Information on EC 1.6.1.1 - NAD(P)+ transhydrogenase (Si-specific)

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

EC NUMBER
COMMENTARY hide
1.6.1.1
-
RECOMMENDED NAME
GeneOntology No.
NAD(P)+ transhydrogenase (Si-specific)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
NADPH + NAD+ = NADP+ + NADH
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
-
NAD metabolism
-
-
NAD/NADH phosphorylation and dephosphorylation
-
-
Nicotinate and nicotinamide metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
NADPH:NAD+ oxidoreductase (Si-specific)
The enzyme from Azotobacter vinelandii is a flavoprotein (FAD). It is Si-specific with respect to both NAD+ and NADP+. Also acts on deamino coenzymes [cf. EC 1.6.1.2 NAD(P)+ transhydrogenase (Re/Si-specific)].
CAS REGISTRY NUMBER
COMMENTARY hide
9014-18-0
not distinguished from EC 1.6.1.2
9072-60-0
not distinguished from EC 1.6.1.2
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Azotobacter agilis
-
-
-
Manually annotated by BRENDA team
Chromatium sp.
-
-
-
Manually annotated by BRENDA team
pea
-
-
Manually annotated by BRENDA team
potato tubers
-
-
Manually annotated by BRENDA team
formerly Pseudomonas natriegens
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
two pyridine nucleotide transhydrogenases: the energy-independent soluble pyridine nucleotide transhydrogenase (STH or UdhA) (EC 1.6.1.1) and the membrane-bound, energy-dependent pyridine nucleotide transhydrogenase (TH or PntAB) (EC 1.6.1.2). PntAB is widely distributed in the mitochondria and some bacteria, while STH is found only in certain Gammaproteobacteria and gram-positive bacteria
physiological function
the soluble pyridine nucleotide transhydrogenase, STH, is an energy-independent flavoprotein that directly catalyzes hydride transfer between NAD(H) and NADP(H) to maintain homeostasis of these two redox cofactors
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
NAD(P)H + 2,6-dichlorophenolindophenol
NAD(P)+ + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
-
-
-
?
NAD(P)H + K4Fe(CN)6
NAD(P)+ + K3Fe(CN)6
show the reaction diagram
-
-
-
?
NADH + 2'-NADP+
NAD+ + 2'-NADPH
show the reaction diagram
-
-
-
?
NADH + 3'-NADP+
NAD+ + 3'-NADPH
show the reaction diagram
-
-
-
?
NADH + NADP+
NADPH + NAD+
show the reaction diagram
-
-
-
?
NADH + thio-NAD+
NAD+ + thio-NADH
show the reaction diagram
-
-
-
?
NADH + thio-NADP+
NAD+ + thio-NADPH
show the reaction diagram
NADP+ + NADH
NADPH + NAD+
show the reaction diagram
NADPH + 3-acetylpyridine-NAD+
NADP+ + 3-acetylpyridine-NADH
show the reaction diagram
NADPH + deamino-NAD+
NADP+ + deamino-NADH
show the reaction diagram
NADPH + NAD+
NADP+ + NADH
show the reaction diagram
-
-
-
r
NADPH + pyridine aldehyde-NAD+
NADP+ + pyridine aldehyde-NADH
show the reaction diagram
NADPH + thio-NAD+
NADP+ + thio-NADH
show the reaction diagram
NADPH + thio-NAD+ + H+[side 1]
NADP+ + thio-NADH + H+[side 2]
show the reaction diagram
-
-
-
r
NADPH + thio-NADP+
NADP+ + thio-NADPH
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
NADH + NADP+
NADPH + NAD+
show the reaction diagram
-
-
-
?
NADPH + NAD+
NADP+ + NADH
show the reaction diagram
P27306
-
-
-
r
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
flavin
flavoprotein
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
EDTA
-
slight activating at low buffer concentrations
K+
-
no full activation compared to Ca2+
Mn2+
-
compared to Ca2+ 10 times higher concetrations are required to obtain the same degree of activation
additional information
not influenced by Na+, Rb+, K+, Li+, and Mg2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-AMP
2-mercaptoethanol
-
5'-AMP
-
5 mM, 92% inhibition of NADP+ reduction, complete inhibition of NAD+ reduction
ADP
-
5 mM, 94% inhibition of NADP+ reduction, complete inhibition of NAD+ reduction
arsenate
-
complete inhibition of activity in either direction
ATP
-
5 mM, 81% inhibition of NADP+ reduction, complete inhibition of NAD+ reduction
beta-mercaptoethanol
72% residual activity at 0.2% (v/v)
Ca2+
91.2% residual activity at 2 mM; slightly inhibitory
CTP
-
5 mM, 86% inhibition of NADP+ reduction
Cu2+
; complete inhibition at 2 mM
deoxycholate
-
-
diphosphate
-
5 mM, 91% inhibition of NADP+ reduction
dithiothreitol
75.3% residual activity at 2 mM
DMSO
slightly inhibitory
EDTA
; 72.6% residual activity at 2 mM
GTP
-
5 mM, 89% inhibition of NADP+ reduction
Mn2+
; 67% residual activity at 2 mM
NAD+
-
competitive to thio-NAD+, uncompetitive with respect to NADPH
NADPH
EcSTH activity is strongly inhibited by excess NADPH, but not by thio-NAD+; the enzyme is strongly inhibited by excess NADPH
Ni2+
; 7.4% residual activity at 2 mM
p-Aminophenylarsenoxide
-
0.1 mM, 40-60% inhibition in the absence of either phosphate or magnesium ions, reduction of NAD+ by NADPH in cell-free extracts is rapidly and completely inhibited in the presence of 20 mM phosphate
p-chloromercuribenzoate
-
0.044 mM, 40-50% inhibition after 30 min, activity can be restored by adding 2-mercaptoethanol
p-hydroxymercuribenzoate
phosphate
phosphoenolpyruvate
-
87% inhibition of NADP+ reduction
pyridoxal 5'-phosphate
-
5 mM, 91% inhibition of NADP+ reduction
TTP
-
2 mM, 71% inhibition of NADP+ reduction
