Information on EC 1.6.1.5 - proton-translocating NAD(P)+ transhydrogenase

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

EC NUMBER
COMMENTARY hide
1.6.1.5
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RECOMMENDED NAME
GeneOntology No.
proton-translocating NAD(P)+ transhydrogenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
NADPH + NAD+ + H+[side 1] = NADP+ + NADH + H+[side 2]
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
NAD phosphorylation and dephosphorylation
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NAD phosphorylation and transhydrogenation
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SYSTEMATIC NAME
IUBMB Comments
NADPH:NAD+ oxidoreductase (H+-transporting)
The enzyme is a membrane bound proton-translocating pyridine nucleotide transhydrogenase that couples the reversible reduction of NADP by NADH to an inward proton translocation across the membrane. In the bacterium Escherichia coli the enzyme provides a major source of cytosolic NADPH. Detoxification of reactive oxygen species in mitochondria by glutathione peroxidases depends on NADPH produced by this enzyme.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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transhydrogenase PntAB is a major source of NADPH that is required for biosynthesis in Escherichia coli
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,N6-etheno-NADPH + NAD+ + H+[side 1]
1,N6-etheno-NADP+ + + NADH + H+[side 2]
show the reaction diagram
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r
1,N6-etheno-NADPH + oxidized acetyl pyridine adenine dinucleotide + H+[side 1]
1,N6-etheno-NADP+ + reduced acetyl pyridine adenine dinucleotide + H+[side 2]
show the reaction diagram
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r
deamino-NADPH + NAD+ + H+[side 1]
deamino-NADP+ + + NADH + H+[side 2]
show the reaction diagram
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-
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r
deamino-NADPH + oxidized acetyl pyridine adenine dinucleotide + H+[side 1]
deamino-NADP+ + reduced acetyl pyridine adenine dinucleotide + H+[side 2]
show the reaction diagram
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-
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r
NADPH + 3-acetylpyridine-NAD+ + H+[side 1]
NADP+ + 3-acetylpyridine-NADH + H+[side 2]
show the reaction diagram
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?
NADPH + APAD+ + H+[side 1]
NADP+ + APADH + H+[side 2]
show the reaction diagram
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?
NADPH + NAD+ + H+/in
NADP+ + NADH + H+/out
show the reaction diagram
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r
NADPH + NAD+ + H+[side 1]
NADP+ + NADH + H+[side 2]
show the reaction diagram
NADPH + oxidized 3-acetylpyridin-adenine dinucleotide + H+[side 1]
NADP+ + reduced 3-acetylpyridin-adenine dinucleotide + H+[side 2]
show the reaction diagram
NADPH + oxidized 3-acetylpyridine adenine dinucleotide + H+[side 1]
NADP+ + reduced 3-acetylpyridine adenine dinucleotide + H+[side 2]
show the reaction diagram
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r
NADPH + oxidized acetyl pyridine adenine dinucleotide + H+/in
NADP+ + reduced acetyl pyridine adenine dinucleotide + H+/out
show the reaction diagram
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r
NADPH + oxidized acetyl pyridine adenine dinucleotide + H+[side 1]
NADP+ + reduced acetyl pyridine adenine dinucleotide + H+[side 2]
show the reaction diagram
thio-NADP+ + NADH + H+[side 2]
thio-NADPH + NAD+ + H+[side 1]
show the reaction diagram
thio-NADPH + NAD+ + H+[side 1]
thio-NADP+ + NADH + H+[side 2]
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
NADPH + NAD+ + H+/in
NADP+ + NADH + H+/out
show the reaction diagram
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r
NADPH + NAD+ + H+[side 1]
NADP+ + NADH + H+[side 2]
show the reaction diagram
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cd2+
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low concentrations of Cd2+ strongly inhibit revers transhydrogenation
N,N'-dicyclohexylcarbodiimide
N-cyclohexyl-N'-(4-dimethylaminonaphthyl)carbodiimide
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about 25% residual activity after 200 min at 0.5 mM
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NADH
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high concentrations of NADH lead to substrate inhibition
oxidized 3-acetylpyridin-adenine dinucleotide
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high concentrations of oxidized 3-acetylpyridin-adenine dinucleotide lead to substrate inhibition
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valinomycin
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Zn2+
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low concentrations of Zn2+ strongly inhibit revers transhydrogenation
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
carbonylcyanide-4-trifluoromethoxyphenyl hydrazone
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0.002 mM stimulates the rate of the forward reaction by 8fold and of the reverse reaction by 10fold
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0013
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unpurified native enzyme, at pH 7.5, temperature not specified in the publication
0.0052
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unpurified recombinant enzyme, at pH 7.5, temperature not specified in the publication
0.022
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purified native enzyme, at pH 7.5, temperature not specified in the publication
0.0299
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purified recombinant enzyme, at pH 7.5, temperature not specified in the publication
0.11
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with oxidized acetyl pyridine adenine dinucleotide and deamino-NADPH as substrates, at pH 6.0 and 25C
0.47
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with oxidized acetyl pyridine adenine dinucleotide and 1-N6-etheno-NADPH as substrates, at pH 6.0 and 25C
1.46
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with oxidized acetyl pyridine adenine dinucleotide and NADPH as substrates, at pH 6.0 and 25C
6
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purified enzyme, at pH 7.0 and 20C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.3
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pH optimum for the reduction of thio-NADP+ by NADH
7.1
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pH optimum for the reduction of oxidized acetyl pyridine adenine dinucleotide by NADPH
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
84000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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2 * 40300, calculated from amino acid sequence
heterodimer
heterotetramer
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2 * 54000 + 2 * 48000, SDS-PAGE
homodimer
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2 * 40000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
NAD(H)-binding domain I in the absence and in the presence of NAD+ or NADH, hanging drop vapor diffusion method, using 0.2 M ammonium acetate, 0.1 M tri-sodium citrate dihydrate (pH 5.6) and 30% (w/v) PEG 4000
mutant enzymes are crystallized by the hanging drop vapor diffusion method, using in 16-24% (w/v) 8K-PEG, 20-150 mM (NH4)2SO4, 100mM MES, pH 6.0, and 10% (v/v) glycerol in the presence of 50 mM NAD+ and 5 mM NADP+
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
-15
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the enzyme remains stable for more than 3 months at -15C
