Information on EC 1.18.1.5 - putidaredoxin-NAD+ reductase

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The expected taxonomic range for this enzyme is: Pseudomonas putida

EC NUMBER
COMMENTARY hide
1.18.1.5
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RECOMMENDED NAME
GeneOntology No.
putidaredoxin-NAD+ reductase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
reduced putidaredoxin + NAD+ = oxidized putidaredoxin + NADH + H+
show the reaction diagram
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SYSTEMATIC NAME
IUBMB Comments
putidaredoxin:NAD+ oxidoreductase
Requires FAD. The enzyme from Pseudomonas putida reduces putidaredoxin. It contains a [2Fe-2S] cluster. Involved in the camphor monooxygenase system (see EC 1.14.15.1, camphor 5-monooxygenase).
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2 ferricytochrome c + NADH
2 ferrocytochrome c + NAD+ + H+
show the reaction diagram
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-
-
-
?
Fe(CN)62- + NAD+
Fe(CN)63- + NADH + H+
show the reaction diagram
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-
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NADH + H+ + Fe(CN)63-
NAD+ + Fe(CN)62-
show the reaction diagram
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-
-
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?
NADH + H+ + oxidized 2,6-dichlorophenolindophenol
NAD+ + reduced 2,6-dichlorophenolindophenol
show the reaction diagram
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-
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?
putidaredoxin + NADH
?
show the reaction diagram
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?
reduced 2,6-dichlorophenolindophenol + NAD+
oxidized 2,6-dichlorophenolindophenol + NADH + H+
show the reaction diagram
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reduced putidaredoxin + ferricytochrome c
oxidized putidaredoxin + ferrocytochrome c
show the reaction diagram
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the physiological electron acceptor, putidaredoxin, can be used to transfer electrons to ferri-cytochrome c in a putidaredoxin-dependent cytochrome c reductase assay
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?
reduced putidaredoxin + NAD+
oxidized putidaredoxin + NADH + H+
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
iron-sulfur centre
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[2Fe-2S] cluster
NADH
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dependent
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.036 - 0.143
Fe(CN)63-
0.017 - 0.05
NADH
0.029 - 0.2
oxidized 2,6-dichlorophenolindolphenol
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
126 - 671
Fe(CN)62-
543 - 1485
Fe(CN)63-
650 - 820
NADH
203 - 617
oxidized 2,6-dichlorophenolindolphenol
264
reduced putidaredoxin
Pseudomonas putida
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pH 8.0, 25C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
34.4
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pH 8.0, temperature not specified in the publication
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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both WT and His6-Pdr undergo a monomer-dimer association-dissociation
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
computational modeling based on crystal strucutures of putidaredoxin Ptxand putidaredoxin reductase PdR. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and [2Fe-2S] of 14.6 A. This mode of interaction allows Pdx to easily adjust and optimize orientation of its cofactor relative to Pdr. The key residues of Pdx located at the center are Asp38 and Trp106, and at the edge of the protein-protein interface are Tyr33 and Arg66
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crystal structure of a covalently linked putidaredoxin reductase (Pdr)-putidaredoxin (Pdx) complex. Residues R65 and R310 are the key elements required for the formation of a productive electron transfer complex with Pdx. The C-terminal lysine cluster assists in Pdx docking by fine-tuning Pdr-Pdx interactions to achieve the optimal geometry between the redox centers, and the basic surface residues in Pdr-like ferredoxin reductases not only define specificity for the redox partner but also may facilitate its dissociation
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crystal structures of C73S/C85S and C73S mutants, to 1.47 A and 1.65 A resolution, respectively, are nearly identical and very similar to those of bovine adrenodoxin and Escherichia coli ferredoxin. In particular, formation of a hydrogen bond between the side-chain of Y51 and the carbonyl oxygen atom of E77 and the presence of two well-ordered water molecules linking the interaction domain and the C-terminal peptide to the core of the molecule are unique to Pdx. The folding topology of the NMR model is similar to that of the X-ray structure of Pdx. W106, important in the Pdr-to-Pdx and Pdx-to-P450cam electron transfer reactions, is in a position to regulate and/or mediate electron transfer to or from the [2Fe2S] center of Pdx
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mutant C73G, to 1.9 A resolution. The C2 crystal contains three putidaredoxin molecules in the asymmetric unit. Findings show a unanimous structure in some regions crucial for electron-transfer interactions, including the cluster-binding loop 39-48 and the cytochrome-interaction region of Asp38 and Trp106. In addition, the Cys45 amide group donates a hydrogen bond to cluster sulfur S1, with Ala46 adopting an Lalpha conformation
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sitting drop vapour diffusion method with 1.5 M lithium sulfate, 0.15 M lithium acetate, 0.1 M lithium formate, 2% (v/v) glycerol, 1 mM dithiothreitol, and 0.1 M Bis-Tris-propane (pH 8.0)
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
simple purification procedure
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the genes responsible for early steps of D-camphor degradation, i.e. 5-exo-hydroxycamphor dehydrogenase, camD gene, cytochrome P-450cam, camC, NADH-putidaredoxin reductase camA, and putidaredoxin camB form an operon, camDCAB, and are under negative control by the gene camR located immediately upstream from the camD gene
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C73G
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surface mutation facilitating crystallization without affecting cluster ligation and with only minor effects on activity
C73S
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mutation improves protein stability. Decreasing order of stability is C73S/C85S, C73S, C85S, wild-type Pdx
C73S/C85S
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mutation improves protein stability. Decreasing order of stability is C73S/C85S, C73S, C85S, wild-type Pdx
C85S
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mutation improves protein stability. Decreasing order of stability is C73S/C85S, C73S, C85S, wild-type Pdx
D38A
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mutation does not affect assembly of the [2Fe-2S] cluster and results in a marginal change in the redox potential of Pdx. 45% of wild-type activity
D38N
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mutation does not affect assembly of the [2Fe-2S] cluster and results in a marginal change in the redox potential of Pdx. 33% of wild-type activity
K339A
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moderate decrease in the binding affinity and reduction of Pdx
K387A
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moderate decrease in the binding affinity and reduction of Pdx
K409A
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moderate decrease in the binding affinity and reduction of Pdx
N384A
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mutation has no effect on the Pdr-Pdx interaction
R310A
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mutation lowers the interprotein electron tranfer rate by 20-30fold without perturbing the Pdx association step
R310E
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mutation decreases both the Pdr-to-Pdx ET and partner binding affinity by 100- and 8fold, respectively
R65A
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mutation lowers the interprotein electron tranfer rate by 20-30fold without perturbing the Pdx association step
R66A
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mutation does not affect assembly of the [2Fe-2S] cluster and results in a marginal change in the redox potential of Pdx. 25% of wild-type activity
R66E
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mutation does not affect assembly of the [2Fe-2S] cluster and results in a marginal change in the redox potential of Pdx. 21% of wild-type activity
W106A
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mutation does not affect assembly of the [2Fe-2S] cluster and results in a marginal change in the redox potential of Pdx. 54% of wild-type activity
W106Delta
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mutation does not affect assembly of the [2Fe-2S] cluster and results in a marginal change in the redox potential of Pdx. 102% of wild-type activity
W106F
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mutation does not affect assembly of the [2Fe-2S] cluster and results in a marginal change in the redox potential of Pdx. 83% of wild-type activity
Y33A
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mutation does not affect assembly of the [2Fe-2S] cluster and results in a marginal change in the redox potential of Pdx. 26% of wild-type activity
Y33F
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mutation does not affect assembly of the [2Fe-2S] cluster and results in a marginal change in the redox potential of Pdx. 21% of wild-type activity
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis