Information on EC 1.5.1.30 - flavin reductase (NADPH)

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

EC NUMBER
COMMENTARY hide
1.5.1.30
-
RECOMMENDED NAME
GeneOntology No.
flavin reductase (NADPH)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
reduced riboflavin + NADP+ = riboflavin + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
-
Riboflavin metabolism
-
-
flavin biosynthesis
-
-
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.
CAS REGISTRY NUMBER
COMMENTARY hide
56626-29-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
ISW 1214.
-
-
Manually annotated by BRENDA team
strain WU-S2B
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
gene fre-1
-
-
Manually annotated by BRENDA team
strain NIH:200
-
-
Manually annotated by BRENDA team
strain NIH:200
-
-
Manually annotated by BRENDA team
New Zealand white rabbits, ca. 1.5 kg each
-
-
Manually annotated by BRENDA team
strain AS1.1737
-
-
Manually annotated by BRENDA team
strain AS1.1737
-
-
Manually annotated by BRENDA team
2a strain 2457T
-
-
Manually annotated by BRENDA team
stain 7
-
-
Manually annotated by BRENDA team
stain 7
-
-
Manually annotated by BRENDA team
strain MAV or B392.
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,3-dinitrobenzene + NADH + H+
? + NAD+
show the reaction diagram
-
10.6% activity compared to FMN
-
-
?
1,4-benzoquinone + NADH + H+
1,4-benzoquinol + NAD+
show the reaction diagram
-
18.6% activity compared to FMN
-
-
?
2 ferricyanide + NADH
2 ferrocyanide + NAD+ + H+
show the reaction diagram
2 ferricytochrome c + NADH
2 ferrocytochrome c + NAD+ + H+
show the reaction diagram
3-nitrophenol + NADH + H+
? + NAD+
show the reaction diagram
-
8.95% activity compared to FMN
-
-
?
4-nitroacetophenone + NADH + H+
? + NAD+
show the reaction diagram
-
17.9% activity compared to FMN
-
-
?
4-nitroaniline + NADH + H+
? + NAD+
show the reaction diagram
-
4.91% activity compared to FMN
-
-
?
4-nitrobenzoate + NADH + H+
? + NAD+
show the reaction diagram
-
21.2% activity compared to FMN
-
-
?
4-nitrophenol + NADH + H+
? + NAD+
show the reaction diagram
-
8.07% activity compared to FMN
-
-
?
4-nitrotoluene + NADH + H+
? + NAD+
show the reaction diagram
-
8.83% activity compared to FMN
-
-
?
FAD + NADH + H+
FADH2 + NAD+
show the reaction diagram
-
58.1% activity compared to FMN
-
-
?
FAD + NADPH
FADH2 + NADP+
show the reaction diagram
FAD + NADPH + H+
FADH2 + NADP+
show the reaction diagram
FMN + NAD(P)H
FMNH2 + NAD(P)+
show the reaction diagram
-
-
-
-
r
FMN + NADH
FMNH2 + NAD+
show the reaction diagram
FMN + NADH
FMNH2 + NADP+
show the reaction diagram
-
preference for NADPH over NADH, rate of reduction is 80 times faster with NADPH
-
-
?
FMN + NADH + H+
FMNH2 + NAD+
show the reaction diagram
FMN + NADPH
FMNH2 + NADP+
show the reaction diagram
FMN + NADPH
FMNH2 + NADP+ + H+
show the reaction diagram
-
-
-
-
?
FMN + NADPH + H+
FMNH2 + NADP+
show the reaction diagram
galactoflavin + NADPH
reduced galactoflavin + NADP+
show the reaction diagram
lumiflavin + NADH + H+
reduced lumiflavin + NAD+
show the reaction diagram
-
14.1% activity compared to FMN
-
-
?
menadione + NADH + H+
menadiol + NAD+
show the reaction diagram
-
19.0% activity compared to FMN
-
-
?
methyl-4-nitrobenzoate + NADH + H+
? + NAD+
show the reaction diagram
-
10.7% activity compared to FMN
-
-
?
methylene blue + NADH + H+
reduced methylene blue + NAD+
show the reaction diagram
-
29.0% activity compared to FMN
-
-
?
NADH + H+ + oxidized 2,6-dichlorophenolindophenol
NAD+ + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
nitrofurantoin + NADH + H+
? + NAD+
show the reaction diagram
-
10.1% activity compared to FMN
-
-
?
nitrofurazone + NADH + H+
reduced nitrofurazone + NAD+
show the reaction diagram
-
13.5% activity compared to FMN
-
-
?
nitrofurazone + NADPH + 4 H+
5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
show the reaction diagram
oxidized 2,6-dichlorophenolindophenol + NADPH + H+
reduced 2,6-dichlorophenolindophenol + NADP+
show the reaction diagram
-
-
-
-
?
oxidized methylene blue + NADPH + H+
reduced methylene blue + NADP+
show the reaction diagram
-
-
-
-
?
oxidized riboflavin + NADPH + H+
reduced riboflavin + NADP+
show the reaction diagram
riboflavin + NAD(P)H
reduced riboflavin + NAD(P)+
show the reaction diagram
-
-
-
-
r
riboflavin + NAD(P)H
reduced riboflavin + NADP+
show the reaction diagram
-
redox potential of the irreversible reductive half-reaction
-
-
?
riboflavin + NADH + H+
reduced riboflavin + NAD+
show the reaction diagram
-
29.0% activity compared to FMN
-
-
?
riboflavin + NADPH
reduced riboflavin + NADP+
show the reaction diagram
riboflavin + NADPH + H+
reduced riboflavin + NADP+
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
FMN + NADH + H+
FMNH2 + NAD+
show the reaction diagram
FMN + NADPH
FMNH2 + NADP+ + H+
show the reaction diagram
-
-
-
-
?
FMN + NADPH + H+
FMNH2 + NADP+
show the reaction diagram
riboflavin + NADPH
reduced riboflavin + NADP+
show the reaction diagram
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 hide
LITERATURE
IMAGE
2-thio-FMN
NADPH
riboflavin
-
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CaCl2
-
with 1 mM 84.5% activity
FeCl2
-
with 1 mM 90.0% activity
MgCl2
-
with 1 mM 92.6% activity
MnCl2
-
with 1 mM 97.0% activity
NiCl2
-
with 1 mM 86.6% activity
ZnCl2
-
with 1 mM 96.8% activity
additional information
-
Fe2+, Fe3+, Mg2+, and Ca2+ do not affect the enzyme activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
-
-
2,3-diphosphoglycerate
-
-
5,5'-dithiobis(2-nitrobenzoic acid)
-
with 0.1 mM 28.8% activity
7-hydroxycoumarin
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-
Acrinol
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-
AgNO3
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with 1 mM 1.05% activity
Amaranth
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the presence of 50 mM amaranth reduces the NADPH oxidation rate 2.6fold
Bathocuproine sulfonate
-
-
Cibacron Marine
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azo dye, competitive to NADPH and FMN
CuCl2
-
with 1 mM 11.2% activity
HgCl2
lumichrome
competitive inhibitor against FMN and a mixed inhibitor of NADPH
mesobiliverdin XIIIa
competitive kinetics with FMN and mixed inhibition kinetics with NADPH
N-ethylmaleimide
-
with 1 mM 16.3% activity
NADP+
NEM
-
when the enzyme is preincubated with NEM and NADH in the absence of FMN
p-chloromercuribenzoic acid
PCMB
-
-
Ponceau BS
-
the presence of 50 mM Ponceau BS reduces the NADPH oxidation rate 3.7fold
proflavin hemisulfate
-
-
SeO32-
-
-
Zn2+
-
-
additional information
-
with 1 mM ethylenediaminetetraacetic acid 99.8% activity, with 1 mM 2,2'-bipyridyl 81.8% activity
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.007
2-thioFMN
-
enzyme reconstituted from apoenzyme and 2-thioFMN
0.0036 - 0.125
FAD
0.000016 - 14.2
FMN
0.04
galactoflavin
-
-
0.208 - 0.65
NADH
0.000019 - 0.71
NADPH
0.0163
nitrofurazone
-
-
0.053
oxidized riboflavin
pH 7.5, 25°C
0.0013 - 0.04
riboflavin
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.7
Cibacron Marine
Bacillus subtilis
-
pH 7.5, 37°C
0.3 - 51
FAD
0.099 - 60
FMN
5.2 - 70
NADPH
0.3
oxidized riboflavin
Homo sapiens
P30043
pH 7.5, 25°C
6.7
riboflavin
Streptomyces viridifaciens
-
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.4
FAD
Homo sapiens
P30043
pH 7.5, 25°C
20
0.0012 - 1.9
FMN
56
12 - 2670
NADPH
5
5.6
oxidized riboflavin
Homo sapiens
P30043
pH 7.5, 25°C
41645
0.021
riboflavin
Escherichia coli
-
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
351
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00338
7-hydroxycoumarin
-
-
0.003 - 0.015
Cibacron Marine
0.076
lumichrome
pH 7.5, 25°C
0.00059
mesobiliverdin XIIIa
pH 7.5, 25°C
0.0139
NADP+
-
25°C, single-enzyme assay
0.0035 - 0.0039
NADPH
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.37
-
pH 7.0, 25°C
42.1
-
FMN reduction with NADH, pH 6.0, 50°C
85.5
-
FMN reduction with NADPH, pH 6.0, 50°C
133
-
flavin reductase from recombinant Escherichia coli
141
-
without N-terminal His-tag
