Information on EC 1.5.1.40 - 8-hydroxy-5-deazaflavin:NADPH oxidoreductase

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

EC NUMBER
COMMENTARY hide
1.5.1.40
-
RECOMMENDED NAME
GeneOntology No.
8-hydroxy-5-deazaflavin:NADPH oxidoreductase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
reduced coenzyme F420 + NADP+ = oxidized coenzyme F420 + NADPH + H+
show the reaction diagram
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
reduced coenzyme F420:NADP+ oxidoreductase
The enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate [1].
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
T28A mutant shows 3fold increased kinetic efficiency compared with the wild-type enzyme when NADPH is the substrate
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
1,5-dihydro-8-hydroxy-5-deazaflavin + NADP+
8-hydroxy-5-deazaflavin + NADPH + H+
show the reaction diagram
5'-O-methyl-7,8-didemethyl-8-hydroxyflavin + NADPH + H+
8-hydroxypyrimido[4,5-b]-2,4-(3H,10H)-dione + NADP+
show the reaction diagram
-
the enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate
-
-
r
5-deaza-8-hydroxy-10-methylisoalloxazine + NADPH + H+
? + NADP+
show the reaction diagram
5-deaza-8-hydroxyisoalloxazine + NADPH + H+
8-hydroxypyrimido[4,5-b]-2,4-(3H,10H)-dione + NADP+
show the reaction diagram
-
the enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate
-
-
?
7,8-didemethyl-8-hydroxy-5-deazariboflavin 5'-phosphate + NADPH + H+
1-deoxy-1-(8-hydroxy-2,4-dioxo-1,3,4,5-tetrahydropyrimido[4,5-b]quinolin-10(2H)-yl)-5-O-phospho-D-ribitol + NADP+
show the reaction diagram
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the enzyme has an absolute requirement for both the 5-deazaflavin structure and the presence of an 8-hydroxy group in the substrate
-
-
?
coenzyme F0 + NADPH + H+
reduced coenzyme F0 + NADP+
show the reaction diagram
coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
show the reaction diagram
oxidized coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
show the reaction diagram
reduced coenzyme F420 + NADP+
coenzyme F420 + NADPH + H+
show the reaction diagram
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
show the reaction diagram
additional information
?
-
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
coenzyme F420 + NADPH + H+
reduced coenzyme F420 + NADP+
show the reaction diagram
-
the main function of this oxidoreductase is probably to provide cells with reduced 8-hydroxy-5-deazaflavin to be used in specific reduction reactions
-
-
?
reduced coenzyme F420 + NADP+
coenzyme F420 + NADPH + H+
show the reaction diagram
reduced coenzyme F420 + NADP+
oxidized coenzyme F420 + NADPH + H+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
NADPH
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-Chloromercuriphenyl sulfonate
-
-
iodoacetamide
-
-
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0028
1,5-dihydro-8-hydroxy-5-deazaflavin
-
pH 7.0, 20°C
0.0135
5'-O-methyl-7,8-didemethyl-8-hydroxyflavin
-
pH 6.0, 22°C
0.0193
5-deaza-8-hydroxyisoalloxazine
-
pH 6.0, 22°C
0.0155
7,8-didemethyl-8-hydroxy-5-deazariboflavin 5'-phosphate
-
pH 6.0, 22°C
0.008
8-hydroxy-5-deazaflavin
-
pH 7.0, 20°C
0.0775
8-hydroxypyrimido[4,5-b]-2,4-(3H,10H)-dione
-
pH 6.0, 22°C
0.0057
coenzyme F0
-
pH 6.0, 22°C
0.0034 - 0.0625
coenzyme F420
0.0137 - 0.37
NADP+
0.00027 - 500
NADPH
0.0036 - 4
oxidized coenzyme F420
0.0077 - 0.15
reduced coenzyme F420
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7.37
1,5-dihydro-8-hydroxy-5-deazaflavin
-
pH 7.0, 20°C
6.5
5'-O-methyl-7,8-didemethyl-8-hydroxyflavin
-
pH 6.0, 22°C
4.4
5-deaza-8-hydroxyisoalloxazine
-
pH 6.0, 22°C
35.57
7,8-didemethyl-8-hydroxy-5-deazariboflavin 5'-phosphate
-
pH 6.0, 22°C
175
8-hydroxy-5-deazaflavin
-
pH 7.0, 20°C
10.23
8-hydroxypyrimido[4,5-b]-2,4-(3H,10H)-dione
-
pH 6.0, 22°C
12.15
coenzyme F0
-
pH 6.0, 22°C
17.22
coenzyme F420
-
pH 6.0, 22°C
7.37
NADP+
-
pH 7.0, 20°C
0.11 - 175
NADPH
0.7 - 5.3
oxidized coenzyme F420
3
reduced coenzyme F420
-
pH 6.0, 22°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
48
5'-O-methyl-7,8-didemethyl-8-hydroxyflavin
-
pH 6.0, 22°C
23
5-deaza-8-hydroxyisoalloxazine
