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Literature summary extracted from

  • Zenno, S.; Kobori, T.; Tanokura, M.; Saigo, K.
    Purification and characetrizatio of NfrA1, a Bacillus subtilis nitro/flavin reductase capable of interacting with the bacterial luciferase (1998), Biosci. Biotechnol. Biochem., 62, 1978-1987 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.30 gene ipa-43d, DNA and amino acid sequence determination and analysis, genetic structure and sequence comparison, recombinant overexpression in Escherichia coli J83 Bacillus subtilis
1.7.1.B3 gene ipa-43d, DNA and amino acid sequence determination and analysis, genetic structure and sequence comparison, recombinant overexpression in Escherichia coli J83 Bacillus subtilis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.1.30 additional information
-
 additional information enzyme kinetic analysis, overview Bacillus subtilis
1.5.1.30 0.0039
-
 NADPH pH 7.0, 23°C, recombinant enzyme, with FMN Bacillus subtilis
1.5.1.30 0.0047
-
 FMN pH 7.0, 23°C, recombinant enzyme Bacillus subtilis
1.7.1.B3 additional information
-
 additional information enzyme kinetic analysis, overview Bacillus subtilis
1.7.1.B3 0.00085
-
 NADPH pH 7.0, 23°C, recombinant enzyme, with nitrofurazone Bacillus subtilis
1.7.1.B3 0.0163
-
 nitrofurazone pH 7.0, 23°C, recombinant enzyme Bacillus subtilis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.5.1.30 53000
-
 recombinant untagged enzyme, gel filtration Bacillus subtilis
1.7.1.B3 53000
-
 recombinant untagged enzyme, gel filtration Bacillus subtilis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.5.1.30 FAD + NADPH + H+ Bacillus subtilis
-
 reduced FAD + NADP+
-
 ?
1.5.1.30 FAD + NADPH + H+ Bacillus subtilis ISW 1214
-
 reduced FAD + NADP+
-
 ?
1.5.1.30 FMN + NADPH + H+ Bacillus subtilis
-
 reduced FMN + NADP+
-
 ?
1.5.1.30 FMN + NADPH + H+ Bacillus subtilis ISW 1214
-
 reduced FMN + NADP+
-
 ?
1.5.1.30 lumiflavin + NADPH + H+ Bacillus subtilis
-
 reduced lumiflavin + NADP+
-
 ?
1.5.1.30 lumiflavin + NADPH + H+ Bacillus subtilis ISW 1214
-
 reduced lumiflavin + NADP+
-
 ?
1.5.1.30 riboflavin + NADPH + H+ Bacillus subtilis
-
 reduced riboflavin + NADP+
-
 ?
1.5.1.30 riboflavin + NADPH + H+ Bacillus subtilis ISW 1214
-
 reduced riboflavin + NADP+
-
 ?
1.7.1.B3 nitrofurazone + NADPH + H+ Bacillus subtilis
-
 5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.30 Bacillus subtilis
-
-
-
1.5.1.30 Bacillus subtilis ISW 1214
-
-
-
1.7.1.B3 Bacillus subtilis
-
-
-
1.7.1.B3 Bacillus subtilis ISW 1214
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.30 recombinant enzyme 23fold from Escherichia coli to homogeneity by dialysis, anion exchange chromatography, dialysis, two steps of Blue Sepharose affinity chromatography, and again dialysis Bacillus subtilis
1.7.1.B3 recombinant enzyme 23fold from Escherichia coli to homogeneity by dialysis, anion exchange chromatography, dialysis, two steps of Blue Sepharose affinity chromatography, and again dialysis Bacillus subtilis

