Information on EC 1.5.1.38 - FMN reductase (NADPH)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.5.1.38
-
RECOMMENDED NAME
GeneOntology No.
FMN reductase (NADPH)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
FMNH2 + NADP+ = FMN + NADPH + H+
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
bacterial bioluminescence
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two-component alkanesulfonate monooxygenase
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Riboflavin metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
FMNH2:NADP+ oxidoreductase
The enzymes from bioluminescent bacteria contain FMN [4], while the enzyme from Escherichia coli does not [8]. The enzyme often forms a two-component system with monooxygenases such as luciferase. Unlike EC 1.5.1.39, this enzyme does not use NADH as acceptor [1,2]. While FMN is the preferred substrate, the enzyme can also use FAD and riboflavin with lower activity [3,6,8].
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
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a general catalytic cycle is proposed for two-component reductases of the flavodoxin-like superfamily, by which the enzyme can potentially provide FMNH2 to its partner monooxygenase by different routes depending on the FMN concentration and the presence of a partner monooxygenase SsueD, overview
physiological function
additional information
-
at least a dimeric association is required for the function of enzyme SsuE, FMN binding site structure, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-thioFMN + NADPH + H+
2-thioFMNH2 + NADP+
show the reaction diagram
-
the holoenzyme reconstituted with 2-thioFMN is catalytically active in using either FMN or 2-thioFMN as a substrate
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-
?
FAD + NADPH + H+
FADH2 + NADP+
show the reaction diagram
FMN + NADH + H+
FMNH2 + NAD+
show the reaction diagram
when NADH is the pyrimidinic substrate, a distinct activity maximum is obtained at an FMN concentration of 0.5 mM, whereas concentrations higher than 2.5 mM led to more than 60% decrease in specific activity
-
-
?
FMN + NADPH + H+
FMNH2 + NADP+
show the reaction diagram
FMNH2 + NADP+
FMN + NADPH + H+
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 + NADPH + H+
FMNH2 + NADP+
show the reaction diagram
FMNH2 + NADP+
FMN + NADPH + H+
show the reaction diagram
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-
-
-
r
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-thio-FMN
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binds to the cofactor site of the apoenzyme with an affinity similar to that for FMN binding. The holoenzyme reconstituted with 2-thioFMN is catalytically active in using either FMN or 2-thioFMN as a substrate
NADH
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kcat/KM for NADPH is 335fold higher compared to kcat/KM for NADH
NADP+
NADPH
additional information
-
the enzyme does not contain any bound flavin cofactor
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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0.005 M, 14% inhibition
AMP
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0.01 M, 28% inhibition
dicoumarol
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0.000325 M, 15% inhibition
FMN
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when NADPH is supplied as pyrimidinic substrate, maximal reductase activity is obtained with 2.5-10 mM FMN, while higher FMN concentration led to 15% decrease in SsuE activity. When NADH is the pyrimidinic substrate, a distinct activity maximum is obtained at an FMN concentration of 0.5 mM, whereas concentrations higher than 2.5 mM led to more than 60% decrease in specific activity
KCN
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0.01 M, 10% inhibition
N-ethylmaleimide
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0.001 M, 26% inhibition
p-hydroxymercuribenzoate
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0.001 M, 80% inhibition
rotenone
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0.0004 M, 30% inhibition
additional information
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FMN at concentrations over 0.002 mM significantly inhibits the coupled reaction in both light intensity and quantum yield, and shows apparent noncompetitive and competitive inhibition patterns against NADPH and luciferase, respectively. No inhibition of the NADPH oxidation is detected under identical conditions
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.007
2-thioFMN
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pH 7.0, 23C, enzyme derivative reconstituted from apoenzyme and 2-thioFMN
0.0027 - 0.019
FAD
0.000054 - 0.018
FMN
0.5555
NADH
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pH 7.9, 30C, 0.0005 mM FMN
0.003 - 0.2
NADPH
0.025
riboflavin
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pH 7.0, 23C, native enzyme
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.5 - 34
NADPH
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.28
FAD
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pH 7.9, 30C
0.0079
FMN
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pH 7.9, 30C
0.00167
NADH
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pH 7.9, 30C, 0.0005 mM FMN
0.56 - 2670
NADPH
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.85
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pH 6.8, 23C
51
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pH 7.0, 23C
88.4
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23C, pH not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8
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assay at
7.5
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assay at
7.9
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
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the enzyme exhibits a maximum activity at pH 5.5 which drops to a broad shoulder from pH 6.5 to pH 8.5 with an activity 75% that of maximum at pH 7.0. About 60% of maximal activity at pH 5.0, about 50% of maximal activity at pH 10.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
30
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assay at
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Pseudomonas putida (strain ATCC 47054 / DSM 6125 / NCIMB 11950 / KT2440)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23700