-
pH 6.0, 22°C
2295
7,8-didemethyl-8-hydroxy-5-deazariboflavin 5'-phosphate
-
pH 6.0, 22°C
132
8-hydroxypyrimido[4,5-b]-2,4-(3H,10H)-dione
-
pH 6.0, 22°C
2132
coenzyme F0
-
pH 6.0, 22°C
5065
coenzyme F420
-
pH 6.0, 22°C
0.12 - 3400
NADPH
194.4 - 1325
oxidized coenzyme F420
233
reduced coenzyme F420
-
pH 6.0, 22°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.04
-
pH 8.0, 55°C, cell extract
0.06
-
pH 8.0, 37°C, cell extract
0.3
-
pH 8.0, 37°C, cell extract
1.79
-
pH 7.0, 20°C
11.15
-
pH 6.0, 22°C
15.1
pH 8.0, 30°C, cell extract
363
-
pH 5.4, 22°C
660
-
pH 8.0, 80°C
998
pH 8.0, 30°C, purified enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.5
reduction of oxidized coenzyme F420 with NADPH
4 - 6
oxidation of NADPH, broad optimum
4.8
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reaction with 8-hydroxy-5-deazaflavin + NADPH
5.9
-
reduction of 8-hydroxy-5-azaflavins
6
-
reduction of coenzyme F420
8.5
-
reduction of NADP+
8.5 - 9
reduction of NADP+
9.2
pH optimum for reduction of NADP+ with reduced coenzyme F420
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.3 - 6.7
-
pH 4.3: about 50% of maximal activity, pH 6.7: about 50% of maximal activity
4.5 - 6.5
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pH 4.5: about 45% of maximal activity, pH 6.5: about 35% of maximal activity, reduction of oxidized coenzyme F420 with NADPH
7 - 9
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pH 7.0: about 60% of maximal activity, pH 9.0: about 50% of maximal activity, reduction of NADP+ with reduced coenzyme F420
7.5 - 10
pH 7.5: about 50% of maximal activity, pH 10.0: about 90% of maximal activity, reduction of NADP+ with reduced coenzyme F420
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 50
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15°C: about 45% of maximal activity, 50°C: about 45% of maximal activity
25 - 90
the enzyme displays highest activity between 60°C and 70°C. The activity at 65°C is almost 4fold higher than that at 25°C. The apparent melting temperature of Tfu-FNO is 75°C, about 50% of maximal activity at 40°C and 90°C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23500
4 * 23500, calculated from sequence
42000
-
gel filtration
43000
-
2 * 43000, SDS-PAGE
45000
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1 * 60000 (alpha) + 1 * 50000 (beta) + 1 * 45000 (gamma), SDS-PAGE
50000
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1 * 60000 (alpha) + 1 * 50000 (beta) + 1 * 45000 (gamma), SDS-PAGE
52000
-
sedimentation analysis
57000
-
gel filtration
60000
-
1 * 60000 (alpha) + 1 * 50000 (beta) + 1 * 45000 (gamma), SDS-PAGE
85000
-
gel filtration
90000
gradient gel electrophoresis under nondenaturating conditions
148000
-
gel filtration, native PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
trimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour diffusion method, crystal structure of the enzyme bound with coenzyme F420. The structure resolved to 1.65 A contains two domains: an N-terminal domain characteristic of a dinucleotide-binding Rossmann fold and a smaller C-terminal domain. The nicotinamide and the deazaflavin part of the two coenzymes are bound in the cleft between the domains such that the Si-faces of both face each other at a distance of 3.1 A, which is optimal for hydride transfer
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 90
the enzyme displays highest activity between 60°C and 70°C. The activity at 65°C is almost 4fold higher than that at 25°C. The apparent melting temperature of Tfu-FNO is 75°C, about 50% of maximal activity at 40°C and 90°C
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
in 0.08 M NaCl a half-life of 9 h is found which increased to greater than 6000 h in 1 M NaCl (both in the presence of 2% ethylene glycol)
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neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme
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precipitation with (NH4)2SO4 reduces the stability of the enzyme
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ethylene glycol
