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Information on EC 1.18.1.5 - putidaredoxin-NAD+ reductase and Organism(s) Pseudomonas putida and UniProt Accession P16640
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1.18.1.5 putidaredoxin-NAD+ reductaseIUBMB Comments
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).
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The taxonomic range for the selected organisms is: Pseudomonas putida
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
reduced putidaredoxin
+
=
+
+
Synonyms
cama, putidaredoxin reductase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
putidaredoxin reductase
camA
-
-
-
-
putidaredoxin reductase
-
-
-
-
putidaredoxin reductase
component of the soluble cytochrome P450cam-dependent monooxygenase system
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).
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
NADH + H+ + oxidized 2,6-dichlorophenolindophenol
NAD+ + reduced 2,6-dichlorophenolindophenol
-
-
-
?
reduced 2,6-dichlorophenolindophenol + NAD+
oxidized 2,6-dichlorophenolindophenol + NADH + H+
-
-
-
?
reduced putidaredoxin + ferricytochrome c
oxidized putidaredoxin + ferrocytochrome c
-
the physiological electron acceptor, putidaredoxin, can be used to transfer electrons to ferri-cytochrome c in a putidaredoxin-dependent cytochrome c reductase assay
-
-
?
reduced putidaredoxin + NAD+
oxidized putidaredoxin + NADH + H+
-
-
-
-
?
reduced putidaredoxin + NAD+
oxidized putidaredoxin + NADH + H+
-
-
-
?
additional information
?
-
bulky side chains of Tyr33, Arg66, and Trp106 prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr. Transfer of an electron from FAD to [2Fe-2S] can occur with various orientations between the cofactors through multiple electron transfer pathways that do not involve Trp106 but are likely to include Asp38 and Cys39
-
-
?
additional information
?
-
-
bulky side chains of Tyr33, Arg66, and Trp106 prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr. Transfer of an electron from FAD to [2Fe-2S] can occur with various orientations between the cofactors through multiple electron transfer pathways that do not involve Trp106 but are likely to include Asp38 and Cys39
-
-
?
additional information
?
-
the midpoint oxidation-reduction potential of PdR is -369 mV at pH 7.6, which is more negative than the pyridine nucleotide NADH/NAD+. The midpoint potential is a hyperbolic function of increasing NAD+ concentration, such that at concentrations of pyridine nucleotide typically found in an intracellular environment, the midpoint potential would be -230 mV, thereby providing the thermodynamically favorable redox equilibria that enables electron transfer from NADH, with thermodynamic control of electron transfer. The PdRox:NAD+ complex is about 5 orders of magnitude weaker than PdRrd:NAD+ binding. These results support a compulsory ordered pathway to describe the electron-transfer processes
-
-
?
additional information
?
-
wild-type and His6 Pdr are able to function as NAD(H)-dependent dithiol/disulfide oxidoreductases catalyzing both forward and reverse reactions, NAD+-dependent oxidation of thiols, and NADH-dependent reduction of disulfides. This function of the flavoprotein can be dissociated from electron transfer to putidaredoxin
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-
?
additional information
?
-
-
wild-type and His6 Pdr are able to function as NAD(H)-dependent dithiol/disulfide oxidoreductases catalyzing both forward and reverse reactions, NAD+-dependent oxidation of thiols, and NADH-dependent reduction of disulfides. This function of the flavoprotein can be dissociated from electron transfer to putidaredoxin
-
-
?
additional information
?
-
-
reductase is a two-eleetron acceptor with no stable semiquinone intermediate being formed either during reduction or air reoxidation
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
reduced putidaredoxin + NAD+
oxidized putidaredoxin + NADH + H+
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NAD+
both WT and His6-Pdr are partially present as an NAD+-bound form
FAD
-
1 mol FAD per mol of protein. Charge-transfer bands indicate a close association of the pyridine nucleotide and the reduced flavin
NAD+
-
charge-transfer bands indicate a close association of the pyridine nucleotide and the reduced flavin
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.017
NADH
mutant R65A, pH 7.5, 25°C, cosubstrate dichlorophenolindophenol
0.018
NADH
mutant R310A, pH 7.5, 25°C, cosubstrate dichlorophenolindophenol
0.022
NADH
mutant R339A, pH 7.5, 25°C, cosubstrate dichlorophenolindophenol
0.027
NADH
wild-type, pH 7.5, 25°C, cosubstrate dichlorophenolindophenol
0.031
NADH
mutant K409A, pH 7.5, 25°C, cosubstrate dichlorophenolindophenol
0.037
NADH
mutant R310E, pH 7.5, 25°C, cosubstrate dichlorophenolindophenol
0.042
NADH
mutant K387A, pH 7.5, 25°C, cosubstrate dichlorophenolindophenol
0.05
NADH
mutant N384A, pH 7.5, 25°C, cosubstrate dichlorophenolindophenol
0.029
oxidized 2,6-dichlorophenolindolphenol
mutant R65A, pH 7.5, 25°C
0.083
oxidized 2,6-dichlorophenolindolphenol
mutant K387A, pH 7.5, 25°C
0.083
oxidized 2,6-dichlorophenolindolphenol
mutant N384A, pH 7.5, 25°C
0.091
oxidized 2,6-dichlorophenolindolphenol
mutant K409A, pH 7.5, 25°C
0.097
oxidized 2,6-dichlorophenolindolphenol
wild-type, pH 7.5, 25°C
0.125
oxidized 2,6-dichlorophenolindolphenol
mutant R310A, pH 7.5, 25°C
0.125
oxidized 2,6-dichlorophenolindolphenol
mutant R339A, pH 7.5, 25°C
0.2
oxidized 2,6-dichlorophenolindolphenol
mutant R310E, pH 7.5, 25°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
203
oxidized 2,6-dichlorophenolindolphenol
mutant R65A, pH 7.5, 25°C
283
oxidized 2,6-dichlorophenolindolphenol
mutant R310E, pH 7.5, 25°C
288
oxidized 2,6-dichlorophenolindolphenol
mutant R339A, pH 7.5, 25°C
358
oxidized 2,6-dichlorophenolindolphenol
mutant N384A, pH 7.5, 25°C
377
oxidized 2,6-dichlorophenolindolphenol
mutant K409A, pH 7.5, 25°C
408
oxidized 2,6-dichlorophenolindolphenol
mutant K387A, pH 7.5, 25°C
617
oxidized 2,6-dichlorophenolindolphenol
mutant R310A, pH 7.5, 25°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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
UniProt
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
monomer
both WT and His6-Pdr undergo a monomer-dimer association-dissociation
additional information
dimer
both WT and His6-Pdr undergo a monomer-dimer association-dissociation
additional information
Pdr-Pdx complex formation is mainly driven by steric complementarity, bulky side chains of Tyr33, Arg66, and Trp106 prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr
additional information
-
Pdr-Pdx complex formation is mainly driven by steric complementarity, bulky side chains of Tyr33, Arg66, and Trp106 prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr
additional information
-
cross-linking of putidaredoxin and putidaredoxin reductase by 1-ethyl 3-[3-(dimethylamino)propyl]carbodiimide, EDC. EDC promotes formation of stoichiometric Pdr-Pdx complexes only when carboxyl groups on putidaredoxin are activated. The putidaredoxin-putidaredoxin reductase C73S/C85S conjugate protein is more efficient in electron transfer to cytochrome c and, in the presence of saturating levels of P450cam, more effectively supports camphor hydroxylation. The cross-linked complex is physiologically relevant and represents a suitable model for mechanistic studies, and molecular recognition between putidaredoxin and putidaredoxin reductase is redox-controlled and assisted by the putidaredoxin Glu72 and putidaredoxin reducase Lys409 charge-charge interactions
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
computational modeling based on crystal structures 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
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
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
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)
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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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C73S
mutation improves protein stability. Decreasing order of stability is C73S/C85S, C73S, C85S, wild-type Pdx
C73S/C85S
mutation improves protein stability. Decreasing order of stability is C73S/C85S, C73S, C85S, wild-type Pdx
C85S
