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 (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 | 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 Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics of wild-type and mutant enzymes | Pseudomonas putida |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | in heme | Pseudomonas putida |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | P00183 | - |
- |
Pseