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Information on EC 7.1.1.1 - proton-translocating NAD(P)+ transhydrogenase and Organism(s) Escherichia coli and UniProt Accession P07001
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7.1.1.1 proton-translocating NAD(P)+ transhydrogenaseIUBMB Comments
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.
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Word Map
- 7.1.1.1
- nadp+
-
nadph-binding
- 3-acetylpyridine-nad+
- thio-nadp+
-
proteoliposomes
-
hydride
-
cysteine-free
-
membrane-spanning
-
dissipation
-
protrude
-
alpha-helices
-
detergent-dispersed
-
microbalance
- quartz
-
unspecific
-
palmitoyl-coenzyme
The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
proton-pumping nicotinamide nucleotide transhydrogenase, proton-translocating nad(p)+ transhydrogenase, proton translocating nicotinamide nucleotide transhydrogenase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
NNT
-
-
-
-
pntA
pntB
proton-translocating nicotinamide nucleotide transhydrogenase
-
-
proton-translocating transhydrogenase
-
-
pntA
-
-
-
-
pntB
-
-
-
-
PATHWAY SOURCE
PATHWAYS
-
-, -
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.
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
H+/out + NADH + NADP+
H+/in + NAD+ + NADPH
the enzyme couples the redox reaction between NAD(H) and NADP(H) to the inward transport of protons across a membrane
-
-
?
NADPH + oxidized acetyl pyridine adenine dinucleotide + H+[side 1]
NADP+ + reduced acetyl pyridine adenine dinucleotide + H+[side 2]
-
-
-
r
1,N6-etheno-NADPH + NAD+ + H+[side 1]
1,N6-etheno-NADP+ + NADH + H+[side 2]
-
-
-
-
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]
-
-
-
-
r
deamino-NADPH + NAD+ + H+[side 1]
deamino-NADP+ + NADH + H+[side 2]
-
-
-
-
r
deamino-NADPH + oxidized acetyl pyridine adenine dinucleotide + H+[side 1]
deamino-NADP+ + reduced acetyl pyridine adenine dinucleotide + H+[side 2]
-
-
-
-
r
H+/out + NADH + NADP+
H+/in + NAD+ + NADPH
-
the enzyme couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane
-
-
?
NADP+ + NADH
NADPH + NAD+
-
mitochondrial transhydrogenase is stereospecific for the 4A-NADH and 4B-NADPH hydrogens
-
-
?
NADPH + 3-acetylpyridine-NAD+ + H+[side 1]
NADP+ + 3-acetylpyridine-NADH + H+[side 2]
-
-
-
-
?
NADPH + oxidized 3-acetylpyridin-adenine dinucleotide + H+[side 1]
NADP+ + reduced 3-acetylpyridin-adenine dinucleotide + H+[side 2]
-
-
-
-
?
NADPH + oxidized 3-acetylpyridine adenine dinucleotide + H+[side 1]
NADP+ + reduced 3-acetylpyridine adenine dinucleotide + H+[side 2]
-
-
-
-
r
NADPH + oxidized acetyl pyridine adenine dinucleotide + H+[side 1]
NADP+ + reduced acetyl pyridine adenine dinucleotide + H+[side 2]
NADPH + oxidized acetylpyridine adenine dinucleotide + H+[side 1]
NADP+ + reduced acetylpyridine adenine dinucleotide + H+[side 2]
-
-
-
-
?
additional information
?
-
-
NADP(H) binding leads to perturbation of a deeply buried part of the polypeptide backbone and to protonation of a carboxylic acid residue
-
-
?
NADH + NADP+
NAD+ + NADPH
-
-
-
-
r
NADH + NADP+
NAD+ + NADPH
-
reaction is catalyzed by a mixture of recombinant domains dI and dIII of either species or by a hybrid mixture of domains I and III from both species
-
-
?
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the outside in translocation of protons across membrane
-
-
r
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the translocation of protons across membrane
-
-
r
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the translocation of protons across membrane, cellular regeneration of NADPH
-
-
r
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the translocation of protons across membrane, major source of NADPH
-
-
r
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the translocation of protons across membrane, provides NADPH for biosynthesis and reduction of glutathione
-
-
r
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the translocation of protons across membrane, provides NADPH for the reduction of glutathione, reverse reaction results in outward proton translocation and creation of a proton motive force
-
-
r
NADPH + NAD+ + H+[side 1]
NADP+ + NADH + H+[side 2]
-
-
-
-
?
