Literature summary for 1.18.1.2 extracted from

Faro, M.; Frago, S.; Mayoral, T.; Hermoso, J.A.; Sanz-Aparicio, J.; Gomez-Moreno, C.; Medina, M.
Probing the role of glutamic acid 139 of Anabaena ferredoxin-NADP+ reductase in the interaction with substrates (2002), Eur. J. Biochem., 269, 4938-4947.

Cloned(Commentary)

Cloned (Comment)	Organism
expression of wild-type and mutant enzymes	Anabaena sp.

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant mutant E139K, hanging drop vapour diffusion method, 0.002 ml protein solution containing 0.75 mM protein in 10 mM Tris-HCl, pH 8.0, plus 0.001 ml reservoir solution containing 5% w/v beta-octylglycoside, 18% PEG 6000, 20 mM ammonium sulfate, and 0.1 M MES/NaOH, pH 5.5, equilibration against 1 ml reservoir solution at 20°C, phase separation due to detergent, X-ray diffraction structure determination and analysis at 2.5 A resolution	Anabaena sp.

Crystallization (Comment)

Organism

purified recombinant mutant E139K, hanging drop vapour diffusion method, 0.002 ml protein solution containing 0.75 mM protein in 10 mM Tris-HCl, pH 8.0, plus 0.001 ml reservoir solution containing 5% w/v beta-octylglycoside, 18% PEG 6000, 20 mM ammonium sulfate, and 0.1 M MES/NaOH, pH 5.5, equilibration against 1 ml reservoir solution at 20°C, phase separation due to detergent, X-ray diffraction structure determination and analysis at 2.5 A resolution

Anabaena sp.

Protein Variants

Protein Variants	Comment	Organism
E139D	site-directed mutagenesis, altered conformation compared to the wild-type enzyme, slightly reduced activity compared to the wild-type enzyme	Anabaena sp.
E139K	site-directed mutagenesis, mutant enzyme shows increased interaction with ferredoxin and reduces the appropriate orientation of flavodoxin, altered conformation compared to the wild-type enzyme, increased activity compared to the wild-type enzyme	Anabaena sp.
E139Q	site-directed mutagenesis, mutant enzyme shows increased interaction with ferredoxin and reduces the appropriate orientation of flavodoxin, altered conformation compared to the wild-type enzyme, increased activity compared to the wild-type enzyme	Anabaena sp.

Inhibitors

Inhibitors	Comment	Organism	Structure
additional information	activity decreases with elevated ionic strength	Anabaena sp.

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	stopped-flow kinetics measurements, pH 8.0, 13°C, steady-state kinetics for wild-type and mutant enzymes dependent on ionic strength, overview	Anabaena sp.
0.00027	-	oxidized ferredoxin	recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
0.0005	-	oxidized ferredoxin	recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 100 mM	Anabaena sp.
0.0009	-	oxidized ferredoxin	recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 200 mM	Anabaena sp.
0.0025	-	oxidized ferredoxin	recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 100 mM	Anabaena sp.
0.0034	-	NADPH	recombinant mutant E139Q, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
0.0035	-	oxidized ferredoxin	recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 200 mM	Anabaena sp.
0.0043	-	oxidized ferredoxin	recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
0.0047	-	NADPH	recombinant mutant E139D, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
0.0058	-	NADPH	recombinant mutant E139K, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
0.006	-	NADPH	recombinant wild-type enzyme, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
0.02	-	oxidized ferredoxin	recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 100 mM	Anabaena sp.
0.021	-	oxidized ferredoxin	recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 200 mM	Anabaena sp.
0.023	-	oxidized ferredoxin	recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 100 mM	Anabaena sp.
0.023	-	oxidized ferredoxin	recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
0.1	-	oxidized ferredoxin	recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
0.4	-	oxidized ferredoxin	recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 200 mM	Anabaena sp.

