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Information on EC 1.5.1.38 - FMN reductase (NADPH) and Organism(s) Escherichia coli and UniProt Accession P80644

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EC Tree
     1 Oxidoreductases
         1.5 Acting on the CH-NH group of donors
             1.5.1 With NAD+ or NADP+ as acceptor
                1.5.1.38 FMN reductase (NADPH)
IUBMB Comments
The enzymes from bioluminescent bacteria contain FMN , while the enzyme from Escherichia coli does not . 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].
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Select one or more organisms in this record:
This record set is specific for:
Escherichia coli
UNIPROT: P80644
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
Synonyms
(NADPH)-dependent flavin mononucleotide reductase, (NADPH)-dependent FMN reductase, BC_1619, flavin reductase P, FMN reductase, FRP, More, NAD(P)H:FMN reductase, NADPH specific FMN reductase, NADPH-flavin oxidoreductase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
(NADPH)-dependent flavin mononucleotide reductase
293918
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(NADPH)-dependent FMN reductase
293918
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flavin reductase P
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-
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FMN reductase
293918
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FRP
-
-
-
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NAD(P)H:FMN reductase
293918
-
ssueE
293918
gene name
ycbP
293918
gene name
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
FMNH2 + NADP+ = FMN + NADPH + H+
show the reaction diagram
reaction mechanism, overview
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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].
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
FAD + NADPH + H+
FADH2 + NADP+
show the reaction diagram
FMN is the preferred flavin substrate of SsuE but FAD and riboflavin are also reduced at significant rates, whereas lumiflavin is not
-
-
?
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
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-
?
FMN + NADPH + H+
FMNH2 + NADP+
show the reaction diagram
FMN is the preferred flavin substrate of SsuE but FAD and riboflavin are also reduced at significant rates, whereas lumiflavin is not. When NADPH is supplied as pyrimidinic substrate, maximal reductase activity is obtained with 2.5-10 mM FMN, while higher FMN concentration leads 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
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-
?
FMNH2 + NADP+
FMN + NADPH + H+
show the reaction diagram
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-
-
-
r
riboflavin + NADPH + H+
reduced riboflavin + NADP+
show the reaction diagram
FMN is the preferred flavin substrate of SsuE but FAD and riboflavin are also reduced at significant rates, whereas lumiflavin is not
-
-
?
FMN + NADPH + H+
FMNH2 + NADP+
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
FMNH2 + NADP+
FMN + NADPH + H+
show the reaction diagram
-
-
-
-
r
FMN + NADPH + H+
FMNH2 + NADP+
show the reaction diagram
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADH
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kcat/KM for NADPH is 335fold higher compared to kcat/KM for NADH
NADPH
additional information
-
the enzyme does not contain any bound flavin cofactor
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
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
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0027
FAD
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pH 7.9, 30°C
0.000054
FMN
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pH 7.9, 30°C
0.5555
NADH
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pH 7.9, 30°C, 0.0005 mM FMN
0.046
NADPH
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pH 7.9, 30°C, 0.003 mM FMN
additional information
additional information
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steady-state kinetic analysis of wild-type and mutant enzymes, kinetics of FMN binding, overview
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.28
FAD
-
pH 7.9, 30°C
0.0079
FMN
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pH 7.9, 30°C
0.00167
NADH
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pH 7.9, 30°C, 0.0005 mM FMN
0.56
NADPH
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pH 7.9, 30°C, 0.003 mM FMN
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
-
assay at
7.9
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
30
-
assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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
physiological function
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FMN reductase (SsuE) catalyzes the reduction of FMN by NADPH, and the reduced flavin is transferred to the monooxygenase (SsuD)
additional information
-
at least a dimeric association is required for the function of enzyme SsuE, FMN binding site structure, overview
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
Sequence
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-
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-
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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
40900
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dimeric SsueE in presence of flavin, analytical ultracentrifugation
58400
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gel filtration
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
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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2 * 23700, calculated from sequence; 2 * 25400, SDS-PAGE
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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PROTEIN VARIANTS
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
PURIFICATION/commentary
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme
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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
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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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Eichhorn, E.; van der Ploeg, J.R.; Leisinger, T.
Characterization of a two-component alkanesulfonate monooxygenase from Escherichia coli
J. Biol. Chem.
274
26639-26646
1999
Escherichia coli (P80644)
Manually annotated by BRENDA team
Gao, B.; Ellis, H.R.
Mechanism of flavin reduction in the alkanesulfonate monooxygenase system
Biochim. Biophys. Acta
1774
359-367
2007
Escherichia coli
Manually annotated by BRENDA team
Abdurachim, K.; Ellis, H.R.
Detection of protein-protein interactions in the alkanesulfonate monooxygenase system from Escherichia coli
J. Bacteriol.
188
8153-8159
2006
Escherichia coli
Manually annotated by BRENDA team
Gao, B.; Bertrand, A.; Boles, W.H.; Ellis, H.R.; Mallett, T.C.
Crystallization and preliminary X-ray crystallographic studies of the alkanesulfonate FMN reductase from Escherichia coli
Acta Crystallogr. Sect. F
61
837-840
2005
Escherichia coli
Manually annotated by BRENDA team
Driggers, C.; Dayal, P.; Ellis, H.; Andrew Karplus, P.
Crystal structure of Escherichia coli SsuE defining a general catalytic cycle for FMN reductases of the flavodoxin-like superfamily
Biochemistry
53
3509-3519
2014
Escherichia coli (P80644)
Manually annotated by BRENDA team
Musila, J.; Ellis, H.
Transformation of a flavin-free FMN reductase to a canonical flavoprotein through modification of the Pi-helix
Biochemistry
55
6389-6394
2016
Escherichia coli (P80644)
Manually annotated by BRENDA team
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