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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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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: Escherichia coli
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, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
flavin reductase P
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FRP
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SsuE
SsuE
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
FAD + NADPH + H+
FADH2 + NADP+
FMN is the preferred flavin substrate of SsuE but FAD and riboflavin are also reduced at significant rates, whereas lumiflavin is not
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?
FMN + NADH + H+
FMNH2 + NAD+
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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?
riboflavin + NADPH + H+
reduced riboflavin + NADP+
FMN is the preferred flavin substrate of SsuE but FAD and riboflavin are also reduced at significant rates, whereas lumiflavin is not
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?
FMN + NADPH + H+
FMNH2 + NADP+
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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?
FMN + NADPH + H+
FMNH2 + NADP+
approximately one FMN is bound per monomer
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?
FMN + NADPH + H+
FMNH2 + NADP+
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results from single-wavelength analyses at 450 and 550 nm show that reduction of FMN occurs in three distinct phases. Following a possible rapid equilibrium binding of FMN and NADPH to SsuE (MC-1) that occurs before the first detectable step, an initial fast phase (241 s-1) corresponds to the interaction of NADPH with FMN (CT-1). The second phase is a slow conversion (11 s-1) to form a charge-transfer complex of reduced FMNH2 with NADP+ (CT-2), and represents electron transfer from the pyridine nucleotide to the flavin. The third step (19 s-1) is the decay of the charge-transfer complex to SsuE with bound products (MC-2) or product release from the CT-2 complex. Results from isotope studies with [(4R)-2H]NADPH demonstrates a rate-limiting step in electron transfer from NADPH to FMN
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?
additional information
?
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electron transfer to ferricyanide is performed with FMN-bound Y118A SsuE mutant varying concentrations of NADPH, and ferricyanide, the ferricyanide concentration is saturating to maintain pseudo-first-order kinetic conditions at varying NADPH concentrations
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?
additional information
?
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FMN binds tightly in a deeply held site, which makes available a second binding site, in which either a second FMN or the nicotinamide of NADPH can bind. The FMNH2-bound structure shows subtle changes consistent with its binding being weaker than that of FMN
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?
additional information
?
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flavin reductases of two-component systems must transfer reduced flavin successfully to the monooxygenase enzymes for the insertion of single oxygen atom(s) into their respective substrates. Protein-protein interactions between the FMN-bound Y118 SsuE variants and SsuD, overview. A competition assay is performed with SsuE and SsuD. The enzyme also has desulfonation activity
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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
NADH
kcat/KM for NADPH is 335fold higher compared to kcat/KM for NADH
additional information
the enzyme does not contain any bound flavin cofactor
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
steady-state kinetic analysis of wild-type and mutant enzymes, kinetics of FMN binding, overview
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additional information
additional information
steady-state Michaelis-Menten kinetics, and rapid-reaction kinetic analyses of Y118A, DELTAY118 SsuE, and wild-type SsuE, as well as stopped-flow kinetics, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
the Tyr insertional residue of SsuE makes specific contacts across the dimer interface that may assist in the altered mechanistic properties of this enzyme. The Y118F SsuE variant maintains the Pi-Pi stacking interactions at the tetramer interface and has kinetic parameters similar to those of wild-type SsuE. Substitution of the Pi-helical residue (Tyr118) to Ala or Ser transforms the enzymes into flavin-bound SsuE variants that can no longer support flavin reductase and desulfonation activities. These variants exist as dimers and can form protein-protein interactions with SsuD even though flavin transfer is not sustained. The DELRAY118 SsuE variant is flavin-free as purified and does not undergo the tetramer to dimer oligomeric shift with the addition of flavin. The absence of desulfonation activity can be attributed to the inability of DELTAY118 SsuE to promote flavin transfer and undergo the requisite oligomeric changes to support desulfonation. Results from these studies provide insights into the role of the SsuE Pi-helix in promoting flavin transfer and oligomeric changes that support protein-protein interactions with SsuD
