Literature summary for 1.5.1.40 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 .

Cloned(Commentary)

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

Protein Variants

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 [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Thermobifida fusca	Q47RA9	-	-

Purification (Commentary)

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 and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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

Temperature Optimum [°C]

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

Temperature Range [°C]

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 [°C]

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 [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
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

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

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 [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
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