Literature summary for 1.18.1.2 extracted from

Perez-Amigot, D.; Taleb, V.; Boneta, S.; Anoz-Carbonell, E.; Sebastian, M.; Velazquez-Campoy, A.; Polo, V.; Martinez-Julvez, M.; Medina, M.
Towards the competent conformation for catalysis in the ferredoxin-NADP+ reductase from the Brucella ovis pathogen (2019), Biochim. Biophys. Acta, 1860, 148058.

Cloned(Commentary)

Cloned (Comment)	Organism
gene BOV-0348, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)	Brucella ovis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme free and in complex with NADP+, by either hanging-drop or sitting-drop vapour-diffusion method, mixing of 330 nl of 9.5 mg/ml protein in 25 mM Tris-HCl, 150 mM NaCl, pH 7.4, with 330 nl of reservoir solution containing 25% PEG 4000, 0.1 M MES, pH 6.5, and 0.2 mM MgCl2 for the free enzyme, or 2% PEG 400, 0.1 M HEPES, pH 7.5, and 2 M ammonium sulfate for the enzyme complex, followed by equilibration against 0.06 ml of reservoir solution, 18°C, X-ray diffraction structure determination and analysis at 1.40-1.69 A resolution	Brucella ovis

Crystallization (Comment)

Organism

purified recombinant enzyme free and in complex with NADP+, by either hanging-drop or sitting-drop vapour-diffusion method, mixing of 330 nl of 9.5 mg/ml protein in 25 mM Tris-HCl, 150 mM NaCl, pH 7.4, with 330 nl of reservoir solution containing 25% PEG 4000, 0.1 M MES, pH 6.5, and 0.2 mM MgCl2 for the free enzyme, or 2% PEG 400, 0.1 M HEPES, pH 7.5, and 2 M ammonium sulfate for the enzyme complex, followed by equilibration against 0.06 ml of reservoir solution, 18°C, X-ray diffraction structure determination and analysis at 1.40-1.69 A resolution

Brucella ovis

Inhibitors

Inhibitors	Comment	Organism
1-(4-chlorophenyl)-3-[5-(methylsulfanyl)-1,3,4-thiadiazol-2-yl]urea	64% inhibition at 0.370 mM	Brucella ovis
1-(4-fluorophenyl)-3-(5-[(2-oxopropyl)sulfanyl]-1,3,4-thiadiazol-2-yl)urea	25% inhibition at over 1.0 mM	Brucella ovis
1-[5-(4-chlorobenzylsulfanyl)-[1,3,4]thiadiazol-2-yl]-3-(4-chlorophenyl)urea	88% inhibition at 0.5 mM	Brucella ovis
2-[(5-([(4-chlorophenyl)carbamoyl]amino)-1,3,4-thiadiazol-2-yl)sulfanyl]acetic acid	poor inhibition	Brucella ovis
2-[(5-([(4-fluorophenyl)carbamoyl]amino)-1,3,4-thiadiazol-2-yl)sulfanyl]-N-methylacetamide	poor inhibition	Brucella ovis
NADPH	-	Brucella ovis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	Michaelis-Menten pre-steady-state kinetics and steady-state kinetics showing formation of a charge-transfer complex (CTC-1) prior to the hydride transfer (HT), as well as conversion of CTC-1 into a second charge-transfer complex (CTC-2) concomitantly with the HT event. Thus, during catalysis nicotinamide and flavin reacting rings stack. Kinetic data also identify the HT itself as the rate-limiting step in the reduction of BoFPR by NADPH, as well as product release limiting the overall reaction	Brucella ovis
0.0037	-	NADPH	pH 7.4, 25°C, recombinant enzyme	Brucella ovis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2 reduced ferredoxin + NADP+ + H+	Brucella ovis	-	2 oxidized ferredoxin + NADPH	-	r
2 reduced ferredoxin + NADP+ + H+	Brucella ovis ATCC 25840	-	2 oxidized ferredoxin + NADPH	-	r

