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Enzyme Nomenclature
Information on EC 1.5.1.30 - flavin reductase (NADPH)
for references in articles please use BRENDA:EC1.5.1.30
Word Map on EC 1.5.1.30
- 1.5.1.30
- harveyi
- fmnh2
- nitroreductase
- dibenzothiophene
- fischeri
-
desulfurization
-
flavin-dependent
-
oxygen-insensitive
- halogenase
- isoalloxazine
-
nadh:flavin
-
biodesulfurization
-
luxgs
-
single-enzyme
- 2-hydroxybiphenyl
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Specify your search results
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
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EC NUMBER 
COMMENTARY 
1.5.1.30
-
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RECOMMENDED NAME
GeneOntology No.
flavin reductase (NADPH)
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
reduced riboflavin + NADP+ = riboflavin + NADPH + H+
-
-
-
-
reduced riboflavin + NADP+ = riboflavin + NADPH + H+
single-enzyme kinetic assay, SsuE follows ordered sequential mechanism with NADPH as the first substrate to bind and NADP+ as the last product to leave. In presence of monooxygenase SsuD and octanesulfonate mechanism is altered to rapid equilibrium ordered mechanism, and Km-value for FMN increases 10fold
-
reduced riboflavin + NADP+ = riboflavin + NADPH + H+
FRP:luciferase coupled reaction can utilize reduced flavin by both free diffusion and direct transfer
-
reduced riboflavin + NADP+ = riboflavin + NADPH + H+
binding of FMN by apoenzyme is noncooperative, exothermic and primarily enthalpy driven with significant conformational changes in enzyme upon binding. The kinetically deduced ping-pong mechanism is supported by measurement of binding affinities of the oxidized and reduced FMN cofoactors
-
reduced riboflavin + NADP+ = riboflavin + NADPH + H+
bi-bi ternary mechanism, i.e. both FMN and NADPH must bind to the active site before catalysis can occur
-
reduced riboflavin + NADP+ = riboflavin + NADPH + H+
the nicotinamide of NAD+ stacks the isoalloxazine ring of FMN so that NADH can directly transfer hydride. The bound NADP+ also has a compact conformation but is bound in a reverse direction, which is not suitable for hydride transfer
-
reduced riboflavin + NADP+ = riboflavin + NADPH + H+
ping-pong mechanism
-
reduced riboflavin + NADP+ = riboflavin + NADPH + H+
the enzyme catalysis obeys the ping-pong Bi-Bi kinetic mechanism with substrate FMN as well as substrate nitrofurazone, but upon coupling with the bioluminescent reaction of luciferase, it changes to the sequential mechanism
-
reduced riboflavin + NADP+ = riboflavin + NADPH + H+
the enzyme catalysis obeys the ping-pong Bi-Bi kinetic mechanism with substrate FMN as well as substrate nitrofurazone, but upon coupling with the bioluminescent reaction of luciferase, it changes to the sequential mechanism
-
-
reduced riboflavin + NADP+ = riboflavin + NADPH + H+
the nicotinamide of NAD+ stacks the isoalloxazine ring of FMN so that NADH can directly transfer hydride. The bound NADP+ also has a compact conformation but is bound in a reverse direction, which is not suitable for hydride transfer
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
reduction
-
-
-
-
redox reaction
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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SYSTEMATIC NAME
IUBMB Comments
reduced-riboflavin:NADP+ oxidoreductase
The enzyme reduces riboflavin, and, less efficiently, FMN and FAD. NADH is oxidized less efficiently than NADPH.
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CAS REGISTRY NUMBER
COMMENTARY
56626-29-0
-
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
enzyme SsuE is part of alkanesulfonate monooxygenase system, together with a monooxygenase enzyme SsuD
-
-
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
metabolism
physiological function
evolution
-
analysis of evolutionary or molecular mechanism of divergence of the nitroreductase/flavin reductase family, overview. The enzyme is similar to NfsA from Escherichia coli, overview
evolution
-
analysis of evolutionary or molecular mechanism of divergence of the nitroreductase/flavin reductase family, overview. The enzyme is similar to NfsA from Escherichia coli, overview
-
metabolism
-
the flavin reductase couples with dibenzothiophene and dibenzothiophene sulfone monooxygenase in the thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, the flavin reductase exhibits flavin reductase and nitroreductase activities
metabolism
-
CysJ functions as a specific partner of the YcbX molybdoenzyme and provides the reducing equivalents needed for the detoxification reaction at the YcbX molybdocenter
metabolism
Trichomonas vaginalis has two different enzymatic pathways to remove intracellular oxygen, i.e. NADH oxidase, which is inhibited by metronidazole, and flavin reductase
metabolism
-
the flavin reductase couples with dibenzothiophene and dibenzothiophene sulfone monooxygenase in the thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, the flavin reductase exhibits flavin reductase and nitroreductase activities
-
metabolism
-
Trichomonas vaginalis has two different enzymatic pathways to remove intracellular oxygen, i.e. NADH oxidase, which is inhibited by metronidazole, and flavin reductase
-
physiological function
-
provides reducing equivalents required to drive the luciferase genes in Escherichia coli but is not coupled to luciferase
physiological function
-
CysJ is involved in 6-N-hydroxylaminopurine resistance
physiological function
regarding all flavin reductase enzymes in Trichomonas vaginalis, FR1 is responsible for the flavin reductase activity. Hydrogen peroxide is the main if not the single product of FR1. FR1 displays a 10 to 20fold higher affinity to FMN than enzymes FR5 and FR6. Also the affinity of FR1 to FAD is at least 10fold higher than observed with FR5 and FR6 but the Vmax is considerably lower (roughly 33% of the Vmax for FMN). In contrast, FR5 and FR6 display higher affinity for riboflavin than FR1, which is inhibited by riboflavin
physiological function
-
regarding all flavin reductase enzymes in Trichomonas vaginalis, FR1 is responsible for the flavin reductase activity. Hydrogen peroxide is the main if not the single product of FR1. FR1 displays a 10 to 20fold higher affinity to FMN than enzymes FR5 and FR6. Also the affinity of FR1 to FAD is at least 10fold higher than observed with FR5 and FR6 but the Vmax is considerably lower (roughly 33% of the Vmax for FMN). In contrast, FR5 and FR6 display higher affinity for riboflavin than FR1, which is inhibited by riboflavin
-
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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
1,3-dinitrobenzene + NADH + H+
? + NAD+
-
10.6% activity compared to FMN
-
-
?
1,4-benzoquinone + NADH + H+
1,4-benzoquinol + NAD+
-
18.6% activity compared to FMN
-
-
?
4-nitroacetophenone + NADH + H+
? + NAD+
-
17.9% activity compared to FMN
-
-
?
4-nitrobenzoate + NADH + H+
? + NAD+
-
21.2% activity compared to FMN
-
-
?
FMN + NADH
FMNH2 + NADP+
-
preference for NADPH over NADH, rate of reduction is 80 times faster with NADPH
-
-
?
lumiflavin + NADH + H+
reduced lumiflavin + NAD+
-
14.1% activity compared to FMN
-
-
?
menadione + NADH + H+
menadiol + NAD+
-
19.0% activity compared to FMN
-
-
?
methyl-4-nitrobenzoate + NADH + H+
? + NAD+
-
10.7% activity compared to FMN
-
-
?
methylene blue + NADH + H+
reduced methylene blue + NAD+
-
29.0% activity compared to FMN
-
-
?
NADH + H+ + oxidized 2,6-dichlorophenolindophenol
NAD+ + reduced 2,6-dichlorophenolindophenol
nitrofurazone + NADH + H+
reduced nitrofurazone + NAD+
-
13.5% activity compared to FMN
-
-
?
nitrofurazone + NADPH + 4 H+
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
oxidized 2,6-dichlorophenolindophenol + NADPH + H+
reduced 2,6-dichlorophenolindophenol + NADP+
-
-
-
-
?
riboflavin + NAD(P)H
reduced riboflavin + NADP+
-
redox potential of the irreversible reductive half-reaction
-
-
?
riboflavin + NADH + H+
reduced riboflavin + NAD+
-
29.0% activity compared to FMN
-
-
?
