Contains FMN. The enzyme can utilize NADH and NADPH with similar reaction rates. Different from EC 1.5.1.42, FMN reductase (NADH) and EC 1.5.1.38, FMN reductase (NADPH). The luminescent bacterium Vibrio harveyi possesses all three enzymes . Also reduces riboflavin and FAD, but more slowly.
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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
the enzyme FOR can catalyze the oxidation of NADH to NAD+ with the flavin mononucleotide (FMN) functions as the prosthetic group. In the first step, a semiquinone intermediate (FMNH) is formed by the transfer of the hydride from the nicotinamide group of NADH to the N5 in the isoalloxazine moiety of the oxidized FMN. Then, a proton transfer to the N atom near the ribitol moiety of FMNH which may result in the formation of FMNH2. Eventually, the reduced FMNH2 is oxidized to FMN by O2 molecules
two substrate kinetic analysis by double-reciprocal plots yields a series of intersecting lines. This rules out a ping-pong bi-bi mechanism and suggests a sequential mechanism in which both FMN and NADH bind to the enzyme prior to dissociation of either product. FMN binds first, followed by NADH, kinetic mechanism, overview. The affinity of flavin toward the protein decreases only slightly upon reduction. Reduced FMN formed tends to remain bound to the enzyme where it can be re-oxidized by oxygen or, less efficiently, by various artificial electron acceptors. The enzyme-FMN complex is a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide is identified as the main product. Hydride transfer occurs from the pro-S C4 position of the nicotinamide ring and partially limits the overall turnover rate
the enzyme FOR can catalyze the oxidation of NADH to NAD+ with the flavin mononucleotide (FMN) functions as the prosthetic group. In the first step, a semiquinone intermediate (FMNH) is formed by the transfer of the hydride from the nicotinamide group of NADH to the N5 in the isoalloxazine moiety of the oxidized FMN. Then, a proton transfer to the N atom near the ribitol moiety of FMNH which may result in the formation of FMNH2. Eventually, the reduced FMNH2 is oxidized to FMN by O2 molecules
two substrate kinetic analysis by double-reciprocal plots yields a series of intersecting lines. This rules out a ping-pong bi-bi mechanism and suggests a sequential mechanism in which both FMN and NADH bind to the enzyme prior to dissociation of either product. FMN binds first, followed by NADH, kinetic mechanism, overview. The affinity of flavin toward the protein decreases only slightly upon reduction. Reduced FMN formed tends to remain bound to the enzyme where it can be re-oxidized by oxygen or, less efficiently, by various artificial electron acceptors. The enzyme-FMN complex is a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide is identified as the main product. Hydride transfer occurs from the pro-S C4 position of the nicotinamide ring and partially limits the overall turnover rate
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SYSTEMATIC NAME
IUBMB Comments
FMNH2:NAD(P)+ oxidoreductase
Contains FMN. The enzyme can utilize NADH and NADPH with similar reaction rates. Different from EC 1.5.1.42, FMN reductase (NADH) and EC 1.5.1.38, FMN reductase (NADPH). The luminescent bacterium Vibrio harveyi possesses all three enzymes [1]. Also reduces riboflavin and FAD, but more slowly.
