Literature summary for 1.5.1.40 extracted from

Drenth, J.; Yang, G.; Paul, C.E.; Fraaije, M.W.
A tailor-made deazaflavin-mediated recycling system for artificial nicotinamide cofactor biomimetics (2021), ACS Catal., 11, 11561-11569 .

Application

Application	Comment	Organism
synthesis	Tfu-FNO variants can be used as NCB-Recycling systems for TsOYE-mediated conversions. TsOYE is known to efficiently hydrogenate ketoisophorone (2,6,6-trimethyl-2-cyclohexene-1,4-dione) to form the chiral product (R)-2,2,6- trimethylcyclohexane-1,4-dione with high enantiomeric excess values of 94-97%, when several NCBs as more efficient electron donors in comparison to NADPH are used. The Tfu-FNO variants G29Y, G29W, and P89H, which show high activity toward 1-aminoethylnicotinamide (AmNA+), 1-benzylnicotinamide (BNA+), and 1-benzyl-3-acetylpyridine (BAP+), are selected for use in conversion experiments as recycling catalysts for these NCBs. Potential use of these thermostable Tfu- FNO variants as NCB recycling systems at high temperatures	Thermobifida fusca

Protein Variants

Protein Variants	Comment	Organism
G29L	site-directed mutagenesis, the mutant shows altered kinetics and increased catalytic efficiency with nicotinamide cofactor biomimetics compared to wild-type enzyme	Thermobifida fusca
G29S	site-directed mutagenesis, the mutant shows altered kinetics and increased catalytic efficiency with nicotinamide cofactor biomimetics compared to wild-type enzyme	Thermobifida fusca
G29Y	site-directed mutagenesis, the mutant shows altered kinetics and increased catalytic efficiency with nicotinamide cofactor biomimetics compared to wild-type enzyme	Thermobifida fusca
additional information	engineering of the thermostable F420:NADPH oxidoreductase from Thermobifida fusca (Tfu-FNO) by structure-inspired site-directed mutagenesis to accommodate the unnatural N1 substituents of eight nicotinamide cofactor biomimetics (NCBs). The extraordinarily low redox potential of the natural cofactor F420H2 is then exploited to reduce these NCBs. Wild-type enzyme has detectable activity toward all selected NCBs. Saturation mutagenesis at positions Gly29 and Pro89 results in mutants with up to 139times higher catalytic efficiencies, kinetics comparisons, overview. Most mutations significantly decrease the activity toward NADP+ but do not completely inhibit the enzyme for this cosubstrate	Thermobifida fusca
P89H	site-directed mutagenesis, the mutant shows altered kinetics and increased catalytic efficiency with nicotinamide cofactor biomimetics compared to wild-type enzyme	Thermobifida fusca
P89L	site-directed mutagenesis, the mutant shows altered kinetics and increased catalytic efficiency with nicotinamide cofactor biomimetics compared to wild-type enzyme	Thermobifida fusca
P89Y	site-directed mutagenesis, the mutant shows altered kinetics and increased catalytic efficiency with nicotinamide cofactor biomimetics compared to wild-type enzyme	Thermobifida fusca

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
2	3	1-aminoethylnicotinamide	enzyme mutant G29Y, pH 8.0, 25°C	Thermobifida fusca
2	3	1-hydroxyethylnicotinamide	enzyme mutant G29Y, pH 8.0, 25°C	Thermobifida fusca
4.3	-	1-benzylnicotinamide	enzyme mutant G29W, pH 8.0, 25°C	Thermobifida fusca
5.7	-	1-propylnicotinamide	enzyme mutant P89H, pH 8.0, 25°C	Thermobifida fusca
6.4	-	1-benzylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
7.4	-	1-benzyl-3-acetylpyridine	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
9.5	-	1-benzylnicotinamide	enzyme mutant G29Y, pH 8.0, 25°C	Thermobifida fusca
10	-	1-propylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
12	-	1-aminoethylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
14	-	1-aminoethylnicotinamide	enzyme mutant P98H, pH 8.0, 25°C	Thermobifida fusca
18	-	1-propylnicotinamide	enzyme mutant G29L, pH 8.0, 25°C	Thermobifida fusca
18	-	1-aminoethylnicotinamide	enzyme mutant G29S, pH 8.0, 25°C	Thermobifida fusca
20	-	1-hydroxyethylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
20	-	1-hydroxyethylnicotinamide	enzyme mutant P98H, pH 8.0, 25°C	Thermobifida fusca
25	-	1-hydroxypropylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
25	-	1-ethylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
27	-	1-propylnicotinamide	enzyme mutant P89L, pH 8.0, 25°C	Thermobifida fusca
33	-	1-hydroxyethylnicotinamide	enzyme mutant P98Y, pH 8.0, 25°C	Thermobifida fusca
35	-	1-hydroxypropylnicotinamide	enzyme mutant P89Y, pH 8.0, 25°C	Thermobifida fusca
35	-	1-propylnicotinamide	enzyme mutant P89Y, pH 8.0, 25°C	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	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	no activity of wild-type and mutants with 1-benzylnicotinic acid	Thermobifida fusca	?	-	-
reduced coenzyme F420 + 1-aminoethylnicotinamide	-	Thermobifida fusca	oxidized coenzyme F420 + ?	-	?
reduced coenzyme F420 + 1-benzyl-3-acetylpyridine	-	Thermobifida fusca	oxidized coenzyme F420 + ?	-	?
reduced coenzyme F420 + 1-benzylnicotinamide	-	Thermobifida fusca	oxidized coenzyme F420 + ?	-	?
reduced coenzyme F420 + 1-ethylnicotinamide	-	Thermobifida fusca	oxidized coenzyme F420 + ?	-	?
reduced coenzyme F420 + 1-hydroxyethylnicotinamide	-	Thermobifida fusca	oxidized coenzyme F420 + ?	-	?
reduced coenzyme F420 + 1-hydroxypropylnicotinamide	-	Thermobifida fusca	oxidized coenzyme F420 + ?	-	?
reduced coenzyme F420 + 1-propylnicotinamide	-	Thermobifida fusca	oxidized coenzyme F420 + ?	-	?
reduced coenzyme F420 + NADP+	-	Thermobifida fusca	oxidized coenzyme F420 + NADPH + H+	-	r