2 ferricyanide + NADH
2 ferrocyanide + NAD+ + H+
-
15.4% activity compared to FMN
-
-
?
2 ferricyanide + NADH
2 ferrocyanide + NAD+ + H+
-
15.4% activity compared to FMN
-
-
?
2 ferricytochrome c + NADH
2 ferrocytochrome c + NAD+ + H+
-
8.48% activity compared to FMN
-
-
?
2 ferricytochrome c + NADH
2 ferrocytochrome c + NAD+ + H+
-
8.48% activity compared to FMN
-
-
?
FAD + NADPH + H+
FADH2 + NADP+
-
specific activity for the reduction of oxidized riboflavin, FMN and FAD is similar
-
-
?
FMN + NADPH
FMNH2 + NADP+
-
preference for NADPH over NADH, rate of reduction is 80 times faster with NADPH
-
-
?
FMN + NADPH
FMNH2 + NADP+
-
reaction may be coupled with luciferase for bioluminescence
-
-
?
FMN + NADPH + H+
FMNH2 + NADP+
-
203% activity compared to NAD+
-
-
?
FMN + NADPH + H+
FMNH2 + NADP+
-
specific activity for the reduction of oxidized riboflavin, FMN and FAD is similar
-
-
?
NADH + H+ + oxidized 2,6-dichlorophenolindophenol
NAD+ + reduced 2,6-dichlorophenolindophenol
-
23.0% activity compared to FMN
-
-
?
NADH + H+ + oxidized 2,6-dichlorophenolindophenol
NAD+ + reduced 2,6-dichlorophenolindophenol
-
23.0% activity compared to FMN
-
-
?
nitrofurazone + NADPH + 4 H+
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
-
-
-
-
?
nitrofurazone + NADPH + 4 H+
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
-
-
-
-
?
oxidized riboflavin + NADPH + H+
reduced riboflavin + NADP+
-
specific activity for the reduction of oxidized riboflavin, FMN and FAD is similar
-
-
?
riboflavin + NADPH
reduced riboflavin + NADP+
-
redox potential and equilibria in the reversible reductive half-reaction
-
-
?
additional information
?
-
-
enzyme additionally has azoreductase activity cleaving the -N=N- bond in azo dyes
-
-
-
additional information
?
-
-
the enzyme reduces both nitrofurazone and FMN effectively, it is bifunctional as flavin reductase and nitroreductase. Two different FMN molecules are involved in the FMN reduction process: FMN tightly associated to the enzyme as prosthetic group and FMN as a substrate. The enzyme is also active with FAD, riboflavin, and lumiflavin, the two latter give the highest activity
-
-
-
additional information
?
-
-
the enzyme reduces both nitrofurazone and FMN effectively, it is bifunctional as flavin reductase and nitroreductase. Two different FMN molecules are involved in the FMN reduction process: FMN tightly associated to the enzyme as prosthetic group and FMN as a substrate. The enzyme is also active with FAD, riboflavin, and lumiflavin, the two latter give the highest activity
-
-
-
additional information
?
-
-
bifunctional enzyme flavin reductase (NADPH)/biliverdin-IXbeta reductase
-
-
-
additional information
?
-
-
the enzyme is also active with FMN and NADH, cf. EC 1.5.1.36
-
-
-
additional information
?
-
-
the enzyme is also active with FMN and NADH, cf. EC 1.5.1.36
-
-
-
additional information
?
-
-
no catalytic activity is detected with NADH
-
-
-
additional information
?
-
FR is a relevant source of hydrogen peroxide but superoxide radical anions are a minor by-product of the reaction
-
-
-
additional information
?
-
FR is a relevant source of hydrogen peroxide but superoxide radical anions are a minor by-product of the reaction
-
-
-
additional information
?
