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Literature summary for 1.18.1.2 extracted from

  • Tejero, J.; Martinez-Julvez, M.; Mayoral, T.; Luquita, A.; Sanz-Aparicio, J.; Hermoso, J.A.; Hurley, J.K.; Tollin, G.; Gomez-Moreno, C.; Medina, M.
    Involvement of the pyrophosphate and the 2'-phosphate binding regions of ferredoxin-NADP+ reductase in coenzyme specificity (2003), J. Biol. Chem., 278, 49203-49214.
    View publication on PubMed
Search for more literature for 1.18.1.2

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutant enzymes in Escherichia coli strain 0225 and BL21(DE3) Anabaena sp.

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant mutant enzymes L263P, T155G/A160T, and T155G/A160T/L263P, hanging drop method, 0.002 ml of protein solution, containing 0.75 mM protein, 10 mM Tris-HCl, pH 8.0, plus 0.001 ml of unbuffered 5% w/v beta-octyl-glycoside solution, plus 0.002 ml reservoir solution, containing 18-20% w/v PEG 6000, 20 mM ammonium sulfate, 0.1 M MES-NaOH, pH 5.0, equilibration against 1 ml reservoir solution at 20°C, 1-7 days, phase separation caused by detergent, X-ray diffraction structure determination and analysis at 1.63-2.15 Anabaena sp.

Protein Variants

Protein Variants Comment Organism
L263A site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
L263P site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
R224Q/R233L/Y235F site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
R233L/Y235F site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
S223D/R224Q/R233L/Y235F site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
S223D/R233L/Y235F site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
T155G site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
T155G/A160T site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
T155G/A160T/L263P site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
T155G/A160T/S223D/R224Q/R233L/Y235F site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
T155G/A160T/S223D/R224Q/R233L/Y235F/L263P site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
T155G/R224Q/R233L/Y235F site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.
T155G/S223D/R224Q/R233L/Y235F site-directed mutagenesis, altered cofactor specificity compared to the wild-type enzyme Anabaena sp.

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics, wild-type and mutant enzymes Anabaena sp.
0.006
-
 NADPH wild-type enzyme, pH 8.0 Anabaena sp.
0.012
-
 NADPH mutant T155G/A160T/L263P, pH 8.0 Anabaena sp.
0.015
-
 NADPH mutant L263A, pH 8.0 Anabaena sp.
0.019
-
 NADPH mutant L263P, pH 8.0 Anabaena sp.
0.022
-
 NADPH mutant T155G/A160T, pH 8.0 Anabaena sp.
0.023
-
 NADPH mutant T155G, pH 8.0 Anabaena sp.
0.18
-
 NADH mutant T155G, pH 8.0 Anabaena sp.
0.39
-
 NADH mutant T155G/A160T/L263P, pH 8.0 Anabaena sp.
0.51
-
 NADH mutant T155G/A160T, pH 8.0 Anabaena sp.
0.63
-
 NADH mutant L263A, pH 8.0 Anabaena sp.
0.65
-
 NADH mutant L263P, pH 8.0 Anabaena sp.
0.8
-
 NADH wild-type enzyme, pH 8.0 Anabaena sp.
1.5
-
 NADH mutant S223D/R233L/Y235F, pH 8.0 Anabaena sp.
1.7
-
 NADPH mutant R233L/Y235F, pH 8.0 Anabaena sp.
1.8
-
 NADPH above, mutant T155G/A160T/S223D/R224Q/R233L/Y235F/L263P, pH 8.0 Anabaena sp.
2.3
-
 NADH mutant R233L/Y235F, pH 8.0 Anabaena sp.
2.7
-
 NADPH mutant T155G/R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
3
-
 NADH mutant T155G/R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
3.6
-
 NADPH mutant R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
4.3
-
 NADH mutant R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
12
-
 NADH mutant T155G/A160T/S223D/R224Q/R233L/Y235F/L263P, pH 8.0 Anabaena sp.

Organism

Organism UniProt Comment Textmining
Anabaena sp. P21890
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli Anabaena sp.

Reaction

Reaction Comment Organism Reaction ID
2 reduced ferredoxin + NADP+ + H+ = 2 oxidized ferredoxin + NADPH coenzyme recognition and reaction mechanism Anabaena sp.
a

Source Tissue

Source Tissue Comment Organism Textmining

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 activity of wild-type and mutant enzymes with different cofactors Anabaena sp.

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information coenzyme binding causes structural rearrangements of the protein backbone Anabaena sp. ?
-
 ?
NAD(P)H + H+ + oxidized 2,6-dichlorophenolindophenol diaphorase activity, cofactor specificity, overview Anabaena sp. NAD(P)+ + reduced 2,6-dichlorophenolindophenol
-
 ?
oxidized 2,6-dichlorophenolindophenol + NADH
-
 Anabaena sp. NAD+ + reduced 2,6-dichlorophenolindophenol
-
 ?

Synonyms

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

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.02
-
 NADH mutant T155G, pH 8.0 Anabaena sp.
0.02
-
 NADPH mutant T155G/A160T/S223D/R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
0.03
-
 NADPH mutant T155G/A160T/S223D/R224Q/R233L/Y235F/L263P, pH 8.0 Anabaena sp.
0.04
-
 NADH mutant S223D/R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
0.04
-
 NADH mutant T155G/A160T/S223D/R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
0.05
-
 NADH mutant L263P, pH 8.0 Anabaena sp.
0.05
-
 NADPH mutant S223D/R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
0.06
-
 NADPH mutant T155G/S223D/R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
0.06
-
 NADH mutant T155G/S223D/R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
0.07
-
 NADH mutant T155G/A160T, pH 8.0 Anabaena sp.
0.1
-
 NADH mutant S223D/R233L/Y235F, pH 8.0 Anabaena sp.
0.13
-
 NADH mutant L263A, pH 8.0 Anabaena sp.
0.16
-
 NADH wild-type enzyme, pH 8.0 Anabaena sp.
0.2
-
 NADPH mutant T155G/R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
0.28
-
 NADH mutant T155G/R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
0.33
-
 NADH mutant T155G/A160T/L263P, pH 8.0 Anabaena sp.
0.86
-
 NADH mutant R233L/Y235F, pH 8.0 Anabaena sp.
1.2
-
 NADH mutant R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
1.3
-
 NADPH mutant R224Q/R233L/Y235F, pH 8.0 Anabaena sp.
2.1
-
 NADH mutant T155G/A160T/S223D/R224Q/R233L/Y235F/L263P, pH 8.0 Anabaena sp.
17
-
 NADPH mutant L263P, pH 8.0 Anabaena sp.
22
-
 NADPH mutant R233L/Y235F, pH 8.0 Anabaena sp.
60
-
 NADPH mutant L263A, pH 8.0 Anabaena sp.
72
-
 NADPH mutant T155G/A160T, pH 8.0 Anabaena sp.
77
-
 NADPH mutant T155G/A160T/L263P, pH 8.0 Anabaena sp.
81.5
-
 NADPH wild-type enzyme, pH 8.0 Anabaena sp.
97
-
 NADPH mutant T155G, pH 8.0 Anabaena sp.

pH Optimum

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

Cofactor

Cofactor Comment Organism Structure
additional information the coenzyme specificity determining structures are located in the 2'-phosphate NADP+ and pyrophosphate binding region of amino acid residues 155-160, 261-268, and S233, R224, R233, and Y235, coenzyme binding causes structural rearrangements of the protein backbone, binding and interaction mechanism with the enzyme, overview Anabaena sp.
NADH diaphorase activity Anabaena sp.
NADPH diaphorase activity Anabaena sp.