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
-
-
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].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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
-
-
?
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
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
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-thio-FMN
-
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
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
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
-
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
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
pH 7.9, 30C, 0.0005 mM FMN
0.003 - 0.046
NADPH
0.025
riboflavin
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pH 7.0, 23C, native enzyme
additional information
additional information
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the kinetic mechanism of FRP is changed to a sequential pattern with a Km(FMN) of 0.003 mM and a Km(NADPH) of 0.02 mM in a luciferase-coupled assay measuring light emission
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.2 - 34
NADPH
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.28
FAD
Escherichia coli
P80644
pH 7.9, 30C
20
0.0079
FMN
Escherichia coli
P80644
pH 7.9, 30C
56
0.00167
NADH
Escherichia coli
P80644
pH 7.9, 30C, 0.0005 mM FMN
8
0.56 - 2670
NADPH
5
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
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
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Pseudomonas putida (strain KT2440)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000
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gel filtration
58400
gel filtration
63000
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sucrose density gradient centrifugation
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
2 * 23700, calculated from sequence; 2 * 25400, SDS-PAGE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
-
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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SsuE is purified to homogeneity as an N-terminal histidine-tagged fusion protein
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3)pLysS cells
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overexpression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
Show AA Sequence (431 entries)
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