-
-
enzyme transfers reduced riboflavin 5'-phosphate to luciferase by direct channeling in vitro and in vivo, formation of donor-acceptor enzyme complex
-
-
-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
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
FMN + NADPH + H+
FMNH2 + NADP+
-
203% activity compared to NAD+
-
-
?
additional information
?
-
A2GH85
FR is a relevant source of hydrogen peroxide but superoxide radical anions are a minor by-product of the reaction
-
-
-
additional information
?
-
A2GH85
FR is a relevant source of hydrogen peroxide but superoxide radical anions are a minor by-product of the reaction
-
-
-
additional information
?
-
-
enzyme transfers reduced riboflavin 5'-phosphate to luciferase by direct channeling in vitro and in vivo, formation of donor-acceptor enzyme complex
-
-
-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FMN
-
the presence of exogenously added FMN is necessary for activity
FMN
-
contains one FMN cofactor per monomer, the FMN cofactor of FRPVh can be replaced by 2-thioFMN to yield a catalytically active reconstituted reductase
FMN
-
contains one FMN cofactor per monomer, the FMN cofactor of FRPVh can be replaced by 2-thioFMN to yield a catalytically active reconstituted reductase
FMN
-
tightly associated to, two different FMN molecules are involved in the FMN reduction process: FMN tightly associated to the enzyme as prosthetic group and FMN as a substrate
NADPH
-
the FMN reductase activity is highly specific for NADPH, and the activity with NADH is 10% of that with NADPH
additional information
FR1 displays only 3% activity when NADH is added instead of NADPH
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
Fe2+, Fe3+, Mg2+, and Ca2+ do not affect the enzyme activity
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Amaranth
-
the presence of 50 mM amaranth reduces the NADPH oxidation rate 2.6fold
lumichrome
competitive inhibitor against FMN and a mixed inhibitor of NADPH
mesobiliverdin XIIIa
competitive kinetics with FMN and mixed inhibition kinetics with NADPH
NEM
-
when the enzyme is preincubated with NEM and NADH in the absence of FMN
Ponceau BS
-
the presence of 50 mM Ponceau BS reduces the NADPH oxidation rate 3.7fold
riboflavin
FR1 inhibition by possibly either by the semiquinone or the fully reduced form
additional information
-
with 1 mM ethylenediaminetetraacetic acid 99.8% activity, with 1 mM 2,2'-bipyridyl 81.8% activity
-
NADP+
-
0.1 mM, about 50% inhibition at no inhibition by NAD+ at pH 7.5, 12% inhibition at pH 4.8
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
enzyme coexpression with gene dcs-1 is induced by heat shock
-
additional information
enzyme coexpression with gene dcs-1 is induced by heat shock
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.00013
FMN
-
25°C, enzyme in complex with monooxygenase SsuD, presence of octanesulfonate
0.0009
FMN
-
coupled oxidoreductase-luciferase assay, kcat/Km = 73/(M*min), in 100 mM Na+/K+ phosphate buffer with 100 mM NaCl, pH 7.0, 1 microM Fre oxidoreductase, 10 microM decanal, 10 microM NADPH, 5 microM luciferase
0.072
FMN
-
enzyme fused to luciferase, at 23°C in 50 mM phosphate buffer, pH 7.0
0.00007
NADPH
-
mutant enzyme E99K, in 50 mM phosphate buffer, at pH 7.0, at 23°C
0.009
NADPH
-
wild type enzyme, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
0.01
NADPH
-
mutant enzyme R225A, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
0.016
NADPH
-
mutant enzyme R133A, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
0.034
NADPH
-
mutant enzyme N134A, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
0.31
NADPH
-
enzyme fused to luciferase, at 23°C in 50 mM phosphate buffer, pH 7.0
0.0013
riboflavin
-
coupled oxidoreductase-luciferase assay, kcat/Km = 1257/(M*min), in 100 mM Na+/K+ phosphate buffer with 100 mM NaCl, pH 7.0, 1 microM Fre oxidoreductase, 10 microM decanal, 10 microM NADPH, 5 microM luciferase, riboflavin is favoured by Fre oxidoreductase but a poor substrate for bacterial luciferase
additional information
additional information
-
substrate and cofactor binding, reaction kinetics
-
additional information
additional information
-
substrate and cofactor binding, reaction kinetics
-
additional information
additional information
-
Km-value of both NADPH and FMN gradually decreases at increasing concentrations of oxygen, with 0.000104 and 0.000019 mM as the upper limits for Km of FMN and NADPH, resp.