4
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the enzyme remains stable for more than 2 days at 4C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE-Sepharose column chromatography and hydroxyapatite column chromatography
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Ni-NTA resin column chromatography
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phenyl-Sepharose column chromatography and DEAE-Trisacryl M column chromatography
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Q-Sepharose column chromatography and Mono Q column chromatography
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Q-Sepharose column chromatography, and butyl Toyopearl column chromatography
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the recombinant enzyme is purified by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The wild type enzyme is purified by phenyl Sepharose column chromatography, DEAE-Bio-Gel A column chromatography, and NAD+ agarose column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
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expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli C600 and BL21(DE3) cells
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expressed in Escherichia coli JM 109 cells
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expressed in Escherichia coli JM83 cells
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expressed in Escherichia coli strain JM109
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expressed in Saccharomyces cerevisiae strain TN24
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the NADP(H)-binding enzyme portion is expressed in Escherichia coli BL21(DE3) cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A398C
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the mutant with wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
A432C
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the mutant shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
D392C
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the mutant shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
G245L
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the mutation leads to a general inhibition of all enzyme activities
G249L
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the mutation leads to a general inhibition of all enzyme activities
G252L
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the mutation leads to a general inhibition of all enzyme activities
G408C
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the mutant with wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
G430C
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the mutant shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
H91C
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the mutant of the beta subunit is unable to undergo the conformational change that occurs on binding of the substrates NADP+ or NADPH. The mutant retains 12% of the hydride transfer activity while proton translocation is reduced to 7% compared to the wild type enzyme
H91D
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the mutant of the beta subunit retains 15% of the hydride transfer activity while proton translocation is reduced to 9% compared to the wild type enzyme
H91K
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the mutant of the beta subunit is present in the NADP(H)-induced conformation even in the absence of these substrates. The mutant retains 4% of the hydride transfer activity while proton translocation is reduced to 20% compared to the wild type enzyme
H91N
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the mutant of the beta subunit retains 80% of the hydride transfer activity while proton translocation is reduced to 7% compared to the wild type enzyme
H91S
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the mutant of the beta subunit is unable to undergo the conformational change that occurs on binding of the substrates NADP+ or NADPH. The mutant retains 19% of the hydride transfer activity while proton translocation is reduced to 11% compared to the wild type enzyme
H91T
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the mutant of the beta subunit is unable to undergo the conformational change that occurs on binding of the substrates NADP+ or NADPH. The mutant retains 11% of the hydride transfer activity while proton translocation is reduced to 8% compared to the wild type enzyme
I406C
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the mutant with 450% of wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
M409C
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the mutant with 75% of wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
S250C
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the mutation leads to enhanced activities of all enzyme activities
S251C
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the mutation leads to enhanced activities of all enzyme activities
S256C
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the mutation leads to enhanced activities of all enzyme activities
S404C
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the mutant with 75% of wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
T393C
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the mutant shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
V411C
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the mutant with 125% of wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
Y431C
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the mutant with 450% of wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
D135N
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the mutant shows reduced activity compared to the wild type enzyme
E155W/R165A
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the mutations mutation do not significantly affect catalytic activity
M239F
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the Km for APAD+ during reverse transhydrogenation is 6fold greater compared to the wild type. Cyclic transhydrogenation (in membranes and the recombinant system) is substantially more inhibited (84%) than either forward or reverse transhydrogenation
M293I
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the Km for APAD+ during reverse transhydrogenation is 5fold greater compared to the wild type. Cyclic transhydrogenation (in membranes and the recombinant system) is substantially more inhibited (70%) than either forward or reverse transhydrogenation
Q132N
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the mutant shows reduced activity compared to the wild type enzyme
R127A
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the mutant shows reduced activity compared to the wild type enzyme
R127M
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the mutant shows reduced activity compared to the wild type enzyme
R165A
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a higher concentration of the nucleotide is needed to achieve the half-maximal rate compared to with the wild type protein
S138A
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the mutant shows reduced activity compared to the wild type enzyme
W72F
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the mutant shows wild type activity
Y146A
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the mutant binds NADH much more weakly than the wild type enzyme
Y146F
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the mutant shows wild type NADH binding ability
Y171W
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the mutant shows wild type activity