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Literature summary extracted from

  • Kumar, H.; Nguyen, Q.T.; Binda, C.; Mattevi, A.; Fraaije, M.W.
    Isolation and characterization of a thermostable F420 NADPH oxidoreductase from Thermobifida fusca (2017), J. Biol. Chem., 292, 10123-10130 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.5.1.40 gene Tfu-fno, DNA and amino acid sequence determination and analysis, recombinant expression in Escherichia coli Thermobifida fusca

Protein Variants

EC Number Protein Variants Comment Organism
1.5.1.40 R51A site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 R51E/R55A site-directed mutagenesis, the mutant shows similar catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 R51E/R55N site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 R51E/R55S site-directed mutagenesis, the mutant shows similar catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 R51V site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 R51V/R55V site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 R55A site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 R55N site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 R55S site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 R55V site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 S50E site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 S50E/R55A site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 S50E/R55V site-directed mutagenesis, the mutant shows slightly increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 S50Q site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 T28A site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 T28A/R51V site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 T28A/R51V/R55V site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca
1.5.1.40 T28A/R55A site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme Thermobifida fusca

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.5.1.40 additional information
-
 additional information steady-state kinetics Thermobifida fusca
1.5.1.40 2
-
 oxidized coenzyme F420 pH 6.0, 25°C, recombinant enzyme Thermobifida fusca
1.5.1.40 3.2
-
 NADPH pH 6.0, 25°C, recombinant mutant S50E Thermobifida fusca
1.5.1.40 4.4
-
 NADPH pH 6.0, 25°C, recombinant mutant R55S Thermobifida fusca
1.5.1.40 5
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A Thermobifida fusca
1.5.1.40 5.4
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R55A Thermobifida fusca
1.5.1.40 6.3
-
 NADPH pH 6.0, 25°C, recombinant mutant R55N Thermobifida fusca
1.5.1.40 6.5
-
 NADPH pH 6.0, 25°C, recombinant mutant R51E/R55N Thermobifida fusca
1.5.1.40 7
-
 NADPH pH 6.0, 25°C, recombinant mutant R55A Thermobifida fusca
1.5.1.40 7.3
-
 NADPH pH 6.0, 25°C, recombinant wild-type enzyme Thermobifida fusca
1.5.1.40 8.2
-
 NADPH pH 6.0, 25°C, recombinant mutant S50Q Thermobifida fusca
1.5.1.40 8.6
-
 NADPH pH 6.0, 25°C, recombinant mutant R51A Thermobifida fusca
1.5.1.40 8.7
-
 NADPH pH 6.0, 25°C, recombinant mutant R51V Thermobifida fusca
1.5.1.40 9.6
-
 NADPH pH 6.0, 25°C, recombinant mutant R55V Thermobifida fusca
1.5.1.40 9.8
-
 NADPH pH 6.0, 25°C, recombinant mutant S50E/R55V Thermobifida fusca
1.5.1.40 10
-
 NADPH pH 6.0, 25°C, recombinant mutant R51E/R55A Thermobifida fusca
1.5.1.40 10
-
 NADPH pH 6.0, 25°C, recombinant mutant R51V/R55V Thermobifida fusca
1.5.1.40 12
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R51V Thermobifida fusca
1.5.1.40 12
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V Thermobifida fusca
1.5.1.40 14
-
 NADPH pH 6.0, 25°C, recombinant wild-type enzyme Thermobifida fusca
1.5.1.40 19
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A Thermobifida fusca
1.5.1.40 20
-
 NADPH pH 6.0, 25°C, recombinant mutant S50E/R55A Thermobifida fusca
1.5.1.40 29
-
 NADPH pH 6.0, 25°C, recombinant mutant R55A Thermobifida fusca
1.5.1.40 32
-
 NADPH pH 6.0, 25°C, recombinant mutant R51E/R55S Thermobifida fusca
1.5.1.40 49
-
 NADPH pH 6.0, 25°C, recombinant mutant R55V Thermobifida fusca
1.5.1.40 93
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R55A Thermobifida fusca
1.5.1.40 170
-
 NADPH pH 6.0, 25°C, recombinant mutant R55S Thermobifida fusca
1.5.1.40 180
-
 NADPH above, pH 6.0, 25°C, recombinant mutant R51A Thermobifida fusca
1.5.1.40 180
-
 NADPH above, pH 6.0, 25°C, recombinant mutant R51V Thermobifida fusca
1.5.1.40 500
-
 NADPH above, pH 6.0, 25°C, recombinant mutant R51E/R55A Thermobifida fusca
1.5.1.40 500
-
 NADPH above, pH 6.0, 25°C, recombinant mutant R51E/R55N Thermobifida fusca
1.5.1.40 500
-
 NADPH above, pH 6.0, 25°C, recombinant mutant R51E/R55S Thermobifida fusca
1.5.1.40 500
-
 NADPH above, pH 6.0, 25°C, recombinant mutant R51V/R55V Thermobifida fusca
1.5.1.40 500
-
 NADPH above, pH 6.0, 25°C, recombinant mutant R55N Thermobifida fusca
1.5.1.40 500
-
 NADPH above, pH 6.0, 25°C, recombinant mutant S50E Thermobifida fusca
1.5.1.40 500
-
 NADPH above, pH 6.0, 25°C, recombinant mutant S50E/R55A Thermobifida fusca
1.5.1.40 500
-
 NADPH above, pH 6.0, 25°C, recombinant mutant S50E/R55V Thermobifida fusca
1.5.1.40 500
-
 NADPH above, pH 6.0, 25°C, recombinant mutant S50Q Thermobifida fusca
1.5.1.40 500
-
 NADPH above, pH 6.0, 25°C, recombinant mutant T28A/R51V Thermobifida fusca
1.5.1.40 500
-
 NADPH above, pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V Thermobifida fusca