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
reduced ferredoxin + NADP+	Anabaena sp.	-	oxidized ferredoxin + NADPH	-	r

Organism

Organism	UniProt	Comment	Textmining
Anabaena sp.	-	-	-

Reaction

Reaction	Comment	Organism	Reaction ID
2 reduced ferredoxin + NADP+ + H+ = 2 oxidized ferredoxin + NADPH	Glu139 is essential for interaction with the substrate steering ferredoxin, flavodoxin and NADP+/NADPH in appropriate position for docking, Glu139 is not involved in binding	Anabaena sp.	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	NADPH-dependent cytochrome c reductase assay for determination of activity with ferredoxin or flavodoxin as electron carrier	Anabaena sp.	?	-	?
oxidized 2,6-dichlorophenolindophenol + NADP+	diaphorase activity	Anabaena sp.	reduced 2,6-dichlorophenolindophenol + NADPH	-	?
oxidized ferredoxin + NADPH + H+	-	Anabaena sp.	reduced ferredoxin + NADP+	-	r
reduced ferredoxin + NADP+	-	Anabaena sp.	oxidized ferredoxin + NADPH	-	r
reduced ferredoxin + NADP+	hydride transfer of the N5 of the FAD isoalloxazine ring to the NADP+ nicotinamide ring, transfer of 2 electrons via the one-electron-carrier ferredoxin	Anabaena sp.	oxidized ferredoxin + NADPH	-	r
reduced ferredoxin + NADP+	-	Anabaena sp.	oxidized ferredoxin + NADPH + H+	-	r
reduced flavodoxin + NADP+	-	Anabaena sp.	oxidized flavodoxin + NADPH + H+	-	r

Subunits

Subunits	Comment	Organism
More	three-dimensional structure of mutant E139K	Anabaena sp.

Synonyms

Synonyms	Comment	Organism
ferredoxin-NADP+ reductase	-	Anabaena sp.
FNR	-	Anabaena sp.

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Anabaena sp.

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
2	8	reduced flavodoxin	recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 200 mM	Anabaena sp.
14	-	reduced flavodoxin	recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 200 mM	Anabaena sp.
17	-	reduced flavodoxin	recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
19	-	reduced flavodoxin	recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 100 mM	Anabaena sp.
20	-	reduced flavodoxin	recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 200 mM	Anabaena sp.
24	-	reduced flavodoxin	recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
25	-	reduced flavodoxin	recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 100 mM	Anabaena sp.
25	-	reduced flavodoxin	recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
26	-	reduced flavodoxin	recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 100 mM	Anabaena sp.
38	-	reduced flavodoxin	recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
58	-	reduced ferredoxin	recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 100 mM	Anabaena sp.
59	-	NADPH	recombinant mutant E139K, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
70	-	reduced ferredoxin	recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 200 mM	Anabaena sp.
81	-	NADPH	recombinant wild-type enzyme, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
88	-	NADPH	recombinant mutant E139Q, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
89	-	NADPH	recombinant mutant E139D, diaphorase activity with 2,6-dichlorophenolindophenol, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
117	-	reduced ferredoxin	recombinant mutant E139Q, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
120	-	reduced ferredoxin	recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 200 mM	Anabaena sp.
135	-	reduced ferredoxin	recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 200 mM	Anabaena sp.
148	-	reduced ferredoxin	recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 200 mM	Anabaena sp.
155	-	reduced ferredoxin	recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 100 mM	Anabaena sp.
176	-	reduced ferredoxin	recombinant mutant E139K, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
192	-	reduced ferredoxin	recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 100 mM	Anabaena sp.
209	-	reduced ferredoxin	recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 100 mM	Anabaena sp.
225	-	reduced ferredoxin	recombinant wild-type enzyme, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.
280	-	reduced ferredoxin	recombinant mutant E139D, pH 8.0, 25°C, ionic strength of 28 mM	Anabaena sp.

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Anabaena sp.

Cofactor

Cofactor	Comment	Organism	Structure
FAD	flavoenzyme	Anabaena sp.