-
additional information
additional information
-
enzyme kinetic analysis, overview
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.7
FMN
-
25°C, enzyme in complex with monooxygenase SsuD, presence of octanesulfonate
11.7
FMN
-
enzyme fused to luciferase, at 23°C in 50 mM phosphate buffer, pH 7.0
5.2
NADPH
-
mutant enzyme N134A, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
9.2
NADPH
-
mutant enzyme R133A, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
19.2
NADPH
-
mutant enzyme R225A, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
23.8
NADPH
-
wild type enzyme, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.021
riboflavin
-
coupled oxidoreductase-luciferase assay, in 100 mM Na+/K+ phosphate buffer with 100 mM NaCl, pH 7.0, 1 microM Fre oxidoreductase, 10 microM decanal, 10 microM NADPH, 5 microM luciferase, riboflavin is favoured by Fre oxidoreductase but a poor substrate for bacterial luciferase
0.0012
FMN
-
coupled oxidoreductase-luciferase assay, in 100 mM Na+/K+ phosphate buffer with 100 mM NaCl, pH 7.0, 1 microM Fre oxidoreductase, 10 microM decanal, 10 microM NADPH, 5 microM luciferase
12
NADPH
-
mutant enzyme R15A, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
25
NADPH
-
mutant enzyme K167A, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
150
NADPH
-
mutant enzyme N134A, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
570
NADPH
-
mutant enzyme R133A, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
2000
NADPH
-
mutant enzyme R225A, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
2670
NADPH
-
wild type enzyme, in 20 mM N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid buffer, pH 7.0, at 22°C
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0035
NADPH
-
25°C, enzyme in complex with monooxygenase SsuD, presence of octanesulfonate
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4 - 10
-
maximum activity at pH 6.0, more than 80% activity at pH 5.5, 6.5, and 7.0, about 75% activity at pH 7.5, about 30% activity at pH 8.5, less than 20% activity at pH 9-9.5, less than 10% activity at pH 4, 4.5, and 10, pH 4.0-6.0 citrate-phosphate buffer, pH 6.0-7.5 potassium-phosphate buffer, pH 7.5-8.5 Tris-HCl buffer, pH 8.5-10.0 glycine-NaOH buffer
4.5 - 8
-
pH 4.5: about 85% of maximal activity, pH 8.0: about 45% of maximal activity
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
23
25
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20 - 55
-
maxiumum activity at 50°C, about 90% activity at 45°C, about 80% at 40 and 55°C, about 50-60% activity at 35 and 60°C, about 40% activity at 30°C, about 30% activity at 25°C, about 20% activity at 20 and 65°C, no activity at 70°C
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
additional information
-
genes fre-1 and dcs-1 are coordinately expressed through worm development
additional information
analysis of enzyme FR1 expression in metronidazole-susceptible isolates C1, G3, JH31A and Tv2: the highly metronidazole-resistant cell line of strain C1, C1res, and the clinical isolate B7268 lack detectable FRs. The residual FR activity found in CDC085 and LA1 likely results from alternative FRs that are highly expressed
additional information
-
analysis of enzyme FR1 expression in metronidazole-susceptible isolates C1, G3, JH31A and Tv2: the highly metronidazole-resistant cell line of strain C1, C1res, and the clinical isolate B7268 lack detectable FRs. The residual FR activity found in CDC085 and LA1 likely results from alternative FRs that are highly expressed
-
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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PDB
SCOP
CATH
UNIPROT
ORGANISM
Homo sapiens;
Shewanella oneidensis (strain MR-1);
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
22000
26300
28000
28400
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4 * 28400, dynamic light scattering, ArsH in solution at room temperature does exist predominantly in the tetrameric form
40000
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homodimer
homotetramer
monomer
tetramer
dimer
-
dimerization of native enzyme and apoenzyme, equilibrium ultracentrifugation
homodimer
-
2 * 28320, sequence calculation, 2 * 28000, recombinant untagged enzyme, SDS-PAGE
homodimer
