Information on EC 1.5.1.36 - flavin reductase (NADH)

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

EC NUMBER
COMMENTARY hide
1.5.1.36
-
RECOMMENDED NAME
GeneOntology No.
flavin reductase (NADH)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
reduced flavin + NAD+ = flavin + NADH + H+
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Riboflavin metabolism
-
-
Metabolic pathways
-
-
SYSTEMATIC NAME
IUBMB Comments
flavin:NAD+ oxidoreductase
The enzyme from Escherichia coli W catalyses the reduction of free flavins by NADH. The enzyme has similar affinity to FAD, FMN and riboflavin. Activity with NADPH is more than 2 orders of magnitude lower than activity with NADH.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
genes LJ_0548 and LJ_0549, or nfr1 and nfr2, encoding the subunits 1 and 2 of the enzyme
Q74HL7 AND Q74HL8
UniProt
Manually annotated by BRENDA team
genes LJ_0548 and LJ_0549, or nfr1 and nfr2, encoding the subunits 1 and 2 of the enzyme
Q74HL7 AND Q74HL8
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
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
FAD + NAD(P)H
FADH2 + NAD(P)+
show the reaction diagram
FAD + NADH
FADH2 + NAD+
show the reaction diagram
FAD + NADH + H+
FADH2 + NAD+
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 + H+
FMNH2 + NAD+
show the reaction diagram
FMN + NADPH
FMNH2 + NADP+
show the reaction diagram
-
-
-
-
r
NADH + flavin
NAD+ + reduced flavin
show the reaction diagram
riboflavin + NADH
reduced riboflavin + NAD+
show the reaction diagram
riboflavin + NADH + H+
reduced riboflavin + NAD+
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
FAD + NAD(P)H
FADH2 + NAD(P)+
show the reaction diagram
FAD + NADH
FADH2 + NAD+
show the reaction diagram
FAD + NADH + H+
FADH2 + NAD+
show the reaction diagram
FMN + NAD(P)H
FMNH2 + NAD(P)+
show the reaction diagram
-
-
-
-
r
FMN + NADH + H+
FMNH2 + NAD+
show the reaction diagram
NADH + flavin
NAD+ + reduced flavin
show the reaction diagram
riboflavin + NADH + H+
reduced riboflavin + NAD+
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
additional information
no activity with NADPH
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cr3+
-
slight activation by 1 mM Cr3+
Zn2+
-
slight activation by 1 mM Zn2+
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ag+
-
strong inhibition at 1 mM Ag+
AMP
mixed noncompetitive versus FMN, and competitive versus NADH
Cu2+
-
strong inhibition at 1 mM Cu2+
Fe2+
complete inactivation at 0.1 mM, 38% activity remains in presence of 10 mM EDTA
Hg2+
-
strong inhibition at 1 mM Hg2+
HpaB
-
presence and binding of HpaB reduced the enzyme activity of HpaC due to stabilization and reduced oxidation by O2 of FADH2
-
lumichrome
an FMN analogue completely lacking the ribityl side chain, competitive versus FMN, and mixed noncompetitive versus NADH
styrene oxide
slightly activating at 0.1 mM, inhibitory at 0.2 mM
additional information
poor inhibition by H2O2
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
slightly activating
DTT
slightly activating
iodoacetamide
slightly activating
PMSF
slightly activating
styrene oxide
slightly activating at 0.1 mM, inhibitory at 0.2 mM
Thiourea
slightly activating
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0008 - 0.056
FAD
0.0021 - 0.03
FMN
0.008 - 0.183
NADH
0.0026 - 0.011
riboflavin
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
40 - 178
FAD
1.7 - 7.8
FMN
0.0247 - 178
NADH
21
riboflavin
pH 7.5, 25C, recombinant enzyme, with NADH
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0013
FAD
pH 7.5, 25C, recombinant enzyme, with NADH
0.0014
FMN
pH 7.5, 25C, recombinant enzyme, with NADH
0.0027 - 2.94
NADH
0.0019
riboflavin
pH 7.5, 25C, recombinant enzyme, with NADH
additional information
additional information
-
V(max)/Km-values in 1/min*mg: 33.3 for FMN (with NADH as electron donor), 22.7 for riboflavin (with NADH as electron donor), 12.3 for FAD (with NADH as electron donor)
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
29 - 35
AMP
0.049 - 0.11
lumichrome
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.2
Q74HL7 AND Q74HL8
crude cell extract, about, with FAD, or FMN, or riboflavin, and NADH, pH 7.0, 37C
0.318
-
strain W-KO, recombinant enzyme Fre, growth on 4-hydroxyphenylacetate as carbon source
0.6
-
using riboflavin and NADPH as substrates, at pH 7.0 and 55C
0.8
-
using FAD and NADPH as substrates, at pH 7.0 and 55C
1.3
-
using FMN and NADPH as substrates, at pH 7.0 and 55C
25.1
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strain W-KO, recombinant enzyme HpaC, growth on 4-hydroxyphenylacetate as carbon source
36
-
using riboflavin and NADH as substrates, at pH 7.0 and 55C
189
-
using FAD and NADH as substrates, at pH 7.0 and 55C
617
-
using FMN and NADH as substrates, at pH 7.0 and 55C
additional information
-
activity in a coupled assay of HpaB and HpaC
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
-
assay at
7.8
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.3 - 7.5
activity irreversibly drops to under 5% at pH 5.3 indicating denaturation
6.5 - 8
-
the enzyme maintains more than 80% of activity between pH values of 6.5 to 8
6.5 - 7.5
-
optimal range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.12
sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
-