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.9
-
citrate-phosphate buffer
5
sharp optimum, in citrate buffer
7.5
assay at
8
-
enzyme assay
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
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
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pH 4.5: about 85% of maximal activity, pH 8.0: about 45% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
-
enzyme assay
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 55
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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
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
whole hearts
Manually annotated by BRENDA team
additional information
-
genes fre-1 and dcs-1 are coordinately expressed through worm development
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
perinuclear
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20000
-
2 * 20000
21000
-
4 * 21000, SDS-PAGE
21270
-
calculated from sequence of cloned DNA
21500
-
x * 21500, SDS-PAGE
24600
-
2 * 24600, X-ray crystallography
26000
-
SDS-PAGE
26320
-
calculated from sequence of cloned DNA
28320
-
calculated from sequence of cDNA
28400
-
4 * 28400, dynamic light scattering, ArsH in solution at room temperature does exist predominantly in the tetrameric form
30000 - 38000
-
gel filtration
33000
-
gel filtration
36000
-
gel filtration
49200
-
X-ray crystallography
52600
-
X-ray crystallography
53783
-
x * 53783, recombinant fusion protein, sequence calculation
76000
-
gel filtration
86000
-
gel filtration
113600
-
dynamic light scattering
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
homotetramer
monomer
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
selenomethionine derivative of mutant L114M/L165M
-
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
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
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
6 - 8.5
-
-
392304
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
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
86.5
-
melting point of protein
100
-
boiling of the enzyme leads to complete loss of the FMN reductase activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
freezing and thawing causes no appreciable loss of the enzyme activity
-
stable at temperatures below 50°C
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, the purified enzyme is stable at
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
bifunctional enzyme flavin reductase (NADPH)/biliverdin-IXbeta reductase
-
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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FMN-agarose chromatography as a key step
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HiTrap DEAE column chromatography, Mono Q column chromatography, and Superdex 75 gel filtration
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preparation of apoenzyme
-
recombinant enzyme from Escherichia coli strain BL21(DE3) to over 90% purity, recombinant fusion protein from Escherichia coli strain JM109 to over 80% purity
-
recombinant enzyme from Escherichia coli to homogeneity
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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
expressed in Escherichia coli BL21 cells
-
expressed in Escherichia coli BL21(DE3)pLysS cells
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expression of wild-type enzyme and mutant R203A in Escherichia coli
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expression of wild-type enzyme in Escherichia coli
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frb gene inserted into plasmid pET21-a, introduced into Escherichia coli BL21 (DE3), cultivated at 30°C
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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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PCR-amplification, expression in Escherichia coli Bl21 (deltaDE3)
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
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
N134A
-
the mutant shows increased Km and reduced kcat/Km for NADPH
R133A
-
the mutant shows increased Km and reduced kcat/Km for NADPH
R15A
-
the mutant has apparently greatly increased Km and reduced kcat/Km for NADPH
R225A
-
the mutant shows about wild type activity
additional information
-
Fre oxidoreductase deletion strain shows 99% reduced bioluminescence in coupled assay with luciferase
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
-
Show AA Sequence (238 entries)
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