Zn2+
; 10.1% residual activity at 2 mM
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-AMP
ADP
175.5% activity at 2 mM ADP; 75.5% activation at 2 mM
AMP
171.8% activity at 2 mM AMP; 71.8% activation at 2 mM
ATP
153.4% activity at 2 mM ATP; 53.4% activation at 2 mM
p-hydroxymercuribenzoate
-
activation, depending on presence of oxidized or reduced substrates
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.4
deamino-NAD+
-
cosubstrate NADPH
0.0003 - 0.0025
FAD
0.11 - 0.38
NAD+
0.025 - 0.085
NADH
0.015 - 0.0683
NADPH
0.04 - 0.25
thio-NAD+
0.03
thio-NADP+
-
cosubstrate NADH
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
167.9 - 333
NADPH
259.5
thio-NAD+
Escherichia coli
P27306
at pH 7.5 and 35°C; pH 7.5, 35°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2460
NADPH
Escherichia coli
P27306
at pH 7.5 and 35°C
5
1940
thio-NAD+
Escherichia coli
P27306
at pH 7.5 and 35°C
1286
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0094
-
activity in inside-out mitochondrial particles from leaf
0.0131
-
activity in inside-out mitochondrial particles from tuber
24
Chromatium sp.
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
-
around pH 7.0
7
-
NADH formation
9.6
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NADPH formation
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 10.5
-
pH 7.0: about 55% of maximal activity, pH 10.5: about 45% of maximal activity
7 - 9
more than 50% activity between pH 7.0 and 9.0
8.4 - 8.7
-
half maximal activity in this pH range in the absence of Ca2+ or Mg2+, no activity above
9.1 - 9.3
-
half maximal activity in this pH range in the presence of 5 mM Ca2+ or Mg2+, no activity above
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 45
more than 70% activity between 20 and 45°C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
53000
-
SDS-PAGE
421000
1600000
-
sedimentation equilibrium in presence of 2'-AMP
6400000
-
sedimentation equilibrium in presence of NADP+
27000000
-
rod-like polymers, electron micrography
30000000 - 50000000
-
rod-like structure, light scattering
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 51500, calculated from amino acid sequence; x * 52000, SDS-PAGE
octamer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
-
polymeric enzyme stable
392577
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 50
the enzyme retains 50% activity after 5 h at 50°C. The enzyme is stable at 4°C for 25 days and retains 65% activity at 25°C. The enzyme is rapidly inactivated at high temperatures, retaining 80%, 50% and 10% activity after incubation for 30 min at 50, 57 and 62°C, respectively
51
-
25 min, 50% inactivation, accelerated by addition of NADPH, reactivation by FAD
55
-
approx. 50% activity lost after about 2 min, almost complete loss of activity after 20 min, biphasic inactivation: 70% activity lost with a first-order inactivation constant, 30% is lost much more rapidly, rate of thermal inactivation depends on concentration of NAD+, NADP+, NADH, NADPH, free FAD, Mg2+ and phosphate, independent of pH between pH 5 and pH 9, significant acceleration outside this range, addition of 1 mM FAD lowers inactivation rate about 20fold
57
purified enzyme, pH 7.5, 30 min, 50% activity remaining
62
purified enzyme, pH 7.5, 30 min, 10% activity remaining
65
-
15 min, complete inactivation, protection by FAD
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
50% inactivation after 10 min in 1 M guanidinium HCl
-
bovine serum albumin, 0.2%, stabilization of diluted solutions
-
urea, 5 min, 50% inactivation
-
urea, 8 M, no dissociation, complete inactivation of enzyme activity in 5 M urea
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15°C, 50 mM Tris-HCl buffer, pH 7.5, no loss of activity in 12 weeks
-
-20°C, 50 mM Tris-HCl, pH 7.0, 2 mM dithiothreitol, several weeks, no loss of activity, great loss of activity after several months
-
25°C, purified enzyme, 25 days, 65% activity remaining
4°C, 0.1 M phosphate buffer, pH 7.5, 1 mM EDTA, several months, no loss of activity, storage at -20°C yields a partly insoluble enzyme
-
4°C, purified enzyme, 25 days, stable
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity chromatography
-
affinity chromatography on immobilized 2'-AMP
-
affinity chromatography, native and recombinant enzyme
-
TALON metal affinity resin column chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Escherichia coli strain is transformed with a two plasmid system, one encoding the udhA gene and the other one encoding the phb operon
-
expressed in Escherichia coli DH5alpha cells; expression of the soluble enzyme in Escherichia coli strain DH5alpha
expression in Escherichia coli
-
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
inactivation by 4 M guanidinium HCl, 4-6% reactivation after transfer in 100 mM Tris buffer, pH 7.5 containing 100 mM FAD
-
urea inactivation: 3% reactivation after dialysis against buffer containing 0.1 mM FAD, 35% after dialysis against buffer containing 1% mercaptoethanol and 95% reactivation after dialysis against buffer containing both 0.1 mM FAD and 1% mercaptoethanol
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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