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2 * 28320, sequence calculation, 2 * 28000, recombinant untagged enzyme, SDS-PAGE
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tetramer
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4 * 28400, dynamic light scattering, ArsH in solution at room temperature does exist predominantly in the tetrameric form; dimer of dimer, X-ray crystallography
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapour diffusion method, using 0.2 M calcium chloride and 20% (w/v) polyethylene glycol 3350
-
in NAD(P)(+)-free, NAD(+)-bound, and NADP(+)-bound states. The nicotinamide of NAD+ stacks the isoalloxazine ring of FMN so that NADH can directly transfer hydride. The bound NADP+ also has a compact conformation but is bound in a reverse direction, which is not suitable for hydride transfer
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
2 - 12
-
about 80% activity retained after 24 h incubation at pH 2.0-3.5, 90 to less than 100% at pH 4.0-5.5 and pH 8.0-12.0, 100% at pH 6.0-7.5, pH 4.0-6.0 citrate-phosphate buffer, pH 6.0-7.5 potassium-phosphate buffer, pH 7.5-8.5 Tris-HCl buffer, pH 8.5-10.0 glycine-NaOH buffer
699057
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20 - 70
-
30 min incubation at 20°C retains 100% activity, more than 90% activity is retained at 25-45°C, about 80% at 50°C, about 50% at 55°C, about 20% at 60°C, about 10% at 65°C, no activity at 70°C
100
-
boiling of the enzyme leads to complete loss of the FMN reductase activity
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cells harvested by centrifugation, washing twice and suspended in 50 mM Tris-HCl buffer, pH 8.0, with 1 mM dithiothreitol and 10% glycerol, cells disrupted with ultraoscillator (20 kHz), debris removed by centrifugation, enzyme purified from cell-free extracts with ÄKTA explorer, applied to Toyopearl DEAE-650 M column, equilibrated with 50 mM Tris-HCl buffer, pH 8.0, washed, proteins eluted with linear gradient of 0-0.5 M KCl, active fractions (at 0.15-0.2 M KCl) combined and concentrated by ultrafiltration (membrane filter YM-30), enzyme purified with HiLoad 16/60 Superdex 200, equilibrated with 50 mM Tris-HCl buffer, pH 8.0 with 0.15 M NaCl, elution with same buffer
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crude lysate assay with briefly centrifuged cells, resuspended in B-PER bacterial protein extraction reagent, recombinant pZCFRE1 expression product is clarified by centrifugation and applied to a custom nickel affinity column, dialyzed into buffer containing 100 mM Na+/K+ phosphate and 100 mM NaCl, pH 7.0
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HiTrap DEAE column chromatography, Mono Q column chromatography, and Superdex 75 gel filtration
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recombinant enzyme 23fold from Escherichia coli to homogeneity by dialysis, anion exchange chromatography, dialysis, two steps of Blue Sepharose affinity chromatography, and again dialysis
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recombinant enzyme from Escherichia coli strain BL21(DE3) to over 90% purity, recombinant fusion protein from Escherichia coli strain JM109 to over 80% purity
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recombinant N-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinant wild-type and mutant enzyme from Escherichia coli to homogeneity
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the flavin reductase-luciferase fusion protein is purified using DEAE-cellulose DE-52 column chromatography, DEAE-Sepharose column chromatography, and Superdex 200 gel filtration
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ultracentrifugation, G-25 gel filtration, DEAE-Sepharose column chromatography, and Sephadex G25 gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
frb gene inserted into plasmid pET21-a, introduced into Escherichia coli BL21 (DE3), cultivated at 30°C
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gene frd2, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant overexpression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3)
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gene fre-1, DNA and amino acid sequence determination and analysis, transcribed as a bicistronic pre-mRNA together with gene dcs-1, encoding Hint-related 7meGMP-directed hydrolase DCS-1