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.5.1.40 reduced coenzyme F420 + NADP+ Thermobifida fusca
-
 oxidized coenzyme F420 + NADPH + H+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.5.1.40 Thermobifida fusca Q47RA9
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.5.1.40 recombinant enzyme from Escherichia coli by ammonium sulfate fractionation followed by anion exchange chromatography, DNase I treatment during protein purification is essential to remove residual DNA Thermobifida fusca

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.5.1.40 additional information NADP+ binding site structure, overview. A F420-dependent enzyme Thermobifida fusca ?
-
 ?
1.5.1.40 reduced coenzyme F420 + NADP+
-
 Thermobifida fusca oxidized coenzyme F420 + NADPH + H+
-
 r
1.5.1.40 reduced coenzyme F420 + NADP+ FNO catalyzes the NADP+ reduction more efficiently compared to NADPH oxidation Thermobifida fusca oxidized coenzyme F420 + NADPH + H+
-
 r

Synonyms

EC Number Synonyms Comment Organism
1.5.1.40 F420:NADPH oxidoreductase
-
 Thermobifida fusca
1.5.1.40 Tfu-FNO
-
 Thermobifida fusca

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.5.1.40 65
-
-
 Thermobifida fusca

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.5.1.40 25 90 the enzyme displays highest activity between 60°C and 70°C. The activity at 65°C is almost 4fold higher than that at 25°C. The apparent melting temperature of Tfu-FNO is 75°C, about 50% of maximal activity at 40°C and 90°C Thermobifida fusca

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.5.1.40 additional information
-
 F420-dependent enzyme Tfu-FNO is highly thermostable Thermobifida fusca
1.5.1.40 25 90 the enzyme displays highest activity between 60°C and 70°C. The activity at 65°C is almost 4fold higher than that at 25°C. The apparent melting temperature of Tfu-FNO is 75°C, about 50% of maximal activity at 40°C and 90°C Thermobifida fusca

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.5.1.40 1.3
-
 NADPH above, pH 6.0, 25°C, recombinant mutant R51V Thermobifida fusca
1.5.1.40 1.6
-
 NADPH above, pH 6.0, 25°C, recombinant mutant R51A Thermobifida fusca
1.5.1.40 1.6
-
 NADPH pH 6.0, 25°C, recombinant mutant R51E/R55A Thermobifida fusca
1.5.1.40 1.8
-
 NADPH pH 6.0, 25°C, recombinant mutant S50E/R55V Thermobifida fusca
1.5.1.40 2.2
-
 NADPH pH 6.0, 25°C, recombinant wild-type enzyme Thermobifida fusca
1.5.1.40 2.3
-
 NADPH pH 6.0, 25°C, recombinant mutant S50E/R55A Thermobifida fusca
1.5.1.40 2.5
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R55A Thermobifida fusca
1.5.1.40 2.6
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A Thermobifida fusca
1.5.1.40 2.7
-
 NADPH pH 6.0, 25°C, recombinant mutant R51E/R55N Thermobifida fusca
1.5.1.40 2.7
-
 NADPH pH 6.0, 25°C, recombinant mutant S50E Thermobifida fusca
1.5.1.40 2.7
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R51V Thermobifida fusca
1.5.1.40 2.8
-
 NADPH pH 6.0, 25°C, recombinant mutant R51V/R55V Thermobifida fusca
1.5.1.40 2.8
-
 NADPH pH 6.0, 25°C, recombinant mutant R55N Thermobifida fusca
1.5.1.40 3
-
 NADPH pH 6.0, 25°C, recombinant mutant R55A Thermobifida fusca
1.5.1.40 3.2
-
 NADPH pH 6.0, 25°C, recombinant mutant R51A Thermobifida fusca
1.5.1.40 3.2
-
 NADPH pH 6.0, 25°C, recombinant mutant R55V Thermobifida fusca
1.5.1.40 3.3
-
 NADPH pH 6.0, 25°C, recombinant wild-type enzyme Thermobifida fusca
1.5.1.40 3.3
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V Thermobifida fusca
1.5.1.40 3.3
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R55A Thermobifida fusca
1.5.1.40 3.4
-
 NADPH pH 6.0, 25°C, recombinant mutant R51V Thermobifida fusca
1.5.1.40 3.5
-
 NADPH pH 6.0, 25°C, recombinant mutant R55S Thermobifida fusca
1.5.1.40 4.2
-
 NADPH pH 6.0, 25°C, recombinant mutant S50Q Thermobifida fusca
1.5.1.40 4.9
-
 NADPH pH 6.0, 25°C, recombinant mutant R51E/R55S Thermobifida fusca
1.5.1.40 6.9
-
 NADPH pH 6.0, 25°C, recombinant mutant R55S Thermobifida fusca
1.5.1.40 8.8
-
 NADPH pH 6.0, 25°C, recombinant mutant R55A Thermobifida fusca
1.5.1.40 14
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A Thermobifida fusca