NADPH + NAD+ + H+[side 1]
NADP+ + NADH + H+[side 2]
-
-
-
-
r
NADPH + oxidized acetyl pyridine adenine dinucleotide + H+[side 1]
NADP+ + reduced acetyl pyridine adenine dinucleotide + H+[side 2]
-
-
-
-
?
NADPH + oxidized acetyl pyridine adenine dinucleotide + H+[side 1]
NADP+ + reduced acetyl pyridine adenine dinucleotide + H+[side 2]
-
-
-
-
r
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
H+/out + NADH + NADP+
H+/in + NAD+ + NADPH
the enzyme couples the redox reaction between NAD(H) and NADP(H) to the inward transport of protons across a membrane
-
-
?
H+/out + NADH + NADP+
H+/in + NAD+ + NADPH
-
the enzyme couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane
-
-
?
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the outside in translocation of protons across membrane
-
-
r
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the translocation of protons across membrane
-
-
r
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the translocation of protons across membrane, cellular regeneration of NADPH
-
-
r
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the translocation of protons across membrane, major source of NADPH
-
-
r
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the translocation of protons across membrane, provides NADPH for biosynthesis and reduction of glutathione
-
-
r
NADH + NADP+
NAD+ + NADPH
-
links hydride transfer between NAD(H) and NADP(H) to the translocation of protons across membrane, provides NADPH for the reduction of glutathione, reverse reaction results in outward proton translocation and creation of a proton motive force
-
-
r
NADPH + NAD+ + H+[side 1]
NADP+ + NADH + H+[side 2]
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid
-
0.004 mM, 2 h incubation, 75% inhibition of reverse reaction catalyzed by A348C mutant enzyme, 95% inhibition of A390C mutant enzyme after 1 h, 90% inhibition of K424C mutant enzyme after 1 h, 55% inhibition of R425C mutant enzyme after 1h
N,N'-Dicylclohexylcarbodiimide
-
NADH protects from inhibition, NADP+ and to a lesser extent NADPH increase the rate of inhibition
N-cyclohexyl-N'-(4-dimethylaminonaphthyl)carbodiimide
-
about 25% residual activity after 200 min at 0.5 mM
oxidized 3-acetylpyridin-adenine dinucleotide
-
high concentrations of oxidized 3-acetylpyridin-adenine dinucleotide lead to substrate inhibition
N,N'-dicyclohexylcarbodiimide
-
about 12% residual activity after 180 min at 0.5 mM
N,N'-dicyclohexylcarbodiimide
-
modification of the enzyme with N,N'-dicyclohexylcarbodiimide leads to inhibition of the rate of release of NADP+ and NADPH from the enzyme, but has a much smaller effect on the binding and release of NAD+, NADH and their analogues and on the interconversion of the ternary complexes of the enzyme with its substrates
N,N'-dicyclohexylcarbodiimide
-
NADH protects the enzyme against inhibition by N,N'-dicyclohexylcarbodiimide, while NADP+ , and to a lesser extent NADPH increase the rate of inhibition
N-ethylmaleimide
-
0.2 mM, 57% inhibition of reverse reaction in A348C mutant enzyme, 60% inhibition of K424C mutant enzyme, more than 95% inhibition of A390C mutant enzyme after 1h, R425C mutant enzyme is not inhibited
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
carbonylcyanide-4-trifluoromethoxyphenyl hydrazone
-
0.002 mM stimulates the rate of the forward reaction by 8fold and of the reverse reaction by 10fold
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0667
reduced acetylpyridine adenine dinucleotide
-
reverse reaction of wild-type domain III/R. rubrum domain I mixture
0.1
reduced acetylpyridine adenine dinucleotide
-
reverse reaction of A432C mutant domain III/R. rubrum domain I mixture
0.117
reduced acetylpyridine adenine dinucleotide
-
reverse reaction of D393C mutant domain III/R. rubrum domain I mixture
0.167
reduced acetylpyridine adenine dinucleotide
-
reverse reaction of H345C mutant domain III/R. rubrum domain I mixture
0.167
reduced acetylpyridine adenine dinucleotide
-
reverse reaction of R350C mutant domain III/R. rubrum domain I mixture
0.283
reduced acetylpyridine adenine dinucleotide
-
reverse reaction of K424C mutant domain III/R. rubrum domain I mixture
0.283
reduced acetylpyridine adenine dinucleotide
-
reverse reaction of R425C mutant domain III/R. rubrum domain I mixture
0.55
reduced acetylpyridine adenine dinucleotide
