Cloned (Comment) | Organism |
---|---|
gene Tfu-fno, DNA and amino acid sequence determination and analysis, recombinant expression in Escherichia coli | Thermobifida fusca |
Protein Variants | Comment | Organism |
---|---|---|
R51A | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
R51E/R55A | site-directed mutagenesis, the mutant shows similar catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
R51E/R55N | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
R51E/R55S | site-directed mutagenesis, the mutant shows similar catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
R51V | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
R51V/R55V | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
R55A | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
R55N | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
R55S | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
R55V | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
S50E | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
S50E/R55A | site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
S50E/R55V | site-directed mutagenesis, the mutant shows slightly increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
S50Q | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
T28A | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
T28A/R51V | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
T28A/R51V/R55V | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
T28A/R55A | site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type enzyme | Thermobifida fusca |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics | Thermobifida fusca | |
2 | - |
oxidized coenzyme F420 | pH 6.0, 25°C, recombinant enzyme | Thermobifida fusca | |
3.2 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50E | Thermobifida fusca | |
4.4 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55S | Thermobifida fusca | |
5 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A | Thermobifida fusca | |
5.4 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R55A | Thermobifida fusca | |
6.3 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55N | Thermobifida fusca | |
6.5 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51E/R55N | Thermobifida fusca | |
7 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55A | Thermobifida fusca | |
7.3 | - |
NADPH | pH 6.0, 25°C, recombinant wild-type enzyme | Thermobifida fusca | |
8.2 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50Q | Thermobifida fusca | |
8.6 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51A | Thermobifida fusca | |
8.7 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51V | Thermobifida fusca | |
9.6 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55V | Thermobifida fusca | |
9.8 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50E/R55V | Thermobifida fusca | |
10 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51E/R55A | Thermobifida fusca | |
10 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51V/R55V | Thermobifida fusca | |
12 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R51V | Thermobifida fusca | |
12 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V | Thermobifida fusca | |
14 | - |
NADPH | pH 6.0, 25°C, recombinant wild-type enzyme | Thermobifida fusca | |
19 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A | Thermobifida fusca | |
20 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50E/R55A | Thermobifida fusca | |
29 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55A | Thermobifida fusca | |
32 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51E/R55S | Thermobifida fusca | |
49 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55V | Thermobifida fusca | |
93 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R55A | Thermobifida fusca | |
170 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55S | Thermobifida fusca | |
180 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant R51A | Thermobifida fusca | |
180 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant R51V | Thermobifida fusca | |
500 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant R51E/R55A | Thermobifida fusca | |
500 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant R51E/R55N | Thermobifida fusca | |
500 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant R51E/R55S | Thermobifida fusca | |
500 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant R51V/R55V | Thermobifida fusca | |
500 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant R55N | Thermobifida fusca | |
500 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant S50E | Thermobifida fusca | |
500 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant S50E/R55A | Thermobifida fusca | |
500 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant S50E/R55V | Thermobifida fusca | |
500 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant S50Q | Thermobifida fusca | |
500 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant T28A/R51V | Thermobifida fusca | |
500 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V | Thermobifida fusca |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
reduced coenzyme F420 + NADP+ | Thermobifida fusca | - |
oxidized coenzyme F420 + NADPH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermobifida fusca | Q47RA9 | - |
- |
Purification (Comment) | Organism |
---|---|
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | NADP+ binding site structure, overview. A F420-dependent enzyme | Thermobifida fusca | ? | - |
? | |
reduced coenzyme F420 + NADP+ | - |
Thermobifida fusca | oxidized coenzyme F420 + NADPH + H+ | - |
r | |