the purified recombinant LrFOR has both the NADPH and NADH oxidation activity. The optimum FMN concentration for the LrFOR is 0.015 mM. With increasing of FMN concentration from 0.001 to 0.015 mM, the relative activity of LrFOR is also increased from 42% to 100%. When the concentration of FMN is increased to 0.030 mM, LrFOR displays more than 90% of the optimal activity. And the activity retains more than 80% of the optimal value when the FMN concentration is 0.070 mM
the purified recombinant LrFOR has both the NADPH and NADH oxidation activity. The optimum FMN concentration for the LrFOR is 0.015 mM. With increasing of FMN concentration from 0.001 to 0.015 mM, the relative activity of LrFOR is also increased from 42% to 100%. When the concentration of FMN is increased to 0.030 mM, LrFOR displays more than 90% of the optimal activity. And the activity retains more than 80% of the optimal value when the FMN concentration is 0.070 mM
the enzyme-FMN complex is a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide is identified as the main product. The binding affinity for FMN does not dramatically change with flavin reduction. Oxygen behaves as a true substrate interacting with the enzyme only after dissociation of the first product, NAD+. The enzyme exhibits weak activity with ferricyanide, ubiquinone-0, 1,4-naphthoquinone and nitrofurantoin, while almost no activity is detected with hydrogen peroxide or disulfidic Ellman's reagent. The enzyme is also active in reducing the nonfluorescent N-oxide resazurin to the fluorescent resorufin. The initial-rate kinetics of fluorescence increase measured at various concentrations of NADH, and resazurin obeye the ping-pong mechanism as is observed for oxygen. Oxygen act as a competitive inhibitor of the reduction of resazurin, suggesting that these two electron acceptors are competing for the reduced flavin. The catalytic cycle involves an obligatory release of the formed FMNH2 because its back oxidation to FMN cannot occur in the absence of oxygen
the enzyme-FMN complex is a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide is identified as the main product. The binding affinity for FMN does not dramatically change with flavin reduction. Oxygen behaves as a true substrate interacting with the enzyme only after dissociation of the first product, NAD+. The enzyme exhibits weak activity with ferricyanide, ubiquinone-0, 1,4-naphthoquinone and nitrofurantoin, while almost no activity is detected with hydrogen peroxide or disulfidic Ellman's reagent. The enzyme is also active in reducing the nonfluorescent N-oxide resazurin to the fluorescent resorufin. The initial-rate kinetics of fluorescence increase measured at various concentrations of NADH, and resazurin obeye the ping-pong mechanism as is observed for oxygen. Oxygen act as a competitive inhibitor of the reduction of resazurin, suggesting that these two electron acceptors are competing for the reduced flavin. The catalytic cycle involves an obligatory release of the formed FMNH2 because its back oxidation to FMN cannot occur in the absence of oxygen
the enzyme-FMN complex is a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide is identified as the main product. The binding affinity for FMN does not dramatically change with flavin reduction. Oxygen behaves as a true substrate interacting with the enzyme only after dissociation of the first product, NAD+. The enzyme exhibits weak activity with ferricyanide, ubiquinone-0, 1,4-naphthoquinone and nitrofurantoin, while almost no activity is detected with hydrogen peroxide or disulfidic Ellman's reagent. The enzyme is also active in reducing the nonfluorescent N-oxide resazurin to the fluorescent resorufin. The initial-rate kinetics of fluorescence increase measured at various concentrations of NADH, and resazurin obeye the ping-pong mechanism as is observed for oxygen. Oxygen act as a competitive inhibitor of the reduction of resazurin, suggesting that these two electron acceptors are competing for the reduced flavin. The catalytic cycle involves an obligatory release of the formed FMNH2 because its back oxidation to FMN cannot occur in the absence of oxygen
transient-state FMN reduction, overview. Both rapid NADPH binding and hydride transfer to FMN in NfoR. The low relative rate observed is likely a function of the limiting concentration of FMN present or the trapping of the enzyme in an inhibitory product complex (e.g. NfoR-FMNH2-NADPH) in which the release of FMNH2 and/or NADPH is slow. Reoxidation of flavins (FADH2) by chromate and copper
the enzyme has a hemin binding activity, pronbably in the hydrophobic pocket near loops beta4-alpha5 and beta8-beta9, docking of biliverdin IXalpha and FMN , overview
the substrate binding order is investigated by carrying out dead-end inhibition studies using lumichrome (7,8-dimethylalloxazine) and AMP as inhibitory analogues of FMN and NADH. Strains Pd9233 and Pd9311 contain aa3-type or cbb3-type cytochrome c oxidase, respectively, as the only terminal oxidase of the respiratory electron transfer chain, the flux capacity through terminal oxidase is reduced in strain Pd9311 compared to strain Pd9233 or wild-type strain, manifested by the much lower specific activity of TMPD oxidase found in the cytoplasmic membrane fraction. The pden_5119 transcript in Pd9311 cells but not in Pd9233 cells is elevated similarly as observed in the wild-type strain growing in the presence of electron transfer inhibitors. Downregulation of terminal oxidase thus indeed results in a similar outcome as chemical inhibition
the substrate binding order is investigated by carrying out dead-end inhibition studies using lumichrome (7,8-dimethylalloxazine) and AMP as inhibitory analogues of FMN and NADH. Strains Pd9233 and Pd9311 contain aa3-type or cbb3-type cytochrome c oxidase, respectively, as the only terminal oxidase of the respiratory electron transfer chain, the flux capacity through terminal oxidase is reduced in strain Pd9311 compared to strain Pd9233 or wild-type strain, manifested by the much lower specific activity of TMPD oxidase found in the cytoplasmic membrane fraction. The pden_5119 transcript in Pd9311 cells but not in Pd9233 cells is elevated similarly as observed in the wild-type strain growing in the presence of electron transfer inhibitors. Downregulation of terminal oxidase thus indeed results in a similar outcome as chemical inhibition
Michaelis-Menten steady-state kinetics. The kinetic parameters show that the Km values of mutants T29A, T29G, and T29D are similar to that of wild-type enzyme with 0.0882 mM but the Km of mutants are increased. Also, the Vmax of mutant T29C and T29S are increased compared to wild-type, while the T29R mutant shows a significant decrease in Vmax
steady-state kinetics, relative binding affinities for oxidized and reduced flavin, overview. Steady-state kinetic analysis of the oxidase activity of the Pden_5119 protein and substrate kinetic isotope effect (KIE)
steady-state kinetics, relative binding affinities for oxidized and reduced flavin, overview. Steady-state kinetic analysis of the oxidase activity of the Pden_5119 protein and substrate kinetic isotope effect (KIE)
optimum pH for the oxidation of NADH is pH 5.5, whereas the enzyme shows 87.5% and 86.7% of maximum activity at pH 6.0 and 6.5, respectively. 69.3% residual activity is measured at pH 7.5, 36.8% at pH 8.0
NADH: FMN oxidoreductases (FOR) are old yellow enzyme members with a (beta/alpha) 8-barrel structure and can catalyze the oxidation of NADH to NAD+ with the flavin mononucleotide (FMN) functions as the prosthetic group
the Pden_5119 protein is closely related to the SsuE and MsuE FRs that are parts of the two-component flavin-dependent monooxygenase systems involved in oxygenolytic cleavage of alkanesulfonates into aldehyde and sulfite
NADH: FMN oxidoreductases (FOR) are old yellow enzyme members with a (beta/alpha) 8-barrel structure and can catalyze the oxidation of NADH to NAD+ with the flavin mononucleotide (FMN) functions as the prosthetic group
the Pden_5119 protein is closely related to the SsuE and MsuE FRs that are parts of the two-component flavin-dependent monooxygenase systems involved in oxygenolytic cleavage of alkanesulfonates into aldehyde and sulfite
inactivation of the pden_5119 gene increases susceptibility to oxidative stress, decreases growth rate and increases growth yield of Paracoccus denitrificans, growth on lower alkanesulfonates as sulfur sources is not specifically influenced. Changes in growth on alkanesulfonates and sulfate occurring as a result of mutation indicate that the Pden_5119 protein is not an obligatory component in the desulfurization pathway
unlike wild-type SsuE, which crystallizes as a tetramer, the Tyr118 variant structures determined here all crystallize as dimers. The Pi-helices do not contribute to the dimeric assembly, and the variants show no difference at the dimeric interface compared to wild-type. While the Y118A SsuE variant clearly cannot hydrogen bond to Ala78 through the deleted hydroxyl, the Ala78-FMN hydrogen bond remains intact and the loop containing Ala78 has not shifted in conformation. The structure of the loop containing Ala78 is also maintained in the DELTA118 SsuE structure.
