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Literature summary for 1.18.1.2 extracted from

  • Musumeci, M.A.; Botti, H.; Buschiazzo, A.; Ceccarelli, E.A.
    Swapping FAD binding motifs between plastidic and bacterial ferredoxin-NADP(H) reductases (2011), Biochemistry, 50, 2111-2122.
    View publication on PubMed
Search for more literature for 1.18.1.2

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Escherichia coli
expression in Escherichia coli Pisum sativum

Crystallization (Commentary)

Crystallization (Comment) Organism
mutual exchange of the 112-123 beta-hairpin from Pisum sativum plastidic ferredoxinNAD(P)H reductase and the carboxy-terminal tryptophan of he Escherichia coli enzyme. Crystallographic structures of the chimeras show no significant changes in their overall structure, although alterations in the FAD conformations are observed Escherichia coli
mutual exchange of the 112-123 beta-hairpin from Pisum sativum plastidic ferredoxinNAD(P)H reductase and the carboxy-terminal tryptophan of he Escherichia coli enzyme. Crystallographic structures of the chimeras show no significant changes in their overall structure, although alterations in the FAD conformations are observed Pisum sativum

Protein Variants

Protein Variants Comment Organism
additional information mutual exchange of the 112-123 beta-hairpin from Pisum sativum plastidic ferredoxinNAD(P)H reductase and the carboxy-terminal tryptophan of he Escherichia coli enzyme. The plastidic enzyme lacking the beta-hairpin is unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial enzyme, results in an enzyme with decreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the beta-hairpin into the corresponding position of the bacterial enzyme increases FAD affinity but does not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxyterminal tryptophan produces a bacterial chimera emulating the plastidic architecture with an increased kcat and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme's ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras show no significant changes in their overall structure, although alterations in the FAD conformations are observed Pisum sativum
additional information mutual exchange of the 112-123 beta-hairpin from Pisum sativum plastidic ferredoxinNAD(P)H reductase and the carboxy-terminal tryptophan of he Escherichia coli enzyme. The plastidic enzyme lacking the beta-hairpin is unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial enzyme, results in an enzyme with decreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the beta-hairpin into the corresponding position of the bacterial enzyme increases FAD affinity but does not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxyterminal tryptophan produces a bacterial chimera emulating the plastidic architecture with an increased kcat and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme’s ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras show no significant changes in their overall structure, although alterations in the FAD conformations are observed Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0009
-
 oxidized ferredoxin mutant lacking the beta-hairpin, pH 8.0, 30°C Pisum sativum
0.0009
-
 oxidized ferredoxin mutant with additional tryptophan residue at the C-terminus, pH 8.0, 30°C Pisum sativum
0.0014
-
 oxidized ferredoxin wild-type, pH 8.0, 30°C Escherichia coli
0.0018
-
 oxidized ferredoxin mutant with an insertion of the iloop of Pisum sativum enzyme, pH 8.0, 30°C Escherichia coli
0.0022
-
 oxidized ferredoxin wild-type, pH 8.0, 30°C Pisum sativum
0.0024
-
 NADPH mutant DELTAW248, pH 8.0, 30°C Escherichia coli
0.003
-
 NADPH mutant with an insertion of the iloop of Pisum sativum enzyme plus deletion of residue W248, pH 8.0, 30°C Escherichia coli
0.003
-
 NADH mutant with an insertion of the iloop of Pisum sativum enzyme plus deletion of residue W248, pH 8.0, 30°C Escherichia coli
0.0036
-
 NADPH mutant with an insertion of the iloop of Pisum sativum enzyme, pH 8.0, 30°C Escherichia coli
0.0037
-
 NADH mutant lacking the beta-hairpin, pH 8.0, 30°C Pisum sativum
0.0059
-
 NADH mutant with additional tryptophan residue at the C-terminus, pH 8.0, 30°C Pisum sativum
0.0062
-
 oxidized ferredoxin mutant DELTAW248, pH 8.0, 30°C Escherichia coli
0.0065
-
 NADH mutant DELTAW248, pH 8.0, 30°C Escherichia coli
0.0076
-
 oxidized ferredoxin mutant with an insertion of the iloop of Pisum sativum enzyme plus deletion of residue W248, pH 8.0, 30°C Escherichia coli
0.0083
-
 NADPH wild-type, pH 8.0, 30°C Escherichia coli
0.0143
-
 NADH wild-type, pH 8.0, 30°C Pisum sativum
0.0147
-
 NADPH mutant lacking the beta-hairpin, pH 8.0, 30°C Pisum sativum
0.0153
-
 NADPH wild-type, pH 8.0, 30°C Pisum sativum
0.0207
-
 NADPH mutant with additional tryptophan residue at the C-terminus, pH 8.0, 30°C Pisum sativum

Localization

Localization Comment Organism GeneOntology No. Textmining
plastid
-
 Pisum sativum 9536
-

Organism

Organism UniProt Comment Textmining
Escherichia coli P28861
-
-
Pisum sativum P10933
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 ferricytochrome c2 + NADPH
-
 Escherichia coli 2 ferrocytochrome c2 + NADP+ + H+
-
 ?
2 ferricytochrome c2 + NADPH
-
 Pisum sativum 2 ferrocytochrome c2 + NADP+ + H+
-
 ?
2 ferrocytochrome c + NAD+ + H+ NADH, poor substrate Escherichia coli 2 ferricytochrome c + NADH
-
 ?
2 ferrocytochrome c + NAD+ + H+ NADH, poor substrate Pisum sativum 2 ferricytochrome c + NADH
-
 ?
2 oxidized ferredoxin + NADH + H+ NADH, poor substrate Escherichia coli 2 reduced ferredoxin + NAD+
-
 ?
2 oxidized ferredoxin + NADH + H+ NADH, poor substrate Pisum sativum 2 reduced ferredoxin + NAD+
-
 ?
2 oxidized ferredoxin + NADPH + H+ source of ferredoxin: Pisum sativum Escherichia coli 2 reduced ferredoxin + NADP+
-
 ?
2 oxidized ferredoxin + NADPH + H+ source of ferredoxin: Pisum sativum Pisum sativum 2 reduced ferredoxin + NADP+
-
 ?