reduced coenzyme F420 + NADP+ | FNO catalyzes the NADP+ reduction more efficiently compared to NADPH oxidation | Thermobifida fusca | oxidized coenzyme F420 + NADPH + H+ | - |
r |
Synonyms | Comment | Organism |
---|---|---|
F420:NADPH oxidoreductase | - |
Thermobifida fusca |
Tfu-FNO | - |
Thermobifida fusca |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
65 | - |
- |
Thermobifida fusca |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
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 Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
additional information | - |
F420-dependent enzyme Tfu-FNO is highly thermostable | Thermobifida fusca |
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 Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.3 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant R51V | Thermobifida fusca | |
1.6 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant R51A | Thermobifida fusca | |
1.6 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51E/R55A | Thermobifida fusca | |
1.8 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50E/R55V | Thermobifida fusca | |
2.2 | - |
NADPH | pH 6.0, 25°C, recombinant wild-type enzyme | Thermobifida fusca | |
2.3 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50E/R55A | Thermobifida fusca | |
2.5 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R55A | Thermobifida fusca | |
2.6 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A | Thermobifida fusca | |
2.7 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51E/R55N | Thermobifida fusca | |
2.7 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50E | Thermobifida fusca | |
2.7 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R51V | Thermobifida fusca | |
2.8 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51V/R55V | Thermobifida fusca | |
2.8 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55N | Thermobifida fusca | |
3 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55A | Thermobifida fusca | |
3.2 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51A | Thermobifida fusca | |
3.2 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55V | Thermobifida fusca | |
3.3 | - |
NADPH | pH 6.0, 25°C, recombinant wild-type enzyme | Thermobifida fusca | |
3.3 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V | Thermobifida fusca | |
3.3 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R55A | Thermobifida fusca | |
3.4 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51V | Thermobifida fusca | |
3.5 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55S | Thermobifida fusca | |
4.2 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50Q | Thermobifida fusca | |
4.9 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51E/R55S | Thermobifida fusca | |
6.9 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55S | Thermobifida fusca | |
8.8 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55A | Thermobifida fusca | |
14 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A | Thermobifida fusca |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
4 | 6 | oxidation of NADPH, broad optimum | Thermobifida fusca |
8.5 | 9 | reduction of NADP+ | Thermobifida fusca |
General Information | Comment | Organism |
---|---|---|
malfunction | T28A mutant shows 3fold increased kinetic efficiency compared with the wild-type enzyme when NADPH is the substrate | Thermobifida fusca |
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 Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.12 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50E/R55A | Thermobifida fusca | |
0.15 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51E/R55S | Thermobifida fusca | |
0.16 | - |
NADPH | pH 6.0, 25°C, recombinant wild-type enzyme | Thermobifida fusca | |
0.16 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51E/R55A | Thermobifida fusca | |
0.18 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50E/R55V | Thermobifida fusca | |
0.23 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R51V | Thermobifida fusca | |
0.28 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51V/R55V | Thermobifida fusca | |
0.28 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R51V/R55V | Thermobifida fusca | |
0.29 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51V | Thermobifida fusca | |
0.33 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55V | Thermobifida fusca | |
0.37 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51A | Thermobifida fusca | |
0.42 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R51E/R55N | Thermobifida fusca | |
0.42 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55A | Thermobifida fusca | |
0.44 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55N | Thermobifida fusca | |
0.46 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R55A | Thermobifida fusca | |
0.51 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50Q | Thermobifida fusca | |
0.52 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A | Thermobifida fusca | |
0.79 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55S | Thermobifida fusca | |
0.84 | - |
NADPH | pH 6.0, 25°C, recombinant mutant S50E | Thermobifida fusca | |
3.5 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A/R55A | Thermobifida fusca | |
6.2 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant R51A | Thermobifida fusca | |
9.3 | - |
NADPH | above, pH 6.0, 25°C, recombinant mutant R51V | Thermobifida fusca | |
41 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55S | Thermobifida fusca | |
300 | - |
NADPH | pH 6.0, 25°C, recombinant mutant R55A | Thermobifida fusca | |
450 | - |
NADPH | pH 6.0, 25°C, recombinant wild-type enzyme | Thermobifida fusca | |
720 | - |
NADPH | pH 6.0, 25°C, recombinant mutant T28A | Thermobifida fusca |