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SYSTEMATIC NAME
IUBMB Comments
flavin:NAD+ oxidoreductase
The enzyme from Escherichia coli W catalyses the reduction of free flavins by NADH. The enzyme has similar affinity to FAD, FMN and riboflavin. Activity with NADPH is more than 2 orders of magnitude lower than activity with NADH.
deletion of the encoding genes genes nfr1 and nfr2 in Lactobacillus johnsonii leads to a 40fold reduction of hydrogen peroxide formation. H2O2 production in this mutant can only be restored by in trans complementation of both genes
inhibition of Nox causes a noticeable decrease in the microsclerotium yields. Silencing of Nox decreases the microsclerotium yield by 98.5%, H2O2 and virulence decrease it by 38% and 21.5%, respectively
deletion of the encoding genes genes nfr1 and nfr2 in Lactobacillus johnsonii leads to a 40fold reduction of hydrogen peroxide formation. H2O2 production in this mutant can only be restored by in trans complementation of both genes
inhibition of Nox causes a noticeable decrease in the microsclerotium yields. Silencing of Nox decreases the microsclerotium yield by 98.5%, H2O2 and virulence decrease it by 38% and 21.5%, respectively
the 4-hydroxyphenylacetate 3-monooxygenase from Escherichia coli W is a two-component enzyme encoded by the hpaB and hpaC genes and catalyzes the initial reaction in the degradation of 4-hydroxyphenylacetate, i.e., the introduction of a second hydroxyl group into the benzene nucleus at a position ortho to the existing hydroxyl group, giving rise to 3,4-dihydroxyphenylacetate
the enzyme Nox is required for microsclerotium differentiation through regulation of intracellular H2O2 concentration. Additionally Nox has a great impact on the virulence in Nomuraea rileyi in cabbage caterpillar
the enzyme Nox is required for microsclerotium differentiation through regulation of intracellular H2O2 concentration. Additionally Nox has a great impact on the virulence in Nomuraea rileyi in cabbage caterpillar
stabilizing effect of another Paracoccus denitrificans protein, the NAD(P)H:acceptor oxidoreducase FerB, against spontaneous oxidation of the FerA-produced dihydroflavin. The turnover rate for NADH oxidation initiated by the addition of FMN is comparable to that for the native, untagged FerA, indicating that the His tag does not interfere with catalysis. Enzyme active ite structure analysis, overview
stabilizing effect of another Paracoccus denitrificans protein, the NAD(P)H:acceptor oxidoreducase FerB, against spontaneous oxidation of the FerA-produced dihydroflavin. The turnover rate for NADH oxidation initiated by the addition of FMN is comparable to that for the native, untagged FerA, indicating that the His tag does not interfere with catalysis. Enzyme active ite structure analysis, overview
stabilizing effect of another Paracoccus denitrificans protein, the NAD(P)H:acceptor oxidoreducase FerB, against spontaneous oxidation of the FerA-produced dihydroflavin. The turnover rate for NADH oxidation initiated by the addition of FMN is comparable to that for the native, untagged FerA, indicating that the His tag does not interfere with catalysis. Enzyme active ite structure analysis, overview
HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH
although the HpaC enzyme can also use NADPH as a substrate, its specific activities on FMN, FAD, and riboflavin are more than 2 orders of magnitude lower than those observed in the presence of NADH. Vmax/Km is 37% compared to the value for the reaction of FMN + NADH
HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH
the most effective substrates are NADH and FMN. When FMN is added in a 200fold molar excess of the HpaC protein, it becomes completely reduced, suggesting that the flavin dissociates from the protein and behaves as a true substrate rather than as a tightly bound cofactor. Although the HpaC enzyme can also use NADPH as a substrate, its specific activities on FMN, FAD, and riboflavin are more than 2 orders of magnitude lower than those observed in the presence of NADH
HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH
although the HpaC enzyme can also use NADPH as a substrate, its specific activities on FMN, FAD, and riboflavin are more than 2 orders of magnitude lower than those observed in the presence of NADH. Vmax/Km is 70% compared to the value for the reaction of FMN + NADH
