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Literature summary extracted from

  • Johnson, E.O.; Wong, L.L.
    Partial fusion of a cytochrome P450 system by carboxy-terminal attachment of putidaredoxin reductase to P450cam (CYP101A1) (2016), Catal. Sci. Technol., 6, 7549-7560 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
1.14.15.1 synthesis fusion of putidaredoxin reductase PdR to the carboxy-terminus of camphor monooxygenase CYP101A1 (P450cam) via a linker peptide and reconstitution of camphor hydroxylase activity with free putidaredoxin enables the production of the almost fully heme-incorporated CYP-FdR fusion that is catalytically active in vivo and in vitro Pseudomonas putida

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.15.1 gene camC, recombinant expression of wild-type enzyme and chimeric enzyme fusion protein P450cam-PdR in Escherichia coli strain BL21 (DE3) Pseudomonas putida
1.18.1.5 enzyme fused to the carboxy-terminus of CYP101A1 (P450cam), expressed in Escherichia coli BL21(DE3) cells Pseudomonas putida

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.14.15.1 fusion of putidaredoxin reductase PdR to the carboxy-terminus of camphor monooxygenase CYP101A1 (P450cam) via a linker peptide and reconstitution of camphor hydroxylase activity with free putidaredoxin gives a functional system with comparable in vivo camphor oxidation activity as the native system. In vitro, the fused system’s steady state NADH oxidation rate is 2fold faster than that of the native system. In contrast to the native system, NADH oxidation rates for the fusion enzyme show nonhyperbolic dependence on putidaredoxin concentration Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
1.14.15.1 additional information construction of a fusion enzyme composed of enzyme P450cam and putidaroxin reductase, i.e. P450cam-PdR, kinetic model based on two-site binding of putidaredoxin by P450cam-PdR and inactive dimer formation of the fusion protein. Fusion co-expression with putidaredoxin results in a functional system with in vivo camphor oxidation activity comparable to the wild-type system , but P450cam-PdR is a class I P450 fusion protein that exhibits significantly more favorable catalytic behavior than that of the wild-type system. Further oxidation of 5-exo-hydroxycamphor to 5-oxo-camphor by the fusion enzyme is 39% lower than for the native system Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.15.1 Fe2+ [2Fe-2S] putidaredoxin Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.15.1 (+)-camphor + reduced putidaredoxin + O2 Pseudomonas putida
-
 (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?
1.18.1.5 reduced putidaredoxin + NAD+ Pseudomonas putida
-
 oxidized putidaredoxin + NADH + H+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.15.1 Pseudomonas putida P00183
-
-
1.18.1.5 Pseudomonas putida
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.15.1 recombinant wild-type enzyme and chimeric enzyme fusion protein P450cam-PdR from Escherichia coli strain BL21 (DE3) Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.15.1 (+)-camphor + O2 + reduced putidaredoxin
-
 Pseudomonas putida exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?
1.14.15.1 (+)-camphor + reduced putidaredoxin + O2
-
 Pseudomonas putida (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?
1.14.15.1 DL-camphor + reduced putidaredoxin + NADH + H+ + O2
-
 Pseudomonas putida exo-5-hydroxycamphor + oxidized putidaredoxin + NAD+ + H2O
-
 ?
1.14.15.1 additional information comparison of substrate binding and catalytic ability of wild-type enzyme with putidareductase and putidaredoxin (ternary complex), and recombinant fusion enzyme P450cam-putidareductase with putidaredoxin, overview. The wild-type and mutant systems show comparable activity with camphor. Further oxidation of 5-exo-hydroxycamphor to 5-oxo-camphor by the fusion enzyme is 39% lower than for the native system Pseudomonas putida ?
-
 ?
1.18.1.5 reduced putidaredoxin + NAD+
-
 Pseudomonas putida oxidized putidaredoxin + NADH + H+
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.14.15.1 CamC
-
 Pseudomonas putida
1.14.15.1 camphor 5-hydroxylase
-
 Pseudomonas putida
1.14.15.1 class I cytochrome P450
-
 Pseudomonas putida
1.14.15.1 CYP101A1
-
 Pseudomonas putida
1.14.15.1 P450cam
-
 Pseudomonas putida
1.18.1.5 Pdr
-
 Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.15.1 30
-
 assay at Pseudomonas putida

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.14.15.1 56.4
-
 reduced putidaredoxin putidaredoxin reductase-camphor monooxygenase fusion enzyme, pH 7.4, 30°C Pseudomonas putida
1.14.15.1 64
-
 reduced putidaredoxin wild-type, pH 7.4, 30°C Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.15.1 7.4
-
 assay at Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.15.1 cytochrome P450
-
 Pseudomonas putida
1.14.15.1 cytochrome P450 the putidaredoxin reductase-camphor monooxygenase fusion enzyme shows the 450 nm Soret band characteristic of the CYP superfamily Pseudomonas putida
1.14.15.1 NADH
-
 Pseudomonas putida
1.14.15.1 putidaredoxin [2Fe-2S] putidaredoxin, Pdx. Camphor-bound ferric P450cam domain forms a complex with Pdx, modeling Pseudomonas putida
1.18.1.5 FAD dependent on Pseudomonas putida
1.18.1.5 NAD+
-
 Pseudomonas putida