metabolism
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
evolution
enzyme SsuE is part of the flavodoxin-like superfamily. A Pi-helix present at the tetramer building interface of enzyme Ssue is unique to the reductases from two-component monooxygenase systems
evolution
the flavin reductase of the alkanesulfonate monooxygenase system (SsuE) contains a conserved Pi-helix located at the tetramer interface that originates from the insertion of Tyr118 into helix alpha4 of SsuE, the presence of Pi-helices provides an evolutionary gain of function. Residue Tyr118 residue generates the Pi-helix in SsuE
evolution
the SsuE FMN reductase of the alkanesulfonate monooxygenase system belongs to the NAD(P)H:FMN reductase family based on a conserved flavodoxin fold. The subgroup of enzymes in the NAD(P)H:FMN reductase family is comprised of flavin reductases from two-component monooxygenase systems. The diverging structural feature in these FMN reductases is a Pi-helix centrally located at the tetramer interface that is generated by the insertion of an amino acid in a conserved alpha4 helix
physiological function
SsuD is a monooxygenase that catalyzes the desulfonation of alkanesulfonates and requires reduced FMN, which is provided by the NAD(P)H:flavin oxidoreductase SsuE
physiological function
the Pi-helix enables SsuE to effectively utilize flavin as a substrate in the two-component monooxygenase system of SsueE with SsueD, overview
additional information
at least a dimeric association is required for the function of enzyme SsuE, FMN binding site structure, overview
additional information
enzyme structure analysis, structure-function relationship and substrate binding, overview
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
40900
dimeric SsueE in presence of flavin, analytical ultracentrifugation
73100
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
LITERATURE
homodimer
homotetramer
4 * 21300, recombinant wild-type enzyme and mutants DELTAY188 and Y118F enzyme, SDS-PAGE
additional information
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
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
LITERATURE
Y118A
Y118F
site-directed mutagenesis, altered kinetics compared to wild-type.The mutant forms tetramers like the wild-type
Y118S
site-directed mutagenesis, inability of Y118S SsuE to support desulfonation in the coupled assay. The mutant forms dimers in contrast to the wild-type
additional information
generation of a Y118 deletion mutant, DELTAY118, and of point mutants Y118S and Y118F. The Tyr insertional residue of SsuE makes specific contacts across the dimer interface that may assist in the altered mechanistic properties of this enzyme. The Y118F SsuE variant maintains the Pi-Pi stacking interactions at the tetramer interface and has kinetic parameters similar to those of wild-type SsuE. Substitution of the Pi-helical residue (Tyr118) to Ala or Ser transforms the enzymes into flavin-bound SsuE variants that can no longer support flavin reductase and desulfonation activities. These variants exist as dimers and can form protein-protein interactions with SsuD even though flavin transfer is not sustained. The DELTAY118 SsuE variant is flavin-free as purified and does not undergo the tetramer to dimer oligomeric shift with the addition of flavin. The absence of desulfonation activity can be attributed to the inability of DELTAY118 SsuE to promote flavin transfer and undergo the requisite oligomeric changes to support desulfonation. Results from these studies provide insights into the role of the SsuE Pi-helix in promoting flavin transfer and oligomeric changes that support protein-protein interactions with SsuD. A 10fold lower binding affinity for flavin binding is observed with the DELTAY118 SsuE deletion variant than with wild-type SsuE. Although the DELTAY118 SsuE variant is unable to support NADPH oxidase activity, the 10fold decrease in flavin affinity would not account for the absence of activity because the flavin should still bind at the saturating concentrations of FMN used in the flavin reductase assays. Inability of Y118A, Y118S, and DELTAY118 SsuE to support desulfonation in the coupled assay
Y118A
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
Y118A
site-directed mutagenesis, altered kinetics compared to wild-type, inability of Y118A SsuE to support desulfonation in the coupled assay. The mutant forms dimers in contrast to the wild-type
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3)
SsuE is purified to homogeneity as an N-terminal histidine-tagged fusion protein
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene ssueE, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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)
Gao, B.; Ellis, H.R.
Mechanism of flavin reduction in the alkanesulfonate monooxygenase system
Biochim. Biophys. Acta
1774
359-367
2007
Escherichia coli
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
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
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)
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)
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