the enzyme supplies reduced FADH2 for other enzymes, e.g. the 4-hydroxylphenylacetate 3-monooxygenase HpaB, which contains bound FADH2 and which protects FADH2 from being oxidized by O2, HpaC binds to HpaB without substrate channeling
the reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin, that give a similar activity, and is specific for NADH and not NADPH
the reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin, that give a similar activity, and is specific for NADH and not NADPH
the reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin, that give a similar activity, and is specific for NADH and not NADPH
enzyme FerA binds FMN and FAD with comparable affinity in an enthalpically driven, entropically opposed process. The reduced flavin is bound more loosely than the oxidized one, enzyme FerA follows a random-ordered sequence of substrate, NADH and FMN, binding. The primary kinetic isotope effects from stereospecif-ically deuterated nicotinamide nucleotides demonstrate that hydride transfer occurs from the pro-S position and contributes to rate limitation for the overall reaction. Only minor structural changes around Arg106 take place upon FMN binding, role of Arg106 and His146 in binding offlavin and NADH, respectively. Riboflavin (dephosphorylated FMN) also binds to the enzyme
enzyme FerA binds FMN and FAD with comparable affinity in an enthalpically driven, entropically opposed process. The reduced flavin is bound more loosely than the oxidized one, enzyme FerA follows a random-ordered sequence of substrate, NADH and FMN, binding. The primary kinetic isotope effects from stereospecif-ically deuterated nicotinamide nucleotides demonstrate that hydride transfer occurs from the pro-S position and contributes to rate limitation for the overall reaction. Only minor structural changes around Arg106 take place upon FMN binding, role of Arg106 and His146 in binding offlavin and NADH, respectively. Riboflavin (dephosphorylated FMN) also binds to the enzyme
enzyme FerA binds FMN and FAD with comparable affinity in an enthalpically driven, entropically opposed process. The reduced flavin is bound more loosely than the oxidized one, enzyme FerA follows a random-ordered sequence of substrate, NADH and FMN, binding. The primary kinetic isotope effects from stereospecif-ically deuterated nicotinamide nucleotides demonstrate that hydride transfer occurs from the pro-S position and contributes to rate limitation for the overall reaction. Only minor structural changes around Arg106 take place upon FMN binding, role of Arg106 and His146 in binding offlavin and NADH, respectively. Riboflavin (dephosphorylated FMN) also binds to the enzyme
HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH
HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH
HpaC, the small reductase component of the 4-hydroxyphenylacetate 3-monooxygenase. The reductase (HpaC) and the oxygenase component (HpaB) of the monooxygenase are encoded by two different genes. The reductase component uses NADH to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the substrate by molecular oxygen. HpaC that is recombinantly overproduced in Escherichia coli K12 catalyzes the reduction of free flavins by NADH in preference to NADPH
the enzyme supplies reduced FADH2 for other enzymes, e.g. the 4-hydroxylphenylacetate 3-monooxygenase HpaB, which contains bound FADH2 and which protects FADH2 from being oxidized by O2, HpaC binds to HpaB without substrate channeling
HpaC, the small component of the 4-hydroxyphenylacetate 3-monooxygenase, is recombinantly overproduced in Escherichia coli K12 and catalyzes the reduction of free flavins by NADH in preference to NADPH
although the HpaC enzyme can also use NADPH as a substrate, its specific activities on FMN, FAD, and riboflavin are more than 2 orders of magnitude lower than those observed in the presence of NADH
although the HpaC enzyme can also use NADPH as a substrate, its specific activities on FMN, FAD, and riboflavin are more than 2 orders of magnitude lower than those observed in the presence of NADH
V(max)/Km-values in 1/min*mg: 33.3 for FMN (with NADH as electron donor), 22.7 for riboflavin (with NADH as electron donor), 12.3 for FAD (with NADH as electron donor)