mutation improves protein stability. Decreasing order of stability is C73S/C85S, C73S, C85S, wild-type Pdx
D38A
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
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
moderate decrease in the binding affinity and reduction of Pdx
K387A
moderate decrease in the binding affinity and reduction of Pdx
K409A
moderate decrease in the binding affinity and reduction of Pdx
R310A
mutation lowers the interprotein electron tranfer rate by 20-30fold without perturbing the Pdx association step
R310E
mutation decreases both the Pdr-to-Pdx ET and partner binding affinity by 100- and 8fold, respectively
R65A
mutation lowers the interprotein electron tranfer rate by 20-30fold without perturbing the Pdx association step
R66A
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
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
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
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
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
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
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
C73G
-
surface mutation facilitating crystallization without affecting cluster ligation and with only minor effects on activity
additional information
additional information
expression of wild-type as His-tagged protein. Molecular, spectral, and electron transferring properties of recombinant His6-Pdr to artificial and native electron acceptors are similar to those of the wild-type protein. Contrary to wild-type, under anaerobic conditions, NAD+ induces in His6 Pdr spectral changes indicative of flavin reduction and formation of the charge transfer complex between the reduced FAD and NAD+
additional information
-
expression of wild-type as His-tagged protein. Molecular, spectral, and electron transferring properties of recombinant His6-Pdr to artificial and native electron acceptors are similar to those of the wild-type protein. Contrary to wild-type, under anaerobic conditions, NAD+ induces in His6 Pdr spectral changes indicative of flavin reduction and formation of the charge transfer complex between the reduced FAD and NAD+
additional information
for expression in Escherichia coli, change of the rare start codon, GTG, results in an 18fold increase in the level of expression of the protein to 7.4 mg/g wet weight of cells
additional information
-
for expression in Escherichia coli, change of the rare start codon, GTG, results in an 18fold increase in the level of expression of the protein to 7.4 mg/g wet weight of cells
additional information
functional expression in Escherichia coli of tricistronic constructs consisting of P450cam and the auxiliary proteins, Pd and PdR. Transformed bacterial whole cells efficiently oxidize (1R)-(+)-camphor to 5-exo-hydroxycamphor and interestingly limonene to (-)-perillyl alcohol. These bioengineered Escherichia coli cells possess a heterologous selfsufficient P450 catalytic system
additional information
-
functional expression in Escherichia coli of tricistronic constructs consisting of P450cam and the auxiliary proteins, Pd and PdR. Transformed bacterial whole cells efficiently oxidize (1R)-(+)-camphor to 5-exo-hydroxycamphor and interestingly limonene to (-)-perillyl alcohol. These bioengineered Escherichia coli cells possess a heterologous selfsufficient P450 catalytic system
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
enzyme fused to the carboxy-terminus of CYP101A1 (P450cam), expressed in Escherichia coli BL21(DE3) cells
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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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
synthesis
-
omega-hydroxylation of medium-chain n-alkanes and primary alcohols by CYP153 enzymes from Mycobacterium marinum and Polaromonas sp. strain JS666 in presence of putidaredoxin and putidaredoxin reductase. Without putidaredoxin and putidaredoxin reductase, CYP153A16 shows negligible P450 oxidation activity towards n-octane
synthesis
-
stereoselective production of pravastatin from avastatin by Escherichia coli co-expressing putidaredoxin reductase and putidaredoxin from Pseudomonas putida with cytochrome P450 105A3 from Streptomyces carbophilus. Improved active P450 expression leads to 4.1fold increase in pravastatin yield
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Sevrioukova, I.F.; Li, H.; Poulos, T.L.
Crystal structure of putidaredoxin reductase from Pseudomonas putida, the final structural component of the cytochrome P450cam monooxygenase
J. Mol. Biol.
336
889-902
2004
Pseudomonas putida (P16640), Pseudomonas putida
Kim, D.; Ortiz de Montellano, P.R.