Reaction

EC Number Reaction Comment Organism Reaction ID
1.5.1.30 reduced riboflavin + NADP+ = riboflavin + NADPH + H+ the enzyme catalysis obeys the ping-pong Bi-Bi kinetic mechanism with substrate FMN as well as substrate nitrofurazone, but upon coupling with the bioluminescent reaction of luciferase, it changes to the sequential mechanism Bacillus subtilis
a
1.7.1.B3 an aromatic amine + 3 NADP+ + 2 H2O = an aromatic nitrate + 3 NADPH + 3 H+ the enzyme catalysis obeys the ping-pong Bi-Bi kinetic mechanism with substrate FMN as well as substrate nitrofurazone, but upon coupling with the bioluminescent reaction of luciferase, it changes to the sequential mechanism Bacillus subtilis
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.5.1.30 52.5
-
 purified recombinant untagged enzyme, pH 7.0, 23°C Bacillus subtilis
1.7.1.B3 52.5
-
 purified recombinant untagged enzyme, pH 7.0, 23°C Bacillus subtilis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.30 FAD + NADPH + H+
-
 Bacillus subtilis reduced FAD + NADP+
-
 ?
1.5.1.30 FAD + NADPH + H+
-
 Bacillus subtilis ISW 1214 reduced FAD + NADP+
-
 ?
1.5.1.30 FMN + NADPH + H+
-
 Bacillus subtilis reduced FMN + NADP+
-
 ?
1.5.1.30 FMN + NADPH + H+
-
 Bacillus subtilis ISW 1214 reduced FMN + NADP+
-
 ?
1.5.1.30 lumiflavin + NADPH + H+
-
 Bacillus subtilis reduced lumiflavin + NADP+
-
 ?
1.5.1.30 lumiflavin + NADPH + H+
-
 Bacillus subtilis ISW 1214 reduced lumiflavin + NADP+
-
 ?
1.5.1.30 additional information the enzyme reduces both nitrofurazone and FMN effectively, it is bifunctional as flavin reductase and nitroreductase. Two different FMN molecules are involved in the FMN reduction process: FMN tightly associated to the enzyme as prosthetic group and FMN as a substrate. The enzyme is also active with FAD, riboflavin, and lumiflavin, the two latter give the highest activity Bacillus subtilis ?
-
 ?
1.5.1.30 additional information the enzyme reduces both nitrofurazone and FMN effectively, it is bifunctional as flavin reductase and nitroreductase. Two different FMN molecules are involved in the FMN reduction process: FMN tightly associated to the enzyme as prosthetic group and FMN as a substrate. The enzyme is also active with FAD, riboflavin, and lumiflavin, the two latter give the highest activity Bacillus subtilis ISW 1214 ?
-
 ?
1.5.1.30 riboflavin + NADPH + H+
-
 Bacillus subtilis reduced riboflavin + NADP+
-
 ?
1.5.1.30 riboflavin + NADPH + H+
-
 Bacillus subtilis ISW 1214 reduced riboflavin + NADP+
-
 ?
1.7.1.B3 methyl 4-nitrobenzoate + NADPH + H+
-
 Bacillus subtilis ? + NADP+ + H2O
-
 ?
1.7.1.B3 additional information the enzyme reduces both nitrofurazone and FMN effectively, see for EC 1.5.1.38, it is bifunctional as flavin reductase and nitroreductase. Two different FMN molecules are involved in the FMN reduction process: FMN tightly associated to the enzyme as prosthetic group and FMN as a substrate. The enzyme is also active with FAD, riboflavin, and lumiflavin, the two latter give the highest activity Bacillus subtilis ?
-
 ?
1.7.1.B3 nitrofurazone + NADPH + H+
-
 Bacillus subtilis 5-(hydroxyamino)furan-2-carbaldehyde semicarbazone + NADP+ + H2O
-
 ?

Subunits

EC Number Subunits Comment Organism
1.5.1.30 homodimer 2 * 28320, sequence calculation, 2 * 28000, recombinant untagged enzyme, SDS-PAGE Bacillus subtilis
1.7.1.B3 homodimer 2 * 28320, sequence calculation, 2 * 28000, recombinant untagged enzyme, SDS-PAGE Bacillus subtilis

Synonyms

EC Number Synonyms Comment Organism
1.5.1.30 ipa-43d
-
 Bacillus subtilis
1.5.1.30 NADPH-FMN reductase
-
 Bacillus subtilis
1.5.1.30 NADPH-nitrofurazone reductase
-
 Bacillus subtilis
1.5.1.30 NfrA1
-
 Bacillus subtilis
1.5.1.30 nitro/flavin reductase
-
 Bacillus subtilis
1.5.1.30 non-luminescent-bacterial NfsA/Frp-type enzyme
-
 Bacillus subtilis
1.7.1.B3 ipa-43d
-
 Bacillus subtilis
1.7.1.B3 More cf. EC 1.5.1.30 Bacillus subtilis
1.7.1.B3 NADPH-FMN reductase
-
 Bacillus subtilis
1.7.1.B3 NADPH-nitrofurazone reductase
-
 Bacillus subtilis
1.7.1.B3 NfrA1
-
 Bacillus subtilis
1.7.1.B3 nitro/flavin reductase
-
 Bacillus subtilis
1.7.1.B3 non-luminescent-bacterial NfsA/Frp-type enzyme
-
 Bacillus subtilis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.5.1.30 23
-
 assay at Bacillus subtilis
1.7.1.B3 23
-
 assay at Bacillus subtilis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.5.1.30 7
-
 assay at Bacillus subtilis
1.7.1.B3 7
-
 assay at Bacillus subtilis

Cofactor

EC Number Cofactor Comment Organism Structure
1.5.1.30 FMN tightly associated to, two different FMN molecules are involved in the FMN reduction process: FMN tightly associated to the enzyme as prosthetic group and FMN as a substrate Bacillus subtilis
1.5.1.30 additional information NADPH is much more effective than NADH Bacillus subtilis
1.5.1.30 NADPH dependent on Bacillus subtilis
1.7.1.B3 FMN tightly associated to, two different FMN molecules are involved in the FMN reduction process: FMN tightly associated to the enzyme as prosthetic group and FMN as a substrate Bacillus subtilis
1.7.1.B3 additional information NADPH is much more effective than NADH Bacillus subtilis
1.7.1.B3 NADPH dependent on Bacillus subtilis

General Information

EC Number General Information Comment Organism
1.5.1.30 evolution analysis of evolutionary or molecular mechanism of divergence of the nitroreductase/flavin reductase family, overview. The enzyme is similar to NfsA from Escherichia coli, overview Bacillus subtilis
1.7.1.B3 evolution analysis of evolutionary or molecular mechanism of divergence of the nitroreductase/flavin reductase family, overview. The enzyme is similar to NfsA from Escherichia coli, overview Bacillus subtilis