sp.
-
still requires activation by reduced thioredoxin, activation is almost instantaneous, whereas the native enzyme reaches full activity after 10-20 min of preincubation, the half-saturation concentration for reduced thioredoxin is decreased 2fold
C64S/C69S
Sorghum sp.
-
still requires activation by reduced thioredoxin, activation is almost instantaneous, whereas the native enzyme reaches full activity after 10-20 min of preincubation, the half-saturation concentration for reduced thioredoxin is decreased 2fold
C69S
Sorghum sp.
-
still requires activation by reduced thioredoxin, activation is almost instantaneous, whereas the native enzyme reaches full activity after 10-20 min of preincubation, the half-saturation concentration for reduced thioredoxin is decreased 2fold
D201A
Sorghum sp.
-
only slightly active, 80fold increased Km for oxaloacetate, Km-value for NADPH is slightly decreased
D201N
Sorghum sp.
-
only slightly active, 45fold increased Km for oxaloacetate, Km-value for NADPH is slightly decreased
DELTAEV
Sorghum sp.
-
mutant with the two most C-terminal residues deleted, NADP+ does not inhibit activation, activation time course of thioredoxin-dependent activation of both mutant proteins is similar to that of the wild-type protein
DELTAN
Sorghum sp.
-
truncation mutant corresponding to the deletion of the 5' end of the mdh cDNA open reading frame until the 73rd codon
E387Q
Sorghum sp.
-
NADP+ does not inhibit activation, activation time course of thioredoxin-dependent activation of both mutant proteins is similar to that of the wild-type protein
G84D
Sorghum sp.
-
10fold lower Km for NADH, Km for NADPH remains unchanged
G84D/S851I/R87Q/S88A
Sorghum sp.
-
changed cofactor specificity from NADPH to NADH, the activation of the NAD-specific thiol-regulated enzyme is inhibited by NAD+ but no longer by NADP+
H229N
Sorghum sp.
-
no activity
H229Q
Sorghum sp.
-
no activity
S85I/R87Q/S88A
Sorghum sp.
-
7fold increase in the Km-value for NADPH and 4fold decrease in Km-value for NADH
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
renaturation of the guanidine-HCl denatured enzyme is more rapid with the reduced enzyme and occurs with higher yields, 100%, than with the oxidized enzyme, 60-80%
-
solubilization and refolding of recombinant MDH from Escherichia coli inclusion bodies, the enzyme is dissolved in 6 M guanidine-HCl and gradually refolded to the active enzyme through dilution of the denaturant, conditions: refolding buffer 1.5 L of 0.1 M Tris-HCl, pH 7.5, containing 2 mM EDTA and 0.4 M L-arginine, incubation for 36 h at 4°C
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
-
changes in malate concentration and activity of NADP-dependent malate dehydrogenase are the effect of Botrytis cinerea infection of C3 or CAM-performing Mesembryanthemum crystallinum plants. Biotic stress applied on C3 plants leads to increase in malate concentration during the night and in consequence lead to increase in malate day/night fluctuations in infected leaves on the 2nd day post infection. It corresponds with induction of an additional isoform of NADP-dependent malate dehydrogenase, NADP-ME3. On the contrary, CAM-performing plants exhibit a decrease in malate concentration and a decay in its diurnal fluctuations as a reaction to Botrytis cinerea infection. This correlates with significant decrease in activities of NADP-dependent malate dehydrogenase isoforms