Synonyms

Synonyms	Comment	Organism
F420:NADPH oxidoreductase	-	Thermobifida fusca
Tfu-FNO	-	Thermobifida fusca
Tfu_0970	-	Thermobifida fusca

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Thermobifida fusca

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
25	50	the activity of a selection of variants toward 5 mM of 1-aminoethylnicotinamide (AmNA+), 1-benzylnicotinamide (BNA+), and 1-benzyl-3-acetylpyridine (BAP+) at both 25°C and 50°C shows that the activity increases at elevated temperature	Thermobifida fusca

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0015	-	1-propylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.0042	-	1-hydroxypropylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.0042	-	1-benzyl-3-acetylpyridine	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.0045	-	1-benzylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.0045	-	1-propylnicotinamide	enzyme mutant P89H, pH 8.0, 25°C	Thermobifida fusca
0.006	-	1-propylnicotinamide	enzyme mutant G29L, pH 8.0, 25°C	Thermobifida fusca
0.0073	-	1-ethylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.0073	-	1-propylnicotinamide	enzyme mutant P89L, pH 8.0, 25°C	Thermobifida fusca
0.0098	-	1-propylnicotinamide	enzyme mutant P89Y, pH 8.0, 25°C	Thermobifida fusca
0.0118	-	1-hydroxypropylnicotinamide	enzyme mutant P89Y, pH 8.0, 25°C	Thermobifida fusca
0.0233	-	1-aminoethylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.0233	-	1-hydroxyethylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.042	-	1-aminoethylnicotinamide	enzyme mutant P98H, pH 8.0, 25°C	Thermobifida fusca
0.048	-	1-hydroxyethylnicotinamide	enzyme mutant P98H, pH 8.0, 25°C	Thermobifida fusca
0.053	-	1-aminoethylnicotinamide	enzyme mutant G29Y, pH 8.0, 25°C	Thermobifida fusca
0.083	-	1-hydroxyethylnicotinamide	enzyme mutant P98Y, pH 8.0, 25°C	Thermobifida fusca
0.103	-	1-hydroxyethylnicotinamide	enzyme mutant G29Y, pH 8.0, 25°C	Thermobifida fusca
0.145	-	1-benzylnicotinamide	enzyme mutant G29Y, pH 8.0, 25°C	Thermobifida fusca
0.16	-	1-aminoethylnicotinamide	enzyme mutant G29S, pH 8.0, 25°C	Thermobifida fusca
0.417	-	1-benzylnicotinamide	enzyme mutant G29W, pH 8.0, 25°C	Thermobifida fusca

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Thermobifida fusca

Cofactor

Cofactor	Comment	Organism	Structure
NADP+	-	Thermobifida fusca
NADPH	-	Thermobifida fusca

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.00015	-	1-propylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.00017	-	1-hydroxypropylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.00027	-	1-propylnicotinamide	enzyme mutant P89L, pH 8.0, 25°C	Thermobifida fusca
0.00028	-	1-propylnicotinamide	enzyme mutant P89Y, pH 8.0, 25°C	Thermobifida fusca
0.00029	-	1-ethylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.00033	-	1-propylnicotinamide	enzyme mutant G29L, pH 8.0, 25°C	Thermobifida fusca
0.00034	-	1-hydroxypropylnicotinamide	enzyme mutant P89Y, pH 8.0, 25°C	Thermobifida fusca
0.00057	-	1-benzyl-3-acetylpyridine	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.0007	-	1-benzylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.00079	-	1-propylnicotinamide	enzyme mutant P89H, pH 8.0, 25°C	Thermobifida fusca
0.0012	-	1-hydroxyethylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.00194	-	1-aminoethylnicotinamide	wild-type enzyme, pH 8.0, 25°C	Thermobifida fusca
0.0023	-	1-aminoethylnicotinamide	enzyme mutant G29Y, pH 8.0, 25°C	Thermobifida fusca
0.0024	-	1-hydroxyethylnicotinamide	enzyme mutant P98H, pH 8.0, 25°C	Thermobifida fusca
0.0025	-	1-hydroxyethylnicotinamide	enzyme mutant P98Y, pH 8.0, 25°C	Thermobifida fusca
0.003	-	1-aminoethylnicotinamide	enzyme mutant P98H, pH 8.0, 25°C	Thermobifida fusca
0.0045	-	1-hydroxyethylnicotinamide	enzyme mutant G29Y, pH 8.0, 25°C	Thermobifida fusca
0.0088	-	1-aminoethylnicotinamide	enzyme mutant G29S, pH 8.0, 25°C	Thermobifida fusca
0.0153	-	1-benzylnicotinamide	enzyme mutant G29Y, pH 8.0, 25°C	Thermobifida fusca
0.097	-	1-benzylnicotinamide	enzyme mutant G29W, pH 8.0, 25°C	Thermobifida fusca