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Literature summary for 1.14.15.1 extracted from

  • Verras, A.; Alian, A.; de Montellano, P.R.
    Cytochrome P450 active site plasticity: attenuation of imidazole binding in cytochrome P450(cam) by an L244A mutation (2006), Protein Eng. Des. Sel., 19, 491-496.
    View publication on PubMed

Application

Application Comment Organism
additional information P450cam is not an antifungal target, but is an excellent model in which to study the phenomenon of drug resistance Pseudomonas putida

Cloned(Commentary)

Cloned (Comment) Organism
expression of wild-type and mutant enzymes in Escherichia coli Pseudomonas putida

Crystallization (Commentary)

Crystallization (Comment) Organism
recombinant wild-type enzyme and mutant L244A/C334A in complex with imidazole or 1-methylimidazole, hanging drop vapour diffusion method, purified recombinant protein in 50 mM potassium phosphate, 250 mM KCl, 50 mM DTT, and 36-52% ammonium sulfate, mixing with an equal volume of 0.001 ml of mother liquor containing 25 mM imidazole or 1-methylimidazole, room temperature, 2 days, cryoprotectant is 50 mM KCl, 25% ammonium sulfate and 30% glycerol, X-ray diffraction structure determination and analysis at 1.5-2.15 A resolution Pseudomonas putida

Protein Variants

Protein Variants Comment Organism
L244A/C334A site-directed mutagenesis, mutation C334A prevents adventitious dimerization to facilitate crystallization but has no further effect on structure or activity of the enzyme, the L244A mutation leads to a highly increased Km and reduced activity for imidazole, but not for for 1-methylimidazole, and altered binding of imidazole to the active site and the active site heme involving residue Val247, overview Pseudomonas putida

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0013
-
(+)-Camphor recombinant wild-type enzyme Pseudomonas putida
0.0016
-
(+)-Camphor recombinant mutant L244A/C334A Pseudomonas putida
0.0024
-
1-Methylimidazole recombinant wild-type enzyme Pseudomonas putida
0.0075
-
imidazole recombinant wild-type enzyme Pseudomonas putida
0.0559
-
1-Methylimidazole recombinant mutant L244A/C334A Pseudomonas putida
0.3
-
imidazole recombinant mutant L244A/C334A Pseudomonas putida

Metals/Ions

Metals/Ions Comment Organism Structure
Iron heme iron Pseudomonas putida

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(+)-camphor + O2 + reduced putidaredoxin Pseudomonas putida
-
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
?

Organism

Organism UniProt Comment Textmining
Pseudomonas putida P00183
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli by anion exchange chromatography and gel filtration to homogeneity Pseudomonas putida

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(+)-camphor + O2 + reduced putidaredoxin
-
Pseudomonas putida (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
?
1-methylimidazole + O2 + reduced putidaredoxin
-
Pseudomonas putida ?
-
?
imidazole + O2 + reduced putidaredoxin
-
Pseudomonas putida ?
-
?

Synonyms

Synonyms Comment Organism
cytochrome P450(cam)
-
Pseudomonas putida

Cofactor

Cofactor Comment Organism Structure
putidaredoxin
-
Pseudomonas putida