2 * 18600, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, calculated from sequence; 2 * 20000, HpaC, the recombinant small component of the 4-hydroxyphenylacetate 3-monooxygenase is a homodimer, SDS-PAGE
overproduction of the small HpaC component in Escherichia coli K-12 cells facilitates the purification of the protein, which is a homodimer that catalyzes the reduction of free flavins by NADH in preference to NADPH
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme in apoform or in complex with FMN, sitting drop vapour diffusion method, crystallization from 0.1 M HEPES, pH 7.4, 10% v/v 2-propanol, and 20% w/v PEG 4000, a few days at 20°C, method optimization, X-ray diffraction structure determination and analysis at 1.53-1.85 A resolution, small angle X-ray scattering, molecular replacement method using structure PDB ID 1RZ0 as search model
refolded recombinant enzyme by anion exchange chromatography and gel filtration, or by nickel affinity chromatography and gel filtration, when a His10-tagged enzyme is expressed
Escherichia coli strain BL21(DE3)pLysS effectively produces an active and soluble form of StyB as about 9% of the total protein content, when cultivated at 20°C with 0.5 mM IPTG
gene frd2, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant overexpression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3)
recombinant overexpression of His-tagged wild-type enzymes HpaC and Fre in strain BL21(DE3), complementation of the inactivation mutant by transient expression of different gene hpaC variants, overview
subcloned into an IPTG-inducible expression vector, pBaiH2.2. Escherichia coli DH5a cells transformed with pBaiH2.2 express 10fold higher levels of NADH:FOR upon induction with IPTG than did Eubacterium sp. VPI 12708 cells induced with cholic acid
the NADH-dependent flavin reductase is encoded by two highly similar genes, LJ_0548 and LJ_0549, or ,nfr1 and nfr2, encoding the subunits 1 and 2 of the enzyme, DNA and amino acid sequence determination and analysis, a plasmid with the promoter region of the LJ_0045 lactate dehydrogenase gene and a bidirectional terminator is used for overexpression of the LJ_0548 and LJ_0549 genes
improvement of the intracellular environment for enhancing L-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply. Construction of mutants of gene frd2, strain 5-5(frd2) and deletion strains 5-5DELTAfrd2 and 5-5DELTAfrd12. The extracellular H2O2 concentrations of mutant 5-5DELTAfrd12 are lower than that of the wild-type strain SYPA5-5, and the extracellular H2O2 concentrations of mutant 5-5(frd2) is increased compared to the wild-type. Flavin reductase activities in frd1 and frd2 overexpression and deletion strains with NADH and FAD, overview
improvement of the intracellular environment for enhancing L-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply. Construction of mutants of gene frd2, strain 5-5(frd2) and deletion strains 5-5DELTAfrd2 and 5-5DELTAfrd12. The extracellular H2O2 concentrations of mutant 5-5DELTAfrd12 are lower than that of the wild-type strain SYPA5-5, and the extracellular H2O2 concentrations of mutant 5-5(frd2) is increased compared to the wild-type. Flavin reductase activities in frd1 and frd2 overexpression and deletion strains with NADH and FAD, overview
refolding of recombinant enzyme from Escherichia coli strain BL21(DE3) (pLysS) inclusion bodies, with Triton X100 used in the washing buffer, refolding from solubilization buffer containing 8 M urea, 10 mM DTT, 50 mM Tris-HCl, pH 8.0, per mg pellet at 50°C for 30 min, refolding in a 20fold volume of refolding buffer.containing 50 mM TrisHCl, pH 8.0, 240 mM NaCl, 10 mM KCl, 1 mM Na2EDTA, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, and 0.05 % w/v PEG 4000, followed by ultrafiltration
Functional analysis of the small component of the 4-hydroxyphenylacetate 3-monooxygenase of Escherichia coli W: a prototype of a new flavin:NAD(P)H reductase subfamily
Chakraborty, S.; Ortiz-Maldonado, M.; Entsch, B.; Ballou, D.P.
Studies on the mechanism of p-hydroxyphenylacetate 3-hydroxylase from Pseudomonas aeruginosa: a system composed of a small flavin reductase and a large flavin-dependent oxygenase
Man, Z.; Rao, Z.; Xu, M.; Guo, J.; Yang, T.; Zhang, X.; Xu, Z.
Improvement of the intracellular environment for enhancing L-arginine production of Corynebacterium glutamicum by inactivation of H2O2-forming flavin reductases and optimization of ATP supply