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2 * 23700, calculated from sequence
25400
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2 * 25400, SDS-PAGE
26312
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1 * 26312, calculated from sequence
28000
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1 * 28000, SDS-PAGE
40000
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gel filtration
40900
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dimeric SsueE in presence of flavin, analytical ultracentrifugation
58400
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gel filtration
60000
recombinant His-tagged enzyme, gel filtration
63000
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sucrose density gradient centrifugation
73100
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tetrameric SsuE enzyme in the absence of flavin, analytical ultracentrifugation
additional information
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formation of a stable complex between the flavin mononucleotide (FMN) reductase (SsuE) and monooxygenase (SsuD) of the alkanesulfonate monooxygenase system. The stoichiometry for protein-protein interactions is proposed to involve a 1:1 monomeric association of SsuE with SsuD. Interactions between the two proteins do not lead to overall conformational changes in protein structure
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
monomer
tetramer
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dimer of dimers, 4 * 21300, analytical ultracentrifugation
additional information
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analytical ultracentrifugation studies of SsuE confirm a dimer-tetramer equilibrium exists in solution, with FMN binding favoring the dimer. The active site includes residues from both subunits
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme in apoform, or complexed with FMN or FMNH2, hanging drop vapour diffusion method, mixing 0.004 ml of 10 mg/ml of protein in 10 mM HEPES, pH 7.0, with 0.002 ml of reservoir solution containing 7.5% w/v PEG 3350 and 0.1 M sodium citrate, at room temperature, for complexed protein, the crystals are soaked in an AML containing 1 mM FMN solution, X-ray diffraction structure determination and analysis at 1.9-2.3 A resolution
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vapor-diffusion technique yields single crystals that grow as hexagonal rods and diffract to 2.9 A resolution using synchrotron X-ray radiation. The protein crystallizes in the primitive hexagonal space group P622. Substitution of two leucine residues (Leu114 and Leu165) to methionine is performed to obtain selenomethionine-containing SsuE for MAD phasing. The selenomethionine derivative of SsuE has been expressed and purified and crystals of the protein have been obtained with and without bound FMN
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the 1.8 A crystal structure of flavin reductase P from Vibrio harVeyi is solved by multiple isomorphous replacement and reveals that the enzyme is a unique dimer of interlocking subunits, with 9352 A(2) of surface area buried in the dimer interface. Each subunit comprises two domains
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
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5 min, complete loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, considerable loss of activity of reductase preparations
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4C, considerable loss of activity of reductase preparations
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE-Sepharose column chromatography, Mono Q column chromatography, and Superdex 75 gel filtration
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recombinant enzyme
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recombinant His-tagged enzyme from Escherichia coli strain DH5alpha by nickel affinity chromatography and desalting gel filtration
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3)
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SsuE is purified to homogeneity as an N-terminal histidine-tagged fusion protein
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3)pLysS cells
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gene BC_1619, DNA and amino acid sequence determination and analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain DH5alpha
gene ssueE, phylogenetic tree, recombinant expression
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gene ssueE, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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overexpression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Y118A
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site-directed mutagenesis, the SsuE variant converts the typically flavin-free enzyme to a flavin-bound form. The Y118A SsuE FMN cofactor is reduced with approximately 1 equiv of NADPH in anaerobic titration experiments, and the flavin remains bound following reduction. No measurable sulfite product is formed in a coupled assays with the Y118A SsuE variant and SsuD, demonstrating that flavin transfer is no longer supported
K167A
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the mutant has apparently greatly increased Km and severely reduced kcat/Km values for NADPH and leads to a slight increase in kcat/Km for NADH
N134A
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the mutant shows strongly decreased kcat/Km for NADPH
R133A
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the mutant shows strongly decreased kcat/Km for NADPH
R15A
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the mutant has apparently greatly increased Km and severely reduced kcat/Km values for NADPH
R225A
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the mutant shows decreased kcat/Km for NADPH
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
insolubleoverexpressed enzyme protein is resuspended in a resuspension buffer containing 20 ml, 20 mM Tris-HCL, 0.5 M NaCl, pH 8.0, sonicated for 50 s discontinuously, spun down at 8000 rpm (5400 x g) at 4C for 10 min, pellets resuspended in isolation buffer containing 15 ml, 20 mM Tris-HCL, 0.5 M NaCl, 2% Triton X-100, and 12% w/v urea, pH 8.0
Show AA Sequence (643 entries)
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