1.5.1.38: FMN reductase (NADPH)
This is an abbreviated version!
For detailed information about FMN reductase (NADPH), go to the full flat file.
Reaction
Synonyms
(NADPH)-dependent flavin mononucleotide reductase, (NADPH)-dependent FMN reductase, BC_1619, EC 1.5.1.29, EC 1.6.8.1, flavin reductase P, FMN reductase, FRP, More, NAD(P)H:FMN reductase, NADPH specific FMN reductase, NADPH-flavin oxidoreductase, NADPH-FMN oxidoreductase, NADPH:FMN oxidoreductase, NADPH:FMN reductase, SsuE, ssueE, ycbP, ydgI
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Substrates Products
Substrates Products on EC 1.5.1.38 - FMN reductase (NADPH)
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REACTION DIAGRAM
2-thioFMN + NADPH + H+
2-thioFMNH2 + NADP+
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the holoenzyme reconstituted with 2-thioFMN is catalytically active in using either FMN or 2-thioFMN as a substrate
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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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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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FAD + NADPH + H+
FADH2 + NADP+
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Vmax/KM for riboflavin is 6fold lower compared to Vmax/Km for FMN
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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+
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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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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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FMN + NADPH + H+
FMNH2 + NADP+
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the affinity of the NADPH-specific reductase for NADPH is 1000 times greater than for NADH
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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+
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the NADPH specific FMN reductase does not utilize NADH
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FMN + NADPH + H+
FMNH2 + NADP+
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the NADPH specific FMN reductase does not utilize NADH
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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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riboflavin + NADPH + H+
reduced riboflavin + NADP+
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Vmax/KM for riboflavin is 13fold lower compared to Vmax/Km for FMN
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the nitroreductase of Bacillus cereus strain ATCC 14579 encoded by gene BC_1619 also shows NADPH-dependent FMN reductase activity, and interacts with the prodrug 5-(1-aziridinyl)-2,4-dinitrobenzamide, i.e. CB1954, converting it to either the toxic 2'- or 4'-hydroxylamine metabolites with cofactor NAD(P)H, overview
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additional information
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the nitroreductase of Bacillus cereus strain ATCC 14579 encoded by gene BC_1619 also shows NADPH-dependent FMN reductase activity, and interacts with the prodrug 5-(1-aziridinyl)-2,4-dinitrobenzamide, i.e. CB1954, converting it to either the toxic 2'- or 4'-hydroxylamine metabolites with cofactor NAD(P)H, overview
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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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additional information
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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
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additional information
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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
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