-
reverse reaction of A348C mutant domain III/R. rubrum domain I mixture
0.567
reduced acetylpyridine adenine dinucleotide
-
reverse reaction of G430C mutant domain III/R. rubrum domain I mixture
0.917
reduced acetylpyridine adenine dinucleotide
-
reverse reaction of D392C mutant domain III/R. rubrum domain I mixture
10
reduced acetylpyridine adenine dinucleotide
-
cyclic reaction of R425C mutant domain III/R. rubrum domain I mixture
11.7
reduced acetylpyridine adenine dinucleotide
-
cyclic reaction of G430C mutant domain III/R. rubrum domain I mixture
15
reduced acetylpyridine adenine dinucleotide
-
cyclic reaction of D392C mutant domain III/R. rubrum domain I mixture
20.8
reduced acetylpyridine adenine dinucleotide
-
cyclic reaction of H345C mutant domain III/R. rubrum domain I mixture
33.3
reduced acetylpyridine adenine dinucleotide
-
cyclic reaction of R350C mutant domain III/R. rubrum domain I mixture
36.7
reduced acetylpyridine adenine dinucleotide
-
cyclic reaction of K424C mutant domain III/R. rubrum domain I mixture
40
reduced acetylpyridine adenine dinucleotide
-
cyclic reaction of D393C mutant domain III/R. rubrum domain I mixture
51.7
reduced acetylpyridine adenine dinucleotide
-
cyclic reaction of A432C mutant domain III/R. rubrum domain I mixture
55
reduced acetylpyridine adenine dinucleotide
-
cyclic reaction of A348C mutant domain III/R. rubrum domain I mixture
81.7
reduced acetylpyridine adenine dinucleotide
-
cyclic reaction of wild-type domain III/R. rubrum domain I mixture
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.0013
-
unpurified native enzyme, at pH 7.5, temperature not specified in the publication
0.0052
-
unpurified recombinant enzyme, at pH 7.5, temperature not specified in the publication
0.022
-
purified native enzyme, at pH 7.5, temperature not specified in the publication
0.0299
-
purified recombinant enzyme, at pH 7.5, temperature not specified in the publication
0.1
0.11
-
with oxidized acetyl pyridine adenine dinucleotide and deamino-NADPH as substrates, at pH 6.0 and 25°C
0.2
-
reduction of NADP+ by NADH driven by electron transport, cysteine-free enzyme reconstituted in membrane vesicles
0.3
-
membrane bound mutant enzyme with a 18 residues long linker between alpha and beta subunits
0.4
-
purified mutant enzyme with a 18 residues long linker between alpha and beta subunits, forward reaction
0.42
-
reduction of NADP+ by NADH driven by electron transport, wild-type enzyme reconstituted in membrane vesicles
0.47
-
with oxidized acetyl pyridine adenine dinucleotide and 1-N6-etheno-NADPH as substrates, at pH 6.0 and 25°C
0.6
-
membrane bound mutant enzyme with a 32 residues long linker between alpha and beta subunits
0.7
-
purified mutant enzyme with a 32 residues long linker between alpha and beta subunits, forward reaction
0.8
-
purified mutant enzyme with a direct linker between alpha and beta subunits, reverse reaction
1.46
-
with oxidized acetyl pyridine adenine dinucleotide and NADPH as substrates, at pH 6.0 and 25°C
1.9
-
reduction of acetylpyridine adenine dinucleotide by NADPH, cysteine-free enzyme reconstituted in membrane vesicles
10.8
3
-
reduction of acetylpyridine adenine dinucleotide by NADPH, wild-type enzyme reconstituted in membrane vesicles
3.9
-
purified mutant enzyme with a 18 residues long linker between alpha and beta subunits, reverse reaction
42
-
purified mutant enzyme with a 32 residues long linker between alpha and beta subunits, cyclic reaction
46
-
purified mutant enzyme with a 18 residues long linker between alpha and beta subunits, cyclic reaction
5.5
-
purified mutant enzyme with a 32 residues long linker between alpha and beta subunits, reverse reaction
7
-
purified mutant enzyme with a direct linker between alpha and beta subunits, cyclic reaction
0.1
-
membrane bound mutant enzyme with a direct linker between alpha and beta subunits
0.1
-
purified mutant enzyme with a direct linker between alpha and beta subunits, forward reaction
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5.5
-
H91E mutant enzyme, optima for forward reaction below pH 5.5, 7% of wild-type enzyme activity at pH 6.0
6
-
reverse reaction catalyzed by H91E mutant enzyme, 20% of wild-type enzyme activity
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
