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Information on EC 1.5.1.38 - FMN reductase (NADPH) and Organism(s) Vibrio harveyi and UniProt Accession Q56691
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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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The taxonomic range for the selected organisms is: Vibrio harveyi
The expected taxonomic range for this enzyme is: Bacteria, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Archaea
Synonyms
nadph:fmn reductase, nad(p)h:fmn reductase, nadph specific fmn reductase, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
flavin reductase P
FRP
SsuE
-
-
-
-
flavin reductase P
-
-
-
-
FRP
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
FMNH2 + NADP+ = FMN + NADPH + H+
the first step in catalysis, which is hydride transfer from C4 of NADPH to cofactor FMN, involves addition to the re face of the FMN, probably at the N5 position. The limited accessibility of the FMN binding pocket and the extensive FMN-protein hydrogen bond network are consistent with the observed ping-pong bisubstrate-biproduct reaction kinetics
FMNH2 + NADP+ = FMN + NADPH + H+
the enzyme exhibits double displacement or ping-pong kinetics
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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)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2-thioFMN + NADPH + H+
2-thioFMNH2 + NADP+
-
the holoenzyme reconstituted with 2-thioFMN is catalytically active in using either FMN or 2-thioFMN as a substrate
-
-
?
riboflavin + NADPH + H+
reduced riboflavin + NADP+
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Vmax/KM for riboflavin is 13fold lower compared to Vmax/Km for FMN
-
-
?
additional information
?
-
-
the enzyme also exhibits appreciable activity with some artificial acceptors: menadione, 2,6-dichlorophenolindophenol, KFeCN6 or 5,5'-dithiobis(2-nitrobenzoic acid). Low activity with methylene blue as acceptor
-
-
?
FAD + NADPH + H+
FADH2 + NADP+
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Vmax/KM for riboflavin is 6fold lower compared to Vmax/Km for FMN
-
-
?
FMN + NADPH + H+
FMNH2 + NADP+
-
-
-
-
?
FMN + NADPH + H+
FMNH2 + NADP+
-
the affinity of the NADPH-specific reductase for NADPH is 1000 times greater than for NADH
-
-
?
FMN + NADPH + H+
FMNH2 + NADP+
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the apoenzyme binds one FMN per enzyme monomer with a dissociation constant of 0.2 mM at 23°C. The reconstituted holoenzyme is catalytically as active as the native enzyme. FMN binding results in 87% and 92% of quenching of protein and flavin fluorescence, respectively, indicating a conformational difference between the apoprotein and the holoenzyme. Neither riboflavin nor FAD shows any appreciable binding to the cofactor site of the apoenzyme but both flavins are active substrates for the FMN-containing holoenzyme. The holoenzyme reconstituted with 2-thioFMN is catalytically active in using either FMN or 2-thioFMN as a substrate
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-
?
FMN + NADPH + H+
FMNH2 + NADP+
-
the NADPH specific FMN reductase does not utilize NADH
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
FMN
the enzyme is specific for FMN as cofactor. FMN is recognized and tightly bound by a network of 16 hydrogen bonds, while steric considerations prevent the binding of FAD. A flexible loop containing a Lys and an Arg could account for the NADPH specificity
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
FMN
-
the apoenzyme binds one FMN per enzyme monomer with a dissociation constant of 0.2 mM at 23°C. The reconstituted holoenzyme is catalytically as active as the native enzyme. FMN binding results in 87% and 92% of quenching of protein and flavin fluorescence, respectively, indicating a conformational difference between the apoprotein and the holoenzyme. Neither riboflavin nor FAD shows any appreciable binding to the cofactor site of the apoenzyme but both flavins are active substrates for the FMN-containing holoenzyme
NADPH
-
the affinity of the NADPH-specific reductase for NADPH is 1000 times greater than for NADH
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
LITERATURE
IMAGE
0.007
2-thioFMN
-
pH 7.0, 23°C, enzyme derivative reconstituted from apoenzyme and 2-thioFMN
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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0.008
FMN
-
pH 7.0, 23°C, single-enzyme spectrophotometric assay monitoring the NADPH oxidation
0.003
NADPH
-
pH 7.0, 23°C, enzyme derivative reconstituted from apoenzyme and 2-thioFMN
0.02
NADPH
-
pH 7.0, 23°C, single-enzyme spectrophotometric assay monitoring the NADPH oxidation
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5 - 10
-
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
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
NADPH:FMN oxidoreductase is involved in bioluminescence by providing reduced FMN to luciferase
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
K167A
-
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
R15A
-
the mutant has apparently greatly increased Km and severely reduced kcat/Km values for NADPH
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
DEAE-Sepharose column chromatography, Mono Q column chromatography, and Superdex 75 gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Gerlo, E.; Charlier, J.
Identification of NADH-specific and NADPH-specific FMN reductases in Beneckea harveyi
Eur. J. Biochem.
57
461-467
1975
Vibrio harveyi
Jablonski, E.; DeLuca, M.
Purification and properties of the NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi
Biochemistry
16
2932-2936
1977
Vibrio harveyi, Vibrio harveyi No. 392
Lei, B.; Tu, S.C.
Mechanism of reduced flavin transfer from Vibrio harveyi NADPH-FMN oxidoreductase to luciferase
Biochemistry
37
14623-14629
1998
Vibrio harveyi
Lei, B.; Liu, M.; Huang, S.; Tu, S.C.
Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme
J. Bacteriol.
176
3552-3558
1994
Vibrio harveyi
Tanner, J.J.; Lei, B.; Tu, S.C.; Krause, K.L.
Flavin reductase P: structure of a dimeric enzyme that reduces flavin
Biochemistry
35
13531-13539
1996
Vibrio harveyi (Q56691)
Liu, M.; Lei, B.; Ding, Q.; Lee, J.C.; Tu, S.C.
Vibrio harveyi NADPH:FMN oxidoreductase: preparation and characterization of the apoenzyme and monomer-dimer equilibrium
Arch. Biochem. Biophys.
337
89-95
1997
Vibrio harveyi
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