Synonyms

Synonyms Comment Organism
FNR
-
 Escherichia coli
PETH
-
 Pisum sativum

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2
-
 NADH mutant lacking the beta-hairpin, pH 8.0, 30°C Pisum sativum
3.7
-
 oxidized ferredoxin mutant with an insertion of the iloop of Pisum sativum enzyme plus deletion of residue W248, pH 8.0, 30°C Escherichia coli
4.6
-
 oxidized ferredoxin mutant DELTAW248, pH 8.0, 30°C Escherichia coli
5
-
 NADH mutant with additional tryptophan residue at the C-terminus, pH 8.0, 30°C Pisum sativum
7
-
 NADH wild-type, pH 8.0, 30°C Pisum sativum
19.4
-
 oxidized ferredoxin mutant with an insertion of the iloop of Pisum sativum enzyme, pH 8.0, 30°C Escherichia coli
22.8
-
 oxidized ferredoxin wild-type, pH 8.0, 30°C Escherichia coli
24.5
-
 NADPH mutant with an insertion of the iloop of Pisum sativum enzyme, pH 8.0, 30°C Escherichia coli
32.7
-
 oxidized ferredoxin mutant lacking the beta-hairpin, pH 8.0, 30°C Pisum sativum
35.9
-
 oxidized ferredoxin mutant with additional tryptophan residue at the C-terminus, pH 8.0, 30°C Pisum sativum
38.2
-
 NADPH wild-type, pH 8.0, 30°C Escherichia coli
45.3
-
 NADH mutant with an insertion of the iloop of Pisum sativum enzyme plus deletion of residue W248, pH 8.0, 30°C Escherichia coli
54.1
-
 NADH mutant DELTAW248, pH 8.0, 30°C Escherichia coli
75
-
 oxidized ferredoxin wild-type, pH 8.0, 30°C Pisum sativum
95.5
-
 NADPH mutant DELTAW248, pH 8.0, 30°C Escherichia coli
99
-
 NADPH mutant with an insertion of the iloop of Pisum sativum enzyme plus deletion of residue W248, pH 8.0, 30°C Escherichia coli
251.1
-
 NADPH mutant lacking the beta-hairpin, pH 8.0, 30°C Pisum sativum
360.5
-
 NADPH mutant with additional tryptophan residue at the C-terminus, pH 8.0, 30°C Pisum sativum
374.3
-
 NADPH wild-type, pH 8.0, 30°C Pisum sativum

Cofactor

Cofactor Comment Organism Structure
NADH ratio NADPH/NADH is 49000 Escherichia coli
NADH ratio NADPH/NADH is 49000 Pisum sativum
NADPH ratio NADPH/NADH is 49000 Escherichia coli
NADPH ratio NADPH/NADH is 49000 Pisum sativum

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.5
-
 NADH wild-type, pH 8.0, 30°C Pisum sativum
0.6
-
 NADH mutant lacking the beta-hairpin, pH 8.0, 30°C Pisum sativum
0.8
-
 NADH mutant with additional tryptophan residue at the C-terminus, pH 8.0, 30°C Pisum sativum
8
-
 NADH mutant DELTAW248, pH 8.0, 30°C Escherichia coli
15
-
 NADH mutant with an insertion of the iloop of Pisum sativum enzyme plus deletion of residue W248, pH 8.0, 30°C Escherichia coli
500
-
 oxidized ferredoxin mutant with an insertion of the iloop of Pisum sativum enzyme plus deletion of residue W248, pH 8.0, 30°C Escherichia coli
740
-
 oxidized ferredoxin mutant DELTAW248, pH 8.0, 30°C Escherichia coli
1700
-
 NADPH mutant with additional tryptophan residue at the C-terminus, pH 8.0, 30°C Pisum sativum
4600
-
 NADPH wild-type, pH 8.0, 30°C Escherichia coli
6800
-
 NADPH mutant with an insertion of the iloop of Pisum sativum enzyme, pH 8.0, 30°C Escherichia coli
10700
-
 oxidized ferredoxin mutant with an insertion of the iloop of Pisum sativum enzyme, pH 8.0, 30°C Escherichia coli
16300
-
 oxidized ferredoxin wild-type, pH 8.0, 30°C Escherichia coli
17100
-
 NADPH mutant lacking the beta-hairpin, pH 8.0, 30°C Pisum sativum
24500
-
 NADPH wild-type, pH 8.0, 30°C Pisum sativum
33000
-
 NADPH mutant with an insertion of the iloop of Pisum sativum enzyme plus deletion of residue W248, pH 8.0, 30°C Escherichia coli
34100
-
 oxidized ferredoxin wild-type, pH 8.0, 30°C Pisum sativum
36300
-
 oxidized ferredoxin mutant lacking the beta-hairpin, pH 8.0, 30°C Pisum sativum
39800
-
 NADPH mutant DELTAW248, pH 8.0, 30°C Escherichia coli
39800
-
 oxidized ferredoxin mutant with additional tryptophan residue at the C-terminus, pH 8.0, 30°C Pisum sativum