titration of Escherichia coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
-
392619, 392668, 657468, 658003, 658197, 658712, 659274, 659437, 660435, 672325, 689135, 698677, 733148, 733317, 733369, 733374, 733380, 733850
-
the enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
-
transhydrogenase PntAB is a major source of NADPH that is required for biosynthesis in Escherichia coli
physiological function
overexpression of pyridine nucleotide transhydrogenase, PntAB, results in a significant increase in biomass and glycolic acid titer and yield. Improved redox homeostasis resulting from PntAB overexpression positively affects the anabolic rate of the cell. PntAB overexpression result in 154 and 37% increase in NAD+/NADH ratio, at 48 and 72 h, respectively, while the NADP+ concentrations are 70 and 30% lower at 24 and 72 h. Expression of PntAB in an optimized glycolic acid-producing strain improves the growth and product titer significantly
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
48000
50000
54000
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
heterodimer
tetramer
-
domain I, i.e. alpha1 to alpha404, and III i.e. beta260 to beta462, are exposed to the cytosol and contain the binding sites for NAD(H) and NAD(P)H, respectively, domain II, i.e. alpha405 to alpha 510, spans the membrane
trimer
-
dI2dIII trimers formed in mixtures of recombinant dI and dIII domains
dimer
-
monomers consist of three domains dI, dII, and dIII located on two separate polypeptide chains
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging-drop method, crystal structures of the NAD(H)-binding domain I of transhydrogenase in the absence as well as in the presence of oxidized and reduced substrate. The structures are determined at 1.9-2.0 A resolution
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A348C
-
mutation introduced into a cysteine-free mutant enzyme, mutant shows markedly reduced activity
A398C
-
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
C292T/C339T/C395S/C397T/C435S
-
cysteine of the alpha subunits replaced, similar activity as wild-type
C292T/C339T/C395S/C397T/C435S/C147S/C260S
-
all 7 cysteines of the enzyme, 5 localized in the alpha subunit and 2 in the beta subunit, are replaced, the cysteine-free mutant shows about 5fold more activity in the reduction of acetylpyridine adenine dinucleotide by NADH than wild-type, the cyclic reduction of acetylpyridine adenine dinucleotide by NADH via NADPH is 2-2.5fold more activ
D392C
-
the mutant shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
E124C
-
domian dII, strongly reduced reverse activity, no effect on cyclic activity
G245L
G249L
G252C
G252L
G408C
-
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
H91C
-
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
-
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
H91N
-
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
H91R
-
mutation in domain II, leads to occlusion of NADP(H) at the NADP(H)-binding site of domain III
H91S
-
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
-
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
-
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
K424C
-
mutation introduced into a cysteine-free mutant enzyme, mutant shows markedly reduced activity
M409C
-
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
N222K
-
mutation in domain II, leads to occlusion of NADP(H) at the NADP(H)-binding site of domain III
N222R
-
mutation in domain II, leads to occlusion of NADP(H) at the NADP(H)-binding site of domain III
R425C
S237C
-
domian dII, slightly reduced reverse activity, no efect on cyclic activity
S250C
S251C
S404C
-
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
V411C
-
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
-
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
additional information
-
properties of a variety of mutant enzymes containing modified conserved and semiconserved basic and acidic residues in the beta subunit
A432C
-
mutation in domain III, reverse reaction in the presence of domain I from R. rubrum, 150% higher reaction rate than wild-type domain III/R. rubrum domain I mixture
A432C
-
the mutant shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
G430C
-
mutation in domain III, reverse reaction in the presence of domain I from R. rubrum, 850% higher reaction rate than wild-type domain III/R. rubrum domain I mixture
