Literature summary for 1.7.1.B2 extracted from

Williams, E.M.; Rich, M.H.; Mowday, A.M.; Ashoorzadeh, A.; Copp, J.N.; Guise, C.P.; Anderson, R.F.; Flanagan, J.U.; Smaill, J.B.; Patterson, A.V.; Ackerley, D.F.
Engineering Escherichia coli NfsB To activate a hypoxia-resistant analogue of the PET probe EF5 to enable non-invasive imaging during enzyme prodrug therapy (2019), Biochemistry, 58, 3700-3710 .

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli BL21(DE3) cells	Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
E72Q	the mutation does not increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
E72Q/F108Y	the mutations substantially increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
F108Y	the mutation substantially increases the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
F70A	the mutation substantially increases the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
F70A/E72Q	the mutations substantially increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
F70A/E72Q/F108Y	the mutations substantially increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
F70A/F108Y	the mutations confer high levels of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide activity and also substantially improve activity with 5-(aziridin-1-yl)-2,4-dinitrobenzamide and metronidazole. The mutations substantially increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
F70A/F108Y	the mutations substantially increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
T41L	the mutation does not increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
T41L/F70A	the mutations substantially increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
T41L/F70A/F108Y	the mutations substantially increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
T41L/F70A/N71S	the mutations substantially increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
T41L/F70A/N71S/F108Y	the mutations substantially increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli
T41L/N71S	the mutations do not increase the sensitivity of the Escherichia coli host cell to 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	Escherichia coli

Inhibitors

Inhibitors	Comment	Organism
2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	-	Escherichia coli
5-(aziridin-1-yl)-2,4-dinitrobenzamide	-	Escherichia coli
CB1954	-	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
1.5	-	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	mutant enzyme F70A/F108Y, at pH 7.0 and 23°C	Escherichia coli
3.3	-	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	mutant enzyme F70A, at pH 7.0 and 23°C	Escherichia coli
3.7	-	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	mutant enzyme F108Y, at pH 7.0 and 23°C	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2,4,6-trinitrotoluene + NADPH + H+	Escherichia coli	-	4-hydroxyamino-2,6-dinitrotoluene + NADP+ + H2O	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P38489	-	-

Purification (Commentary)

Purification (Comment)	Organism
His-bind Ni-NTA column chromatography	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2,4,6-trinitrotoluene + NADPH + H+	-	Escherichia coli	4-hydroxyamino-2,6-dinitrotoluene + NADP+ + H2O	-	?
2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide + NADPH + H+	the wild type enzyme is largely inactive with 2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (SN33623) as a substrate	Escherichia coli	?	-	?
2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide + NADPH + H+	-	Escherichia coli	?	-	?
2-[(2-bromoethyl)(2-[(2-hydroxyethyl)carbamoyl]-4,6-dinitrophenyl)amino]ethyl methanesulfonate + NADPH + H+	-	Escherichia coli	?	-	?
5-(aziridin-1-yl)-2,4-dinitrobenzamide + NADPH + H+	-	Escherichia coli	?	-	?
methyl (6-nitro-4-oxoquinolin-1(4H)-yl)acetate + NADPH + H+	-	Escherichia coli	?	-	?
metronidazole + NADPH + H+	-	Escherichia coli	?	-	?
nitrofurazone + NADPH + H+	-	Escherichia coli	?	-	?
pimonidazole + NADPH + H+	-	Escherichia coli	?	-	?
tinidazole + NADPH + H+	-	Escherichia coli	?	-	?

Synonyms

Synonyms	Comment	Organism
NfsB	-	Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.19	-	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	mutant enzyme F70A, at pH 7.0 and 23°C	Escherichia coli
0.2	-	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	mutant enzyme F108Y, at pH 7.0 and 23°C	Escherichia coli
0.27	-	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	mutant enzyme F70A/F108Y, at pH 7.0 and 23°C	Escherichia coli

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor
0.038	-	mutant enzyme F70A/E72Q/F108Y, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.041	-	mutant enzyme F70A/F108Y, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.045	-	mutant enzyme F70A/F108Y, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.07	-	mutant enzyme T41L/F70A/F108Y, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.11	-	mutant enzyme F70A/F108Y, at pH 7.0 and 23°C	Escherichia coli	5-(aziridin-1-yl)-2,4-dinitrobenzamide
0.15	-	mutant enzyme F70A/E72Q, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.17	-	mutant enzyme T41L/F70A/N71S/F108Y, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.19	-	mutant enzyme F70A, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.19	-	mutant enzyme T41L/N71S, at pH 7.0 and 23°C	Escherichia coli	5-(aziridin-1-yl)-2,4-dinitrobenzamide
0.2	-	mutant enzyme T41L/F70A, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.2	-	mutant enzyme T41L/F70A/F108Y, at pH 7.0 and 23°C	Escherichia coli	5-(aziridin-1-yl)-2,4-dinitrobenzamide
0.23	-	mutant enzyme E72Q/F108Y, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.23	-	mutant enzyme T41L/F70A, at pH 7.0 and 23°C	Escherichia coli	5-(aziridin-1-yl)-2,4-dinitrobenzamide
0.25	-	mutant enzyme T41L/F70A/N71S, at pH 7.0 and 23°C	Escherichia coli	5-(aziridin-1-yl)-2,4-dinitrobenzamide
0.26	-	mutant enzyme F108Y, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.29	-	mutant enzyme T41L, at pH 7.0 and 23°C	Escherichia coli	5-(aziridin-1-yl)-2,4-dinitrobenzamide
0.31	-	mutant enzyme T41L/F70A/N71S, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.32	-	mutant enzyme T41L/F70A/N71S/F108Y, at pH 7.0 and 23°C	Escherichia coli	5-(aziridin-1-yl)-2,4-dinitrobenzamide
0.38	-	wild type enzyme, at pH 7.0 and 23°C	Escherichia coli	5-(aziridin-1-yl)-2,4-dinitrobenzamide
0.4	-	mutant enzyme E72Q, IC50 above 0.4 mM, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.4	-	mutant enzyme T41L, IC50 above 0.4 mM, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.4	-	mutant enzyme T41L/N71S, IC50 above 0.4 mM, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide
0.4	-	wild type enzyme, IC50 above 0.4 mM, at pH 7.0 and 23°C	Escherichia coli	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.055	-	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	mutant enzyme F108Y, at pH 7.0 and 23°C	Escherichia coli
0.058	-	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	mutant enzyme F70A, at pH 7.0 and 23°C	Escherichia coli
0.31	-	2-(2-methyl-5-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide	mutant enzyme F70A/F108Y, at pH 7.0 and 23°C	Escherichia coli