Organism

Organism	UniProt	Comment	Textmining
Brucella ovis	-	-	-
Brucella ovis ATCC 25840	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Brucella ovis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2 oxidized ferredoxin + NADPH	-	Brucella ovis	2 reduced ferredoxin + NADP+ + H+	-	r
2 oxidized ferredoxin + NADPH	-	Brucella ovis ATCC 25840	2 reduced ferredoxin + NADP+ + H+	-	r
2 reduced ferredoxin + NADP+ + H+	-	Brucella ovis	2 oxidized ferredoxin + NADPH	-	r
2 reduced ferredoxin + NADP+ + H+	-	Brucella ovis ATCC 25840	2 oxidized ferredoxin + NADPH	-	r
additional information	analysis of NADP+/H interaction and NADPH oxidation by hydride transfer to enzyme BoFPR. The enzyme has neither oxidase nor superoxide reductase activities, it binds NADP+/H and exhibits NADPH oxidoreductase activity	Brucella ovis	?	-	?
additional information	analysis of NADP+/H interaction and NADPH oxidation by hydride transfer to enzyme BoFPR. The enzyme has neither oxidase nor superoxide reductase activities, it binds NADP+/H and exhibits NADPH oxidoreductase activity	Brucella ovis ATCC 25840	?	-	?

Subunits

Subunits	Comment	Organism
?	x * 30000, about, recombinant His-tagged enzyme SDS-PAGE	Brucella ovis

Synonyms

Synonyms	Comment	Organism
BoFPR	-	Brucella ovis
BOV-0348	-	Brucella ovis
ferredoxin-NADP+ reductase	-	Brucella ovis
FPR	-	Brucella ovis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Brucella ovis

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
additional information	-	evaluation of BoFPR enzyme thermal stability, kinetics, overview. The BoFPRox unfolding process of the FAD-binding and NADP+-binding domains are not fully cooperative, with the FAD domain being just slightly more stable, and the presence of NADP+ not having impact on the thermal stability of BoFPRox	Brucella ovis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
14.3	-	NADPH	pH 7.4, 25°C, recombinant enzyme	Brucella ovis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.4	-	-	Brucella ovis

Cofactor

Cofactor	Comment	Organism	Structure
FAD	thermal denaturation confirms that BoFPR has FAD as cofactor	Brucella ovis

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.052	-	NADPH	pH 7.4, 25°C, recombinant enzyme	Brucella ovis

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor
0.15	-	pH 7.4, 25°C, recombinant enzyme	Brucella ovis	1-(4-chlorophenyl)-3-[5-(methylsulfanyl)-1,3,4-thiadiazol-2-yl]urea
0.16	-	pH 7.4, 25°C, recombinant enzyme	Brucella ovis	1-[5-(4-chlorobenzylsulfanyl)-[1,3,4]thiadiazol-2-yl]-3-(4-chlorophenyl)urea
0.32	-	pH 7.4, 25°C, recombinant enzyme	Brucella ovis	2-[(5-([(4-fluorophenyl)carbamoyl]amino)-1,3,4-thiadiazol-2-yl)sulfanyl]-N-methylacetamide
0.43	-	pH 7.4, 25°C, recombinant enzyme	Brucella ovis	1-(4-fluorophenyl)-3-(5-[(2-oxopropyl)sulfanyl]-1,3,4-thiadiazol-2-yl)urea
1.9	-	pH 7.4, 25°C, recombinant enzyme	Brucella ovis	2-[(5-([(4-chlorophenyl)carbamoyl]amino)-1,3,4-thiadiazol-2-yl)sulfanyl]acetic acid

General Information

General Information	Comment	Organism
evolution	Brucella ovis encodes a bacterial subclass 1 ferredoxin-NADP(H) reductase (BoFPR), but BoFPR has the typical spectroscopic features of a member of the plant FNR family	Brucella ovis
additional information	all-atom molecular dynamics simulations indicate that the architecture of the FAD folded conformation in enzyme BoFPR might be key in catalysis, pointing to its adenine as an element to orient the reactive atoms in conformations competent for hydride transfer. Enzyme structure comparisons with other FPR enzymes, three-dimensional structure analysis and modeling, detailed overview	Brucella ovis