G430C
-
the mutant shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
H91K
-
mutation in domain II, leads to occlusion of NADP(H) at the NADP(H)-binding site of domain III
H91K
-
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
R425C
-
mutation introduced into a cysteine-free mutant enzyme, mutant shows markedly reduced activity
R425C
-
mutation in domain III, reverse reaction in the presence of domain I from R. rubrum, 425% higher reaction rate than wild-type domain III/R. rubrum domain I mixture
T393C
-
mutation in domain III, reverse reaction in the presence of domain I from R. rubrum, 175% higher reaction rate than wild-type domain III/R. rubrum domain I mixture
T393C
-
the mutant shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
fusion protein is substantially more stable than wild type enzyme upon storage at 4°C
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, 50 mM Tris-HCl, pH 7.8, 1 mM, EDTA, 1 mM, dithiothreitol, 1 week, 10% loss of activity
-
4°C, fusion protein is substantially more stable than wild type enzyme upon storage
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
DEAE-Sepharose column chromatography and hydroxyapatite column chromatography
-
phenyl-Sepharose column chromatography and DEAE-Trisacryl M column chromatography
-
purification of bacterially expressed, recombinant membrane protein fused with calmodulin-binding domains. This method allows isolation of the protein fusions in a single chromatography step using elution with the calcium chelating agent EDTA. Unlike purification of His-tagged proteins on nickel chelate, it is not sensitive to the presence of strong reducing agents (e.g., DTT). The protocol involves disruption of host bacteria by sonication, sedimentation of membranes by differential centrifugation, solubilization of membrane proteins and affinity chromatography on calmodulin-agarose. To achieve maximum purity and yield, the use of a combination of non-ionic and anionic detergents is suggested. Purification takes two working days, with an overnight wash of the column to increase the purity of the product
-
Q-Sepharose column chromatography, and butyl Toyopearl column chromatography
-
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
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of cysteine mutants A348C, A390C, K424C, and R425C in Escherichia coli
-
expression of wild-type domain III, T393C, R425C, G430C and A432C mutant domain III in Escherichia coli
-
fusion protein with calmodulin-binding peptide and His-tag expressed in Escherichia coli JM109
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
overexpression of pyridine nucleotide transhydrogenase, PntAB, results in a significant increase in biomass and glycolic acid titer and yield. Improved redox homeostasis resulting from PntAB overexpression positively affects the anabolic rate of the cell. PntAB overexpression result in 154 and 37% increase in NAD+/NADH ratio, at 48 and 72 h, respectively, while the the NADP+ concentrations are 70 and 30% lower at 24 and 72 h. Expression of PntAB in an optimized glycolic acid-producing strain improves the growth and product titer significantly
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Jackson, J.B.; Lever, T.M.; Rydström, J.; Persson, B.; Carlenor, E.
Proton-translocating transhydrogenase from photosynthetic bacteria
Biochem. Soc. Trans.
19
573-575
1991
Escherichia coli
Clarke, D.M.; Bragg, P.D.
Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli
Eur. J. Biochem.
149
517-523
1985
Escherichia coli, Escherichia coli W-6
Clarke, D.M.; Bragg, P.D.
Cloning and expression of the transhydrogenase gene of Escherichia coli
J. Bacteriol.
162
367-373
1985
Escherichia coli, Escherichia coli MV-12
Meuller, J.; Zhang, J.; Hou, C.; Bragg, P.D.; Rydstrom, J.
Properties of a cysteine-free proton-pumping nicotinamide nucleotide transhydrogenase
Biochem. J.
324
681-687
1997
Escherichia coli
Fjellström, O.; Axelsson, M.; Bizouarn, T.; Hu, X.; Johansson, C.; Meuller, J.; Rydström, J.
Mapping of residues in the NADP(H)-binding site of proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli
J. Biol. Chem.
274
6350-6359
1999
Escherichia coli, Rhodospirillum rubrum
Hu, X.; Zhang, J.; Fjellstrom, O.; Bizouarn, T.; Rydstrom, J.
Site-directed mutagenesis of charged and potentially proton-carrying residues in the beta subunit of the proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli. Characterization of the beta H91, beta D392, and beta K424 mutants
Biochemistry
38
1652-1658
1999
Escherichia coli
Fjellstrom, O.; Bizouarn, T.; Zhang, J.W.; Rydstrom, J.; Venning, J.D.; Jackson, J.B.
Catalytic properties of hybrid complexes of the NAD(H)-binding and NADP(H)-binding domains of the proton-translocating transhydrogenases from Escherichia coli and Rhodospirillum rubrum
Biochemistry
38
415-422
1999
Escherichia coli, Rhodospirillum rubrum
Bergkvist, A.; Johansson, C.; Johansson, T.; Rydström, J.; Karlsson, g.
Interactions of the NADP(H)-binding domain III of proton-translocating transhydrogenase from Escherichia coli with NADP(H) and the NAD(H)-binding domain I studied by NMR and site-directed mutagenesis
Biochemistry
39
12595-12605
2000
Escherichia coli, Rhodospirillum rubrum
Bizouarn, T.; Fjellstrom, O.; Meuller, J.; Axelsson, M.; Bergkvist, A.; Johansson, C.; Goran Karlsson, B.; Rydstrom, J.
Proton translocating nicotinamide nucleotide transhydrogenase from E. coli. Mechanism of action deduced from its structural and catalytic properties
Biochim. Biophys. Acta
1457
211-228
2000
Escherichia coli
Bragg, P.D.; Hou, C.
The presence of an aqueous cavity in the proton-pumping pathway of the pyridine nucleotide transhydrogenase of Escherichia coli is suggested by the reaction of the enzyme with sulfhydryl inhibitors
Arch. Biochem. Biophys.
380
141-150
2000
Escherichia coli
Meuller, J.; Mjörn, K.; Karlsson, J.; Tigerström, a.; Rydström, J.; Hou, C.; Bragg, P.D.
Properties of a proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli with alpha and beta subunits linked through fused transmembrane helices
Biochim. Biophys. Acta
1506
163-171
2001
Escherichia coli
Oswald, C.; Johansson, T.; Tornroth, S.; Okvist, M.; Krengel, U.
Crystallization and preliminary crystallographic analysis of the NAD(H)-binding domain of Escherichia coli transhydrogenase
Acta Crystallogr. Sect. D
60
743-745
2004
Escherichia coli
Karlsson, J.; Althage, M.; Rydstrom, J.
Roles of individual amino acids in helix 14 of the membrane domain of proton-translocating transhydrogenase from Escherichia coli as deduced from cysteine mutagenesis
Biochemistry
42
6575-6581
2003
Escherichia coli
Althage, M.; Bizouarn, T.; Kindlund, B.; Mullins, J.; Alander, J.; Rydstrom, J.
Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain
Biochim. Biophys. Acta
1659
73-82
2004
Escherichia coli
Jackson, J.B.
Proton translocation by transhydrogenase
FEBS Lett.
545
18-24
2003
Escherichia coli, Rhodospirillum rubrum
Yamaguchi, M.; Stout, C.D.
Essential glycine in the proton channel of Escherichia coli transhydrogenase
J. Biol. Chem.
278
45333-45339
2003
Escherichia coli
Sauer, U.; Canonaco, F.; Heri, S.; Perrenoud, A.; Fischer, E.
The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli
J. Biol. Chem.
279
6613-6619
2004
Escherichia coli
Egorov, M.V.; Tigerstrom, A.; Pestov, N.B.; Korneenko, T.V.; Kostina, M.B.; Shakhparonov, M.I.; Rydstrom, J.
Purification of a recombinant membrane protein tagged with a calmodulin-binding domain: properties of chimeras of the Escherichia coli nicotinamide nucleotide transhydrogenase and the C-terminus of human plasma membrane Ca2+ -ATPase
Protein Expr. Purif.
36
31-39
2004
Escherichia coli
Pedersen, A.; Johansson, T.; Rydstroem, J.; Goeran Karlsson, B.
Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range
Biochim. Biophys. Acta
1707
254-258
2005
Escherichia coli
Bizouarn, T.; van Boxel, G.I.; Bhakta, T.; Jackson, J.B.