Tricistronic overexpression of cytochrome P450cam, putidaredoxin, and putidaredoxin reductase provides a useful cell-based catalytic system
Biotechnol. Lett.
31
1427-1431
2009
Pseudomonas putida (P16640), Pseudomonas putida
Smith, N.; Mayhew, M.; Holden, M.; Kelly, H.; Robinson, H.; Heroux, A.; Vilker, V.; Gallagher, D.
Structure of C73G putidaredoxin from Pseudomonas putida
Acta Crystallogr. Sect. D
60
816-822
2004
Pseudomonas putida
Reipa, V.; Holden, M.J.; Vilker, V.L.
Association and redox properties of the putidaredoxin reductase-nicotinamide adenine dinucleotide complex
Biochemistry
46
13235-13244
2007
Pseudomonas putida (P16640)
Sevrioukova, I.F.; Poulos, T.L.
Arginines 65 and 310 in putidaredoxin reductase are critical for interaction with putidaredoxin
Biochemistry
49
5160-5166
2010
Pseudomonas putida (P16640)
Churbanova, I.Y.; Poulos, T.L.; Sevrioukova, I.F.
Production and characterization of a functional putidaredoxin reductase-putidaredoxin covalent complex
Biochemistry
49
58-67
2010
Pseudomonas putida
Koga, H.; Yamaguchi, E.; Matsunaga, K.; Aramaki, H.; Horiuchi, T.
Cloning and nucleotide sequences of NADH-putidaredoxin reductase gene (camA) and putidaredoxin gene (camB) involved in cytochrome P-450cam hydroxylase of Pseudomonas putida
J. Biochem.
106
831-836
1989
Pseudomonas putida (P16640), Pseudomonas putida
Roome Jr., P.; Philley, J.; Peterson, J.
Purification and properties of putidaredoxin reductase
J. Biol. Chem.
258
2593-2598
1983
Pseudomonas putida
Peterson, J.A.; Lorence, M.C.; Amarneh, B.
Putidaredoxin reductase and putidaredoxin. Cloning, sequence determination, and heterologous expression of the proteins
J. Biol. Chem.
265
6066-6073
1990
Pseudomonas putida (P16640), Pseudomonas putida
Sevrioukova, I.F.; Poulos, T.L.
Putidaredoxin reductase, a new function for an old protein
J. Biol. Chem.
277
25831-25839
2002
Pseudomonas putida (P16640), Pseudomonas putida
Kuznetsov, V.Y.; Blair, E.; Farmer, P.J.; Poulos, T.L.; Pifferitti, A.; Sevrioukova, I.F.
The putidaredoxin reductase-putidaredoxin electron transfer complex: theoretical and experimental studies
J. Biol. Chem.
280
16135-16142
2005
Pseudomonas putida (P16640), Pseudomonas putida
Sevrioukova, I.; Garcia, C.; Li, H.; Bhaskar, B.; Poulos, T.
Crystal structure of putidaredoxin, the [2Fe-2S] component of the P450cam monooxygenase system from Pseudomonas putida
J. Mol. Biol.
333
377-392
2003
Pseudomonas putida (P16640)
Ba, L.; Li, P.; Zhang, H.; Duan, Y.; Lin, Z.
Engineering of a hybrid biotransformation system for cytochrome P450sca-2 in Escherichia coli
Biotechnol. J.
8
785-793
2013
Pseudomonas putida
Scheps, D.; Malca, S.H.; Hoffmann, H.; Nestl, B.M.; Hauer, B.
Regioselective omega-hydroxylation of medium-chain n-alkanes and primary alcohols by CYP153 enzymes from Mycobacterium marinum and Polaromonas sp. strain JS666
Org. Biomol. Chem.
9
6727-6733
2011
Pseudomonas putida
Jo, H.; Park, S.; Le, T.; Ma, S.; Kim, D.; Ahn, T.; Joung, Y.; Yun, C.
Peroxide-dependent oxidation reactions catalyzed by CYP191A1 from Mycobacterium smegmatis
Biotechnol. Lett.
39
1245-1252
2017
Pseudomonas putida
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