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Information on EC 1.5.1.39 - FMN reductase [NAD(P)H]
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1.5.1.39 FMN reductase [NAD(P)H]IUBMB Comments
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
Synonyms
nad(p)h:fmn-oxidoreductase, nadph-dependent fmn reductase, nad(p)h-flavin reductase, pden_5119, nadh fmn oxidoreductase, lrfor, two-component fmn-dependent monooxygenase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
ChuY
EC 1.5.1.29
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formerly, part transferred
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EC 1.6.8.1
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formerly, part transferred
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flavin mononucleotide reductase
FMN reductase
FRG
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H2O-forming FOR
LrFOR
NADH: FMN oxidoreductase
NADPH-dependent FMN reductase
non-specific NAD(P)H-FMN reductase
pden_5119
water forming NADH: FMN oxidoreductase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
FMNH2 + NAD(P)+ = FMN + NAD(P)H + H+
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FMNH2 + NAD(P)+ = FMN + NAD(P)H + H+
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
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FMNH2 + NAD(P)+ = FMN + NAD(P)H + H+
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
FMNH2 + NAD(P)+ = FMN + NAD(P)H + H+
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
Lacticaseibacillus rhamnosus ATCC 53103
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FMNH2 + NAD(P)+ = FMN + NAD(P)H + H+
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.
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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
FMN + NADH + H+
FMNH2 + NAD+
reductive and oxidative half reactions, kinetics, overview
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r
FMN + NADH + H+
FMNH2 + NAD+
reductive and oxidative half reactions, kinetics, overview
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r
FMN + NADPH + H+
FMNH2 + NADP+
reductive and oxidative half reactions, kinetics, overview
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r
FMN + NADPH + H+
FMNH2 + NADP+
reductive and oxidative half reactions, kinetics, overview
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r
FMNH2 + NAD+
FMN + NADH + H+
the enzyme exhibits about the same reaction rates with NADH and NADPH
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?
FMNH2 + NAD+
FMN + NADH + H+
the enzyme exhibits about the same reaction rates with NADH and NADPH
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?
additional information
?
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FMN enzyme binding structure analysis using wild-type and mutant enzymes, overview
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additional information
?
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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
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additional information
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Lacticaseibacillus rhamnosus ATCC 53103
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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
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additional information
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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
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additional information
?
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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
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additional information
?
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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
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additional information
?
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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
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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
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
heme
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
NADH
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NADH is docked into docking into the substrate-binding site of wild-type LrFOR, preferred cofactor
additional information
enzyme ChuY binds nucleotides as cofactors., cofactor binding structure, overview
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additional information
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the purified recombinant LrFOR has both the NADPH and NADH oxidation activity
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additional information
the wild-type enzyme's catalytic efficiency is slightly high with NADH compared to NADPH
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
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Li+ and EDTA have a weak effect on enzyme activity
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
AMP
competitive inhibition against NADH and uncompetitive inhibition against FMN
lumichrome
7,8-dimethylalloxazine, competitive inhibitor versus FMN, and mixed noncompetitive inhibitor with respect to NADH
additional information
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
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additional information
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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
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
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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
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additional information
additional information
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)
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additional information
additional information
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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)
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.028
AMP
versus NADH, pH 7.0, 30°C, recombinant wild-type, competitive
0.047
AMP
versus FMN, pH 7.0, 30°C, recombinant wild-type, uncompetitive
0.055
lumichrome
versus NADH, pH 7.0, 30°C, recombinant wild-type, mixed noncompetitive
0.143
lumichrome
versus FMN, pH 7.0, 30°C, recombinant wild-type, competitive
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5.5 - 8
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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
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
physiological function
additional information
evolution
gene chuY is highly conserved in a broad range of pathogenic bacteria
evolution
comparison of wild-type and Y118A variants of SsuE with related wild-type and mutant H126Y MsuE from Pseudomonas fluorescens (EC 1.5.1.42), overview
evolution
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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
evolution
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
evolution
Lacticaseibacillus rhamnosus ATCC 53103
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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
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evolution
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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
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evolution
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gene chuY is highly conserved in a broad range of pathogenic bacteria
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malfunction
a chuY deletion-insertion strain shows reduced survival potential compared to wild-type and complemented strains in mammalian cells
malfunction
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
malfunction
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.
malfunction
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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
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malfunction
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a chuY deletion-insertion strain shows reduced survival potential compared to wild-type and complemented strains in mammalian cells
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physiological function
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
physiological function
NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase
physiological function
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
physiological function
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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
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physiological function
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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
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additional information
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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
additional information
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
additional information
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
additional information
Lacticaseibacillus rhamnosus ATCC 53103
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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
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Show AA Sequence and Transmembrane Helices (723 entries)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
flavin-bound wild-type enzyme, and enzyme mutant Y118A and DELTAY118
monomer
additional information
monomer
ChuY exists as a monomer in solution, SDS-PAGE and gel filtration
monomer
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ChuY exists as a monomer in solution, SDS-PAGE and gel filtration
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monomer
Lacticaseibacillus rhamnosus ATCC 53103
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1 * 42000, recombinant His-tagged enzyme, SDS-PAGE
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additional information
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
additional information
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
additional information
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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
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additional information
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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)
additional information
Lacticaseibacillus rhamnosus ATCC 53103
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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
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Y118A
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
N173A
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site-directed mutagenesis, the mutant shows 66% reduced activity compared to wild-type enzyme
T29A
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site-directed mutagenesis, the mutant shows 2.91fold increased activity compared to the wild-type enzyme
T29D
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site-directed mutagenesis, the mutant shows 3.9fold increased activity compared to the wild-type enzyme
T29G
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site-directed mutagenesis, the mutant shows 3.7fold increased activity compared to the wild-type enzyme
T29N
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site-directed mutagenesis, the mutant shows 2.2fold increased activity compared to the wild-type enzyme
T29R
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site-directed mutagenesis, the mutant shows 90% reduced activity compared to wild-type enzyme
T29Y
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site-directed mutagenesis, the mutant shows 40% reduced activity compared to wild-type