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enhanced the stability: half-life increased from 9 to 750 h in 18% ethylene glycol (in 0.08 M NaCl)
Tween-80
-
0.17% decreases half-life by a factor of approximately 3
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
stable under oxic conditions
724136
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-75°C, 2 months, at low concentration (0.066 mg/ml), in the presence of ethylene glycol, 20% (v/v), no loss of activity
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4°C or -20°C, enzyme solution is stable for several weeks
4°C, activity in crude extract is stable for a month
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4°C, at low concentration (0.066 mg/ml), in the presence of ethylene glycol, 20% (v/v), 22% of the activity is lost
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4°C, at low concentration (0.066 mg/ml), the purified enzyme loses 84% of its activity in 2 days
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4°C, purified enzyme at a protein concentration of 20 mg/ml is completely stable when stored at 4°C in 50 mM Tricine/KOH pH 8.0 containing 0.2 M KCl
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme from cell-free extracts of Escherichia coli strain C41(DE3) by heat treatment at 90°C for 30 min, ammonium sulfate fractionation with addition of 0.05% polyethylenimine, followed by an ion exchange chromatography, ultrafiltration, and gel filtration
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recombinant enzyme from Escherichia coli by ammonium sulfate fractionation followed by anion exchange chromatography, DNase I treatment during protein purification is essential to remove residual DNA
recombinant wild-type and mutant enzymes from cell-free extracts of Escherichia coli strain C41(DE3) by heat treatment at 90°C for 30 min, ammonium sulfate fractionation with addition of 0.05% polyethylenimine, followed by an ion exchange chromatography, ultrafiltration, and gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning and recombinant expression in Escherichia coli strain C41(DE3)
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gene Tfu-fno, DNA and amino acid sequence determination and analysis, recombinant expression in Escherichia coli
overexpression in Escherichia coli
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain C41(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
I135A
-
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
I135G
-
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
I135V
-
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
R51A
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R51E/R55A
site-directed mutagenesis, the mutant shows similar catalytic efficiency compared to the wild-type enzyme
R51E/R55N
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R51E/R55S
site-directed mutagenesis, the mutant shows similar catalytic efficiency compared to the wild-type enzyme
R51V
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R51V/R55V
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R55A
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R55N
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R55S
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
R55V
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
S50E
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
S50E/R55A
site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type enzyme
S50E/R55V
site-directed mutagenesis, the mutant shows slightly increased catalytic efficiency compared to the wild-type enzyme
S50Q
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
T28A
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
T28A/R51V
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
T28A/R51V/R55V
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
T28A/R55A
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme
additional information
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pre-steady-state data with F420 cofactor and NADPH for the enzyme Fno mutant variants reveal biphasic kinetics with a fast and slow phase, similar with wild-type Fno, overview