enzyme activities with different growth conditions, overview, strain W-KO does not grow on 4-hydroxyphenylacetate as carbon source
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
16989
-
2 * 16989, calculated from amino acid sequence
17000
-
2 * 17000, SDS-PAGE
18000
-
SDS-PAGE
18522
-
2 * 18522, small component of the 4-hydroxyphenylacetate 3-monooxygenase, calculated from sequence
18600
-
2 * 18600, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, calculated from sequence
18679
-
2 * 18679, calculated from sequence
19400
-
x * 19400, calculated from amino acid sequence
39000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme in apoform or in complex with FMN, sitting drop vapour diffusion method, crystallization from 0.1 M HEPES, pH 7.4, 10% v/v 2-propanol, and 20% w/v PEG 4000, a few days at 20C, method optimization, X-ray diffraction structure determination and analysis at 1.53-1.85 A resolution, small angle X-ray scattering, molecular replacement method using structure PDB ID 1RZ0 as search model
complexed with FMN, FAD or riboflavin, sitting drop vapor diffusion method, using 1.6 M ammonium sulfate and 4-5% (v/v) isopropanol
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45 - 60
-
enzyme activity is stable at temperatures below 45C. After incubation at 60C for 30 min, more than 90% of the activity is lost
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 2 months, no significant loss of activity
-
-20C, no significant loss of activity is observed during 2 months of storage at this temperature
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, butyl-Sepharose column chromatography, Source 30Q column chromatography, FMN-Sepharose 6B affinity column chromatography, and Superdex 75 gel filtration
-
ammonium sulfate precipitation, HiTrap Q column chromatography, and Superdex 200 gel filtration; ammonium sulfate precipitation, HiTrap Q column chromatography, and Superdex 200 gel filtration
native enzyme from strain NCC 533 by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration
Q74HL7 AND Q74HL8
Ni-affinity chromatography and heat treatment
-
Ni2+ column chromatography
-
recombinant C-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3)pLysS by nickel affinity chromatography
recombinant enzyme produced in Escherichia coli K-12 cells
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recombinant His-tagged wild-type enzymes HpaC and Fre from strain BL21(DE3)
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recombinant N-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
-
refolded recombinant enzyme by anion exchange chromatography and gel filtration, or by nickel affinity chromatography and gel filtration, when a His10-tagged enzyme is expressed
using Ni-NTA chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Escherichia coli strain BL21(DE3)pLysS effectively produces an active and soluble form of StyB as about 9% of the total protein content, when cultivated at 20C with 0.5 mM IPTG
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expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli Rosetta(DE3) cells
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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 Nox, DNA and amino acid sequence determination and analysis, phylogenetic tree, quantitative real-time PCR enzyme expresison analysis
gene Pden_2689, recombinant overexpression of C-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)pLysS
overexpression in Escherichia coli
-
overproduction of the small HpaC component in Escherichia coli K-12
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overproduction of the small HpaC component of the 4-hydroxyphenylacetate 3-monooxygenase in Escherichia coli K-12 cells
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recombinant overexpression of His-tagged wild-type enzymes HpaC and Fre in strain BL21(DE3), complementation of the inactivation mutant by transient expression of different gene hpaC variants, overview
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sequence comparisons and phylogenetic analysis, cloning in Escherichia coli strain DH5alpha, recombinant expression in Escherichia coli strain BL21(DE3) (pLysS) in inclusion bodies
the NADH-dependent flavin reductase is encoded by two highly similar genes, LJ_0548 and LJ_0549, or ,nfr1 and nfr2, encoding the subunits 1 and 2 of the enzyme, DNA and amino acid sequence determination and analysis, a plasmid with the promoter region of the LJ_0045 lactate dehydrogenase gene and a bidirectional terminator is used for overexpression of the LJ_0548 and LJ_0549 genes
Q74HL7 AND Q74HL8
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
expression level of gene Nox is upregulated and co-related to the intracellular H2O2 concentration during microsclerotium initiation
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
refolding of recombinant enzyme from Escherichia coli strain BL21(DE3) (pLysS) inclusion bodies, with Triton X100 used in the washing buffer, refolding from solubilization buffer containing 8 M urea, 10 mM DTT, 50 mM Tris-HCl, pH 8.0, per mg pellet at 50C for 30 min, refolding in a 20fold volume of refolding buffer.containing 50 mM TrisHCl, pH 8.0, 240 mM NaCl, 10 mM KCl, 1 mM Na2EDTA, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, and 0.05 % w/v PEG 4000, followed by ultrafiltration
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