Nucleotide binding affinities of the intact proton-translocating transhydrogenase from Escherichia coli
Biochim. Biophys. Acta
1708
404-410
2005
Escherichia coli
Iwaki, M.; Cotton, N.P.; Quirk, P.G.; Rich, P.R.; Jackson, J.B.
Molecular recognition between protein and nicotinamide dinucleotide in intact, proton-translocating transhydrogenase studied by ATR-FTIR Spectroscopy
J. Am. Chem. Soc.
128
2621-2629
2006
Escherichia coli
Johansson, T.; Oswald, C.; Pedersen, A.; Toernroth, S.; Okvist, M.; Karlsson, B.G.; Rydstroem, J.; Krengel, U.
X-ray structure of domain I of the proton-pumping membrane protein transhydrogenase from Escherichia coli
J. Mol. Biol.
352
299-312
2005
Escherichia coli (P07001), Escherichia coli
Pestov, N.B.; Rydstroem, J.
Purification of recombinant membrane proteins tagged with calmodulin-binding domains by affinity chromatography on calmodulin-agarose: example of nicotinamide nucleotide transhydrogenase
Nat. Protoc.
2
198-202
2007
Escherichia coli
Pedersen, A.; Karlsson, G.B.; Rydstroem, J.
Proton-translocating transhydrogenase: an update of unsolved and controversial issues
J. Bioenerg. Biomembr.
40
463-473
2008
Escherichia coli, Rhodospirillum rubrum
Anderlund, M.; Nissen, T.L.; Nielsen, J.; Villadsen, J.; Rydstroem, J.; Hahn-Haegerdal, B.; Kielland-Brandt, M.C.
Expression of the Escherichia coli pntA and pntB genes, encoding nicotinamide nucleotide transhydrogenase, in Saccharomyces cerevisiae and its effect on product formation during anaerobic glucose fermentation
Appl. Environ. Microbiol.
65
2333-2340
1999
Escherichia coli
Glavas, N.A.; Hou, C.; Bragg, P.D.
Involvement of histidine-91 of the beta subunit in proton translocation by the pyridine nucleotide transhydrogenase of Escherichia coli
Biochemistry
34
7694-7702
1995
Escherichia coli
Bizouarn, T.; Grimley, R.L.; Cotton, N.P.; Stilwell, S.N.; Hutton, M.; Jackson, J.B.
The involvement of NADP(H) binding and release in energy transduction by proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli
Biochim. Biophys. Acta
1229
49-58
1995
Escherichia coli
Bizouarn, T.; Althage, M.; Pedersen, A.; Tigerstroem, A.; Karlsson, J.; Johansson, C.; Rydstroem, J.
The organization of the membrane domain and its interaction with the NADP(H)-binding site in proton-translocating transhydrogenase from E. coli
Biochim. Biophys. Acta
1555
122-127
2002
Escherichia coli
Jackson J.B, J.J.
A review of the binding-change mechanism for proton-translocating transhydrogenase
Biochim. Biophys. Acta
1817
1839-1846
2012
Escherichia coli, Rhodospirillum rubrum
Hutton, M.; Day, J.M.; Bizouarn, T.; Jackson, J.B.
Kinetic resolution of the reaction catalysed by proton-translocating transhydrogenase from Escherichia coli as revealed by experiments with analogues of the nucleotide substrates
Eur. J. Biochem.
219
1041-1051
1994
Escherichia coli
Johansson, C.; Pedersen, A.; Karlsson, B.G.; Rydstroem, J.
Redox-sensitive loops D and E regulate NADP(H) binding in domain III and domain I-domain III interactions in proton-translocating Escherichia coli transhydrogenase
Eur. J. Biochem.
269
4505-4515
2002
Escherichia coli
Jackson, J.B.; Peake, S.J.; White, S.A.
Structure and mechanism of proton-translocating transhydrogenase
FEBS Lett.
464
1-8
1999
Escherichia coli, Homo sapiens, Rhodospirillum rubrum
Cabulong, R.B.; Valdehuesa, K.N.G.; Banares, A.B.; Ramos, K.R.M.; Nisola, G.M.; Lee, W.K.; Chung, W.J.
Improved cell growth and biosynthesis of glycolic acid by overexpression of membrane-bound pyridine nucleotide transhydrogenase
J. Ind. Microbiol. Biotechnol.
46
159-169
2019
Escherichia coli (P07001 and P0AB67)
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