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Literature summary extracted from
- Biochemical properties and crystal structure of the flavin reductase FerA from Paracoccus denitrificans (2016), Microbiol. Res., 188-189, 9-22 .
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 1.5.1.36 | gene Pden_2689, recombinant overexpression of C-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)pLysS | Paracoccus denitrificans |
Crystallization (Commentary)
| EC Number | Crystallization (Comment) | Organism |
|---|---|---|
| 1.5.1.36 | 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 | Paracoccus denitrificans |
Inhibitors
| EC Number | Inhibitors | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.5.1.36 | AMP | mixed noncompetitive versus FMN, and competitive versus NADH | Paracoccus denitrificans |  |
| 1.5.1.36 | lumichrome | an FMN analogue completely lacking the ribityl side chain, competitive versus FMN, and mixed noncompetitive versus NADH | Paracoccus denitrificans | |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 1.5.1.36 | additional information | - |
additional information | bi-substrate kinetic analysis, stopped-flow kinetic measurements, detailed overview | Paracoccus denitrificans |
Localization
| EC Number | Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|---|
| 1.5.1.36 | cytoplasm | - |
Paracoccus denitrificans | 5737 | - |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.5.1.36 | FAD + NADH + H+ | Paracoccus denitrificans | - |
FADH2 + NAD+ | - |
r | |
| 1.5.1.36 | FAD + NADH + H+ | Paracoccus denitrificans 1222 | - |
FADH2 + NAD+ | - |
r | |
| 1.5.1.36 | FMN + NADH + H+ | Paracoccus denitrificans | - |
FMNH2 + NAD+ | - |
r | |
| 1.5.1.36 | FMN + NADH + H+ | Paracoccus denitrificans 1222 | - |
FMNH2 + NAD+ | - |
r | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 1.5.1.36 | Paracoccus denitrificans | A1B5I2 | - |
- |
| 1.5.1.36 | Paracoccus denitrificans 1222 | A1B5I2 | - |
- |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 1.5.1.36 | recombinant C-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3)pLysS by nickel affinity chromatography | Paracoccus denitrificans |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.5.1.36 | FAD + NADH + H+ | - |
Paracoccus denitrificans | FADH2 + NAD+ | - |
r | |
| 1.5.1.36 | FAD + NADH + H+ | - |
Paracoccus denitrificans 1222 | FADH2 + NAD+ | - |
r | |
| 1.5.1.36 | FMN + NADH + H+ | - |
Paracoccus denitrificans | FMNH2 + NAD+ | - |
r | |
| 1.5.1.36 | FMN + NADH + H+ | - |
Paracoccus denitrificans 1222 | FMNH2 + NAD+ | - |
r | |
| 1.5.1.36 | additional information | 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 | Paracoccus denitrificans | ? | - |
? | |
| 1.5.1.36 | additional information | 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 | Paracoccus denitrificans 1222 | ? | - |
? | |
Subunits
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 1.5.1.36 | ? | x * 19936.4, recombinant enzyme, mass spectrometry, x * 20000, recombinant enzyme, SDS-PAGE, x * 20080.8, sequence calculation | Paracoccus denitrificans |
| 1.5.1.36 | More | crystal structure of FerA reveals a twisted seven-stranded antiparallel beta-barrel, enzyme structure modeling, overview | Paracoccus denitrificans |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 1.5.1.36 | FerA | - |
Paracoccus denitrificans |
| 1.5.1.36 | flavin reductase | - |
Paracoccus denitrificans |
| 1.5.1.36 | NADH:flavin oxidoreductase | - |
Paracoccus denitrificans |
| 1.5.1.36 | Pden2689 | - |
Paracoccus denitrificans |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 1.5.1.36 | 25 | 30 | assay at | Paracoccus denitrificans |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 1.5.1.36 | 7 | 7.4 | assay at | Paracoccus denitrificans |
Cofactor
| EC Number | Cofactor | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.5.1.36 | NAD+ | - |
Paracoccus denitrificans | |
| 1.5.1.36 | NADH | - |
Paracoccus denitrificans | |
Ki Value [mM]
| EC Number | Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 1.5.1.36 | 0.049 | - |
lumichrome | versus FMN, pH 7.4, 30°C | Paracoccus denitrificans | |
| 1.5.1.36 | 0.11 | - |
lumichrome | versus NADH, pH 7.4, 30°C | Paracoccus denitrificans | |
| 1.5.1.36 | 29 | - |
AMP | versus FMN, pH 7.4, 30°C | Paracoccus denitrificans | |
| 1.5.1.36 | 35 | - |
AMP | versus NADH, pH 7.4, 30°C | Paracoccus denitrificans | |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 1.5.1.36 | additional information | 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 | Paracoccus denitrificans |
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