gene frp, functional expression of the enzyme fused to yellow fluorescence protein YFP in a Vibrio harveyi strain lacking the frp gene, subcloning and expression in Escherichia coli strains JM109 and BL21(DE3), the yellow fluorescence protein YFP is a mutant of the green fluorescence protein GFP, with mutations S65G, V68L, S72A, and T203Y
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gene ipa-43d, DNA and amino acid sequence determination and analysis, genetic structure and sequence comparison, recombinant overexpression in Escherichia coli J83
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gene TVAG_517010, DNA and amino acid sequence determination and analysis, quantitative RT-PCR expression analysis of FR1 in metronidazole-susceptible isolates C1, G3, JH31A and Tv2 , re-introduction of FR1 into an aerobically-resistant strain, transfection of the highly metronidazole-resistant isolate B7268 with the FR1 gene restores metronidazole susceptibility
gene fre-1, DNA and amino acid sequence determination and analysis, transcribed as a bicistronic pre-mRNA together with gene dcs-1, encoding Hint-related 7meGMP-directed hydrolase DCS-1
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gene fre-1, DNA and amino acid sequence determination and analysis, transcribed as a bicistronic pre-mRNA together with gene dcs-1, encoding Hint-related 7meGMP-directed hydrolase DCS-1
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
L114M/L165M
-
mutant created to obtain selenomethionine-containing enzyme for crystallization. Activity of mutant is similar to wild-type, circular dichroism spectra identical to wiil-type
E99K
-
the mutation destabilizes the dimer and has an enhanced subunit dissociation as evident from a 44fold higher Kd for the monomer-dimer equilibrium
K167A
-
the mutant has apparently greatly increased Km and reduced kcat/Km for NADPH
R15A
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the mutant has apparently greatly increased Km and reduced kcat/Km for NADPH
R203A
additional information
R203A
-
decrease of NADPH binding potential, critical role of Arg203 in the specific recognition and binding of NADPH
R203A
-
site-directed mutagenesis, no reversibility of the reduction of FMN, the half-reaction
additional information
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improvement of the intracellular environment for enhancing L-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply. Construction of mutants of gene frd2, strain 5-5(frd2) and deletion strains 5-5DELTAfrd2 and 5-5DELTAfrd12. The extracellular H2O2 concentrations of mutant 5-5DELTAfrd12 are lower than that of the wild-type strain SYPA5-5, and the extracellular H2O2 concentrations of mutant 5-5(frd2) is increased compared to the wild-type. Flavin reductase activities in frd1 and frd2 overexpression and deletion strains with NADH and FAD, overview
additional information
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improvement of the intracellular environment for enhancing L-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply. Construction of mutants of gene frd2, strain 5-5(frd2) and deletion strains 5-5DELTAfrd2 and 5-5DELTAfrd12. The extracellular H2O2 concentrations of mutant 5-5DELTAfrd12 are lower than that of the wild-type strain SYPA5-5, and the extracellular H2O2 concentrations of mutant 5-5(frd2) is increased compared to the wild-type. Flavin reductase activities in frd1 and frd2 overexpression and deletion strains with NADH and FAD, overview
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additional information
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Fre oxidoreductase deletion strain shows 99% reduced bioluminescence in coupled assay with luciferase
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Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
soluble denatured mutant enzyme E99K is mixed into 50fold volume of pre-cooled 50 mM phosphate buffer containing 0.1 M FMN and stirred on ice in the dark for 15 min for protein renaturation and the reconstitution of E99K holoenzyme
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LINKS TO OTHER DATABASES (specific for EC-Number 1.5.1.30)
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