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Literature summary for 1.5.1.30 extracted from

  • Takahashi, S.; Furuya, T.; Ishii, Y.; Kino, K.; Kirimura, K.
    Characterization of a flavin reductase from a thermophilic dibenzothiophene-desulfurizing bacterium, Bacillus subtilis WU-S2B (2009), J. Biosci. Bioeng., 107, 38-41.
    View publication on PubMed

Activating Compound

Activating Compound Comment Organism Structure
NADH
-
Bacillus subtilis
NADPH about double activity compared to NADH Bacillus subtilis

Cloned(Commentary)

Cloned (Comment) Organism
frb gene inserted into plasmid pET21-a, introduced into Escherichia coli BL21 (DE3), cultivated at 30°C Bacillus subtilis

Inhibitors

Inhibitors Comment Organism Structure
5,5'-dithiobis(2-nitrobenzoic acid) with 0.1 mM 28.8% activity Bacillus subtilis
AgNO3 with 1 mM 1.05% activity Bacillus subtilis
CuCl2 with 1 mM 11.2% activity Bacillus subtilis
HgCl2 with 1 mM 0.77% activity Bacillus subtilis
additional information with 1 mM ethylenediaminetetraacetic acid 99.8% activity, with 1 mM 2,2'-bipyridyl 81.8% activity Bacillus subtilis
N-ethylmaleimide with 1 mM 16.3% activity Bacillus subtilis
p-chloromercuribenzoic acid with 1 mM 15.6% activity Bacillus subtilis

Metals/Ions

Metals/Ions Comment Organism Structure
CaCl2 with 1 mM 84.5% activity Bacillus subtilis
FeCl2 with 1 mM 90.0% activity Bacillus subtilis
MgCl2 with 1 mM 92.6% activity Bacillus subtilis
MnCl2 with 1 mM 97.0% activity Bacillus subtilis
NiCl2 with 1 mM 86.6% activity Bacillus subtilis
ZnCl2 with 1 mM 96.8% activity Bacillus subtilis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
20000
-
2 * 20000 Bacillus subtilis
40000
-
SDS-PAGE Bacillus subtilis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
FMN + NADH + H+ Bacillus subtilis
-
FMNH2 + NAD+
-
?
FMN + NADH + H+ Bacillus subtilis WU-S2B
-
FMNH2 + NAD+
-
?
FMN + NADPH + H+ Bacillus subtilis 203% activity compared to NAD+ FMNH2 + NADP+
-
?
FMN + NADPH + H+ Bacillus subtilis WU-S2B 203% activity compared to NAD+ FMNH2 + NADP+
-
?

Organism

Organism UniProt Comment Textmining
Bacillus subtilis
-
-
-
Bacillus subtilis WU-S2B
-
-
-

Purification (Commentary)

Purification (Comment) Organism
cells harvested by centrifugation, washing twice and suspended in 50 mM Tris-HCl buffer, pH 8.0, with 1 mM dithiothreitol and 10% glycerol, cells disrupted with ultraoscillator (20 kHz), debris removed by centrifugation, enzyme purified from cell-free extracts with ÄKTA explorer, applied to Toyopearl DEAE-650 M column, equilibrated with 50 mM Tris-HCl buffer, pH 8.0, washed, proteins eluted with linear gradient of 0-0.5 M KCl, active fractions (at 0.15-0.2 M KCl) combined and concentrated by ultrafiltration (membrane filter YM-30), enzyme purified with HiLoad 16/60 Superdex 200, equilibrated with 50 mM Tris-HCl buffer, pH 8.0 with 0.15 M NaCl, elution with same buffer Bacillus subtilis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
42.1
-
FMN reduction with NADH, pH 6.0, 50°C Bacillus subtilis
85.5
-
FMN reduction with NADPH, pH 6.0, 50°C Bacillus subtilis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1,3-dinitrobenzene + NADH + H+ 10.6% activity compared to FMN Bacillus subtilis ? + NAD+
-
?
1,4-benzoquinone + NADH + H+ 18.6% activity compared to FMN Bacillus subtilis 1,4-benzoquinol + NAD+
-
?
2 ferricyanide + NADH 15.4% activity compared to FMN Bacillus subtilis 2 ferrocyanide + NAD+ + H+
-
?
2 ferricyanide + NADH 15.4% activity compared to FMN Bacillus subtilis WU-S2B 2 ferrocyanide + NAD+ + H+
-
?
2 ferricytochrome c + NADH 8.48% activity compared to FMN Bacillus subtilis 2 ferrocytochrome c + NAD+ + H+
-
?
2 ferricytochrome c + NADH 8.48% activity compared to FMN Bacillus subtilis WU-S2B 2 ferrocytochrome c + NAD+ + H+
-
?
3-nitrophenol + NADH + H+ 8.95% activity compared to FMN Bacillus subtilis ? + NAD+
-
?
4-nitroacetophenone + NADH + H+ 17.9% activity compared to FMN Bacillus subtilis ? + NAD+
-
?
4-nitroaniline + NADH + H+ 4.91% activity compared to FMN Bacillus subtilis ? + NAD+
-
?
4-nitrobenzoate + NADH + H+ 21.2% activity compared to FMN Bacillus subtilis ? + NAD+
-
?
4-nitrophenol + NADH + H+ 8.07% activity compared to FMN Bacillus subtilis ? + NAD+
-
?
4-nitrotoluene + NADH + H+ 8.83% activity compared to FMN Bacillus subtilis ? + NAD+
-
?
FAD + NADH + H+ 58.1% activity compared to FMN Bacillus subtilis FADH2 + NAD+
-
?
FMN + NADH + H+
-
Bacillus subtilis FMNH2 + NAD+
-
?
FMN + NADH + H+
-
Bacillus subtilis WU-S2B FMNH2 + NAD+
-
?
FMN + NADPH + H+ 203% activity compared to NAD+ Bacillus subtilis FMNH2 + NADP+
-
?
FMN + NADPH + H+ 203% activity compared to NAD+ Bacillus subtilis WU-S2B FMNH2 + NADP+
-
?
lumiflavin + NADH + H+ 14.1% activity compared to FMN Bacillus subtilis reduced lumiflavin + NAD+
-
?
menadione + NADH + H+ 19.0% activity compared to FMN Bacillus subtilis menadiol + NAD+
-
?
methyl-4-nitrobenzoate + NADH + H+ 10.7% activity compared to FMN Bacillus subtilis ? + NAD+
-
?
methylene blue + NADH + H+ 29.0% activity compared to FMN Bacillus subtilis reduced methylene blue + NAD+
-
?
NADH + H+ + oxidized 2,6-dichlorophenolindophenol 23.0% activity compared to FMN Bacillus subtilis NAD+ + reduced 2,6-dichlorophenolindophenol
-
?
NADH + H+ + oxidized 2,6-dichlorophenolindophenol 23.0% activity compared to FMN Bacillus subtilis WU-S2B NAD+ + reduced 2,6-dichlorophenolindophenol
-
?
nitrofurantoin + NADH + H+ 10.1% activity compared to FMN Bacillus subtilis ? + NAD+
-
?
nitrofurazone + NADH + H+ 13.5% activity compared to FMN Bacillus subtilis reduced nitrofurazone + NAD+
-
?
riboflavin + NADH + H+ 29.0% activity compared to FMN Bacillus subtilis reduced riboflavin + NAD+
-
?

Subunits

Subunits Comment Organism
homodimer 2 * 20000 Bacillus subtilis

Synonyms

Synonyms Comment Organism
flavin reductase
-
Bacillus subtilis
Frb
-
Bacillus subtilis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
-
Bacillus subtilis

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
20 55 maxiumum activity at 50°C, about 90% activity at 45°C, about 80% at 40 and 55°C, about 50-60% activity at 35 and 60°C, about 40% activity at 30°C, about 30% activity at 25°C, about 20% activity at 20 and 65°C, no activity at 70°C Bacillus subtilis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
20 70 30 min incubation at 20°C retains 100% activity, more than 90% activity is retained at 25-45°C, about 80% at 50°C, about 50% at 55°C, about 20% at 60°C, about 10% at 65°C, no activity at 70°C Bacillus subtilis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
-
Bacillus subtilis

pH Range

pH Minimum pH Maximum Comment Organism
4 10 maximum activity at pH 6.0, more than 80% activity at pH 5.5, 6.5, and 7.0, about 75% activity at pH 7.5, about 30% activity at pH 8.5, less than 20% activity at pH 9-9.5, less than 10% activity at pH 4, 4.5, and 10, pH 4.0-6.0 citrate-phosphate buffer, pH 6.0-7.5 potassium-phosphate buffer, pH 7.5-8.5 Tris-HCl buffer, pH 8.5-10.0 glycine-NaOH buffer Bacillus subtilis

pH Stability

pH Stability pH Stability Maximum Comment Organism
2 12 about 80% activity retained after 24 h incubation at pH 2.0-3.5, 90 to less than 100% at pH 4.0-5.5 and pH 8.0-12.0, 100% at pH 6.0-7.5, pH 4.0-6.0 citrate-phosphate buffer, pH 6.0-7.5 potassium-phosphate buffer, pH 7.5-8.5 Tris-HCl buffer, pH 8.5-10.0 glycine-NaOH buffer Bacillus subtilis

Cofactor

Cofactor Comment Organism Structure
FMN
-
Bacillus subtilis
NADH
-
Bacillus subtilis
NADPH
-
Bacillus subtilis

General Information

General Information Comment Organism
metabolism the flavin reductase couples with dibenzothiophene and dibenzothiophene sulfone monooxygenase in the thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, the flavin reductase exhibits flavin reductase and nitroreductase activities Bacillus subtilis