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.5.1.40 4 6 oxidation of NADPH, broad optimum Thermobifida fusca
1.5.1.40 8.5 9 reduction of NADP+ Thermobifida fusca

General Information

EC Number General Information Comment Organism
1.5.1.40 malfunction T28A mutant shows 3fold increased kinetic efficiency compared with the wild-type enzyme when NADPH is the substrate Thermobifida fusca
1.5.1.40 additional information the active site of F420-dependent enzyme Tfu-FNO is located in a hydrophobic pocket between an N-terminal dinucleotide binding domain and a smaller C-terminal domain. Residues interacting with the 2'-phosphate of NADP+, Thr28, Ser50, Arg51, and Arg55, are important for discriminating between NADP+ and NAD+. Molecular recognition of the two cofactor substrates, F420 and NAD(P)H by FNO, overview Thermobifida fusca

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.5.1.40 0.12
-
 NADPH pH 6.0, 25°C, recombinant mutant S50E/R55A Thermobifida fusca
1.5.1.40 0.15
-
 NADPH pH 6.0, 25°C, recombinant mutant R51E/R55S Thermobifida fusca
1.5.1.40 0.16
-
 NADPH pH 6.0, 25°C, recombinant wild-type enzyme Thermobifida fusca
1.5.1.40 0.16
-
 NADPH pH 6.0, 25°C, recombinant mutant R51E/R55A Thermobifida fusca
1.5.1.40 0.18
-
 NADPH pH 6.0, 25°C, recombinant mutant S50E/R55V Thermobifida fusca
1.5.1.40 0.23
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R51V Thermobifida fusca
1.5.1.40 0.28
-
 NADPH pH 6.0, 25°C, recombinant mutant R51V/R55V Thermobifida fusca
1.5.1.40 0.28
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V Thermobifida fusca
1.5.1.40 0.29
-
 NADPH pH 6.0, 25°C, recombinant mutant R51V Thermobifida fusca
1.5.1.40 0.33
-
 NADPH pH 6.0, 25°C, recombinant mutant R55V Thermobifida fusca
1.5.1.40 0.37
-
 NADPH pH 6.0, 25°C, recombinant mutant R51A Thermobifida fusca
1.5.1.40 0.42
-
 NADPH pH 6.0, 25°C, recombinant mutant R51E/R55N Thermobifida fusca
1.5.1.40 0.42
-
 NADPH pH 6.0, 25°C, recombinant mutant R55A Thermobifida fusca
1.5.1.40 0.44
-
 NADPH pH 6.0, 25°C, recombinant mutant R55N Thermobifida fusca
1.5.1.40 0.46
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R55A Thermobifida fusca
1.5.1.40 0.51
-
 NADPH pH 6.0, 25°C, recombinant mutant S50Q Thermobifida fusca
1.5.1.40 0.52
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A Thermobifida fusca
1.5.1.40 0.79
-
 NADPH pH 6.0, 25°C, recombinant mutant R55S Thermobifida fusca
1.5.1.40 0.84
-
 NADPH pH 6.0, 25°C, recombinant mutant S50E Thermobifida fusca
1.5.1.40 3.5
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A/R55A Thermobifida fusca
1.5.1.40 6.2
-
 NADPH above, pH 6.0, 25°C, recombinant mutant R51A Thermobifida fusca
1.5.1.40 9.3
-
 NADPH above, pH 6.0, 25°C, recombinant mutant R51V Thermobifida fusca
1.5.1.40 41
-
 NADPH pH 6.0, 25°C, recombinant mutant R55S Thermobifida fusca
1.5.1.40 300
-
 NADPH pH 6.0, 25°C, recombinant mutant R55A Thermobifida fusca
1.5.1.40 450
-
 NADPH pH 6.0, 25°C, recombinant wild-type enzyme Thermobifida fusca
1.5.1.40 720
-
 NADPH pH 6.0, 25°C, recombinant mutant T28A Thermobifida fusca