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Literature summary for 1.14.15.1 extracted from

  • Aldag, C.; Gromov, I.A.; Garcia-Rubio, I.; von Koenig, K.; Schlichting, I.; Jaun, B.; Hilvert, D.
    Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine (2009), Proc. Natl. Acad. Sci. USA, 106, 5481-5486 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.14.15.1

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain XL-1 Blue from expression plasmids, pSUABC, under control of the salicylate promoter Pseudomonas putida

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant His-tagged wild-type enzyme and enzyme mutants C357U and R365L/E366Q in complex with camphor, X-ray diffraction structure determination and analysis at 1.55-1.83 A resolution, molecular replacement Pseudomonas putida

Protein Variants

Protein Variants Comment Organism
C357U site-directed mutagenesis, the engineered gene contains the requisite UGA codon for selenocysteine, the sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2fold, whereas substrate oxidation becomes partially uncoupled from electron transfer. The structure of mutant C357U, including the active site, is very similar to that of wild-type enzyme and mutant R365L/E366Q. The specific activity of the selenoenzyme mutant C357U is approximately half that of the mutant R365L/E366Q, which is 2fold less active than the wild-type enzyme Pseudomonas putida
E366Q site-directed mutagenesis Pseudomonas putida
R365L site-directed mutagenesis Pseudomonas putida
R365L/E366Q site-directed mutagenesis, the structure of mutant R365L/E366Q is very similar to that of wild-type enzyme and mutant C357U. The specific activity of the selenoenzyme mutant C357U is approximately half that of the mutant R365L/E366Q, which is 2fold less active than the wild-type enzyme Pseudomonas putida

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics of wild-type and mutant enzymes Pseudomonas putida

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ in heme Pseudomonas putida

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(+)-camphor + reduced putidaredoxin + O2 Pseudomonas putida
-
 (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?
(+)-camphor + reduced putidaredoxin + O2 Pseudomonas putida ATCC 12633
-
 (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?

Organism

Organism UniProt Comment Textmining
Pseudomonas putida P00183
-
-
Pseudomonas putida ATCC 12633 P00183
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain XL-1 Blue by nickel affinity chromatography and repeated anion exchange chromatography Pseudomonas putida

Reaction

Reaction Comment Organism Reaction ID
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O proposed catalytic cycle for cytochrome P450, a ferryl-oxo Pi-cation porphyrin radical, is the putative oxidant that reacts directly with substrate, overview. The axial ligand to the heme iron, a cysteine thiolate, is generally believed to control P450 reactivity Pseudomonas putida
a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 activities of wild-type and mutant enzymes Pseudomonas putida

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(+)-camphor + reduced putidaredoxin + NADH + H+ + O2
-
 Pseudomonas putida (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + NAD+ + H2O
-
 ?
(+)-camphor + reduced putidaredoxin + NADH + H+ + O2
-
 Pseudomonas putida ATCC 12633 (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + NAD+ + H2O
-
 ?
(+)-camphor + reduced putidaredoxin + O2
-
 Pseudomonas putida (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?
(+)-camphor + reduced putidaredoxin + O2
-
 Pseudomonas putida ATCC 12633 (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?
additional information catalytic turnover in P450cam requires the enzymes putidaredoxin (Pdx) and putidaredoxin reductase (Pdr), which mediate electron transfer from NADH to heme, the process is tightly coupled to substrate hydroxylation Pseudomonas putida ?
-
 ?
additional information catalytic turnover in P450cam requires the enzymes putidaredoxin (Pdx) and putidaredoxin reductase (Pdr), which mediate electron transfer from NADH to heme, the process is tightly coupled to substrate hydroxylation Pseudomonas putida ATCC 12633 ?
-
 ?

Subunits

Subunits Comment Organism
? x * 47561.6, recombinant mutant R365L/E366Q, mass spectrometry, x * 47607.4, recombinant mutant C357U, mass spectrometry Pseudomonas putida

Synonyms

Synonyms Comment Organism
cytochrome p450cam
-
 Pseudomonas putida
P450cam
-
 Pseudomonas putida

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Pseudomonas putida

pH Optimum

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

Cofactor

Cofactor Comment Organism Structure
cytochrome P450
-
 Pseudomonas putida
heme
-
 Pseudomonas putida
additional information catalytic turnover in P450cam requires the enzymes putidaredoxin (Pdx) and putidaredoxin reductase (Pdr), which mediate electron transfer from NADH to heme, the process is tightly coupled to substrate hydroxylation Pseudomonas putida
NADH
-
 Pseudomonas putida
putidaredoxin
-
 Pseudomonas putida