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Literature summary for 1.5.1.36 extracted from

  • Sedlacek, V.; Klumpler, T.; Marek, J.; Kucera, I.
    Biochemical properties and crystal structure of the flavin reductase FerA from Paracoccus denitrificans (2016), Microbiol. Res., 188-189, 9-22 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene Pden_2689, recombinant overexpression of C-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)pLysS Paracoccus denitrificans

Crystallization (Commentary)

Crystallization (Comment) Organism
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

Inhibitors Comment Organism Structure
AMP mixed noncompetitive versus FMN, and competitive versus NADH Paracoccus denitrificans
lumichrome an FMN analogue completely lacking the ribityl side chain, competitive versus FMN, and mixed noncompetitive versus NADH Paracoccus denitrificans

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information bi-substrate kinetic analysis, stopped-flow kinetic measurements, detailed overview Paracoccus denitrificans

Localization

Localization Comment Organism GeneOntology No. Textmining
cytoplasm
-
Paracoccus denitrificans 5737
-

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
FAD + NADH + H+ Paracoccus denitrificans
-
FADH2 + NAD+
-
r
FAD + NADH + H+ Paracoccus denitrificans 1222
-
FADH2 + NAD+
-
r
FMN + NADH + H+ Paracoccus denitrificans
-
FMNH2 + NAD+
-
r
FMN + NADH + H+ Paracoccus denitrificans 1222
-
FMNH2 + NAD+
-
r

Organism

Organism UniProt Comment Textmining
Paracoccus denitrificans A1B5I2
-
-
Paracoccus denitrificans 1222 A1B5I2
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant C-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3)pLysS by nickel affinity chromatography Paracoccus denitrificans

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
FAD + NADH + H+
-
Paracoccus denitrificans FADH2 + NAD+
-
r
FAD + NADH + H+
-
Paracoccus denitrificans 1222 FADH2 + NAD+
-
r
FMN + NADH + H+
-
Paracoccus denitrificans FMNH2 + NAD+
-
r
FMN + NADH + H+
-
Paracoccus denitrificans 1222 FMNH2 + NAD+
-
r
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 ?
-
?
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

Subunits Comment Organism
? x * 19936.4, recombinant enzyme, mass spectrometry, x * 20000, recombinant enzyme, SDS-PAGE, x * 20080.8, sequence calculation Paracoccus denitrificans
More crystal structure of FerA reveals a twisted seven-stranded antiparallel beta-barrel, enzyme structure modeling, overview Paracoccus denitrificans

Synonyms

Synonyms Comment Organism
FerA
-
Paracoccus denitrificans
flavin reductase
-
Paracoccus denitrificans
NADH:flavin oxidoreductase
-
Paracoccus denitrificans
Pden2689
-
Paracoccus denitrificans

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25 30 assay at Paracoccus denitrificans

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7 7.4 assay at Paracoccus denitrificans

Cofactor

Cofactor Comment Organism Structure
NAD+
-
Paracoccus denitrificans
NADH
-
Paracoccus denitrificans

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.049
-
lumichrome versus FMN, pH 7.4, 30°C Paracoccus denitrificans
0.11
-
lumichrome versus NADH, pH 7.4, 30°C Paracoccus denitrificans
29
-
AMP versus FMN, pH 7.4, 30°C Paracoccus denitrificans
35
-
AMP versus NADH, pH 7.4, 30°C Paracoccus denitrificans

General Information

General Information Comment Organism
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