inactivation of the pden_5119 gene increases susceptibility to oxidative stress, decreases growth rate and increases growth yield of Paracoccus denitrificans, growth on lower alkanesulfonates as sulfur sources is not specifically influenced. Changes in growth on alkanesulfonates and sulfate occurring as a result of mutation indicate that the Pden_5119 protein is not an obligatory component in the desulfurization pathway
ChuY has flavin mononucleotide (FMN) reductase activity, using NAD(P)H as a cofactor, and shows porphyrin ring binding affinity. ChuY acts as a reductase in heme homeostasis to maintain the virulence potential of Escherichia coli strain CFT073
role for the Pden_5119 protein in maintaining the cellular redox state. The Pden_5119 protein confers increased resistance to oxidative stress. Dispensability of the Pden_5119 protein in sulfur acquisition from alkanesulfonates
role for the Pden_5119 protein in maintaining the cellular redox state. The Pden_5119 protein confers increased resistance to oxidative stress. Dispensability of the Pden_5119 protein in sulfur acquisition from alkanesulfonates
ChuY has flavin mononucleotide (FMN) reductase activity, using NAD(P)H as a cofactor, and shows porphyrin ring binding affinity. ChuY acts as a reductase in heme homeostasis to maintain the virulence potential of Escherichia coli strain CFT073
enzyme structure homology modeling and docking using the structure of Thermoanaerobacter pseudethanolicus strain E39 enzyme (PDB ID 3KRZ) as the template, overview. Molecular dynamic simulation
the functioning of NAD(P)H:FMN-oxidoreductase (Red) from Vibrio fischeri is not affected under conditions of macromolecular crowding (MMC) simulated in vitro by adding biopolymers (starch and gelatin). The functioning of Red both under conditions of MMC and in diluted solutions is the same
the Pi-helix located at the tetramer interface of two-component FMN-dependent reductases contributes to the structural divergence from canonical FMN-bound reductases within the NADPH:FMN reductase family. The Pi-helix in the SsuE FMN-dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved alpha4-helix. Enzyme-substrate binding structure analysis. In the wild-type structure, a hydrogen bond forms between the Tyr118 and a carbonyl oxygen of Ala78 from the opposing dimer, which in turn hydrogen bonds to the isoalloxazine ring system of the FMN. This network is hypothesized to aid in communication between the oligomerization interface and FMN binding
enzyme structure homology modeling and docking using the structure of Thermoanaerobacter pseudethanolicus strain E39 enzyme (PDB ID 3KRZ) as the template, overview. Molecular dynamic simulation
the two molecules in the asymmetric unit are related by pseudo 2fold rotation symmetry. ChuY contains six alpha-helices and ten beta-strands. A central beta-sheet, consisting of seven parallel beta-strands, beta1, beta2, beta3, beta4, beta5, beta6, and beta10, is flanked by six alpha-helices, forming alternating beta-strand and alpha-helix repeats, which is a representative feature of Rossmann folds. Three other beta-strands (beta7-beta9) are located on the top of the beta-sheet
the tetramer of enzyme SsuE binds FMN, and dissociates to a dimer. In a flavin-bound SsuE structure, the hydroxyl group of Tyr118 hydrogen bonds to the oxygen atom backbone carbonyl of Ala78 across the tetramer interface
the two molecules in the asymmetric unit are related by pseudo 2fold rotation symmetry. ChuY contains six alpha-helices and ten beta-strands. A central beta-sheet, consisting of seven parallel beta-strands, beta1, beta2, beta3, beta4, beta5, beta6, and beta10, is flanked by six alpha-helices, forming alternating beta-strand and alpha-helix repeats, which is a representative feature of Rossmann folds. Three other beta-strands (beta7-beta9) are located on the top of the beta-sheet
the conserved protein fold of LrFOR is comprised of about eight alpha-helices and eight parallel beta-strands that alternate along the peptide backbones (A (beta/alpha) 8 barrel)
the conserved protein fold of LrFOR is comprised of about eight alpha-helices and eight parallel beta-strands that alternate along the peptide backbones (A (beta/alpha) 8 barrel)
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant His-tagged enzyme, using 0.1 M Bis-Tris, pH 6.5, 50 mM CaCl2, and 30% PEG MME 550 as precipitants, ChuY crystals belong to the primitive monoclinic space group P21 with two molecules forming an asymmetric unit, multi-wavelength anomalous dispersion, using a SeMet-labeled crystal at a resolution of 2.4 A
purified enzyme in apoform or in complex with FMN, sitting drop vapor diffusion method, mixing of 0.006 ml of 45 mg/ml protein in 10 mM NAD+, 50 mM HEPES, and 300 mM NaCl, pH 7.5, with 0.006 ml of well solution containing 30% PEG 4000, 210 mM ammonium acetate, and 100 mM sodium citrate, pH 5.6, 14 days, room temperature, X-ray diffraction structure determination and analysis at 2.02 A resolution
site-directed mutagenesis, the structure of the Y118A SsuE Pi-helix is converted to an alpha-helix, similar to the FMN-bound members of the NADPH:FMN reductase family. Although the Pi-helix is altered, the FMN binding region remains unchanged. The mutant forms dimers in contrast to the wild-type