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Literature summary extracted from

  • Kim, D.; Heo, Y.S.; Ortiz de Montellano, P.R.
    Efficient catalytic turnover of cytochrome P450(cam) is supported by a T252N mutation (2008), Arch. Biochem. Biophys., 474, 150-156.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.15.1 open reading frames for P450cam mutants including a 6 x His carboxyl-terminal tag cloned into the pCW(Ori+) expression vector using the NdeI and XbaI restriction sites, expressed in Escherichia coli Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
1.14.15.1 T252A non-productive H2O2 generation is dominant, does not oxidize camphor, substrate binding affinity is similar to that of the wild-type enzyme Pseudomonas putida
1.14.15.1 T252N has comparable turnover number but higher Km value relative to the wild-type enzyme, due to a decrease in the camphor binding affinity, non-productive H2O2 generation is negligible Pseudomonas putida
1.14.15.1 T252N/V253T has comparable turnover number but higher Km value relative to the wild-type enzyme, due to a decrease in the camphor binding affinity, non-productive H2O2 generation is negligible Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.15.1 0.03
-
(+)-Camphor wild-type Pseudomonas putida
1.14.15.1 0.096
-
(+)-Camphor mutant T252N Pseudomonas putida
1.14.15.1 0.388
-
(+)-Camphor mutant T252N/V253T Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.15.1 Fe2+ Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H2O2 and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species Pseudomonas putida

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.14.15.1 47000
-
gel filtration Pseudomonas putida

Organism

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

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.15.1 soluble proteins separated by ultracentrifugation and purified using Ni2+-nitrilotriacetate column and gel filtration 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
-
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Synonyms

EC Number Synonyms Comment Organism
1.14.15.1 CYP101
-
Pseudomonas putida
1.14.15.1 cytochrome p450cam
-
Pseudomonas putida
1.14.15.1 P450cam
-
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 0.9
-
(+)-Camphor mutant T252N Pseudomonas putida
1.14.15.1 2.067
-
(+)-Camphor wild-type Pseudomonas putida
1.14.15.1 2.75
-
(+)-Camphor mutant T252N/V253T Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.15.1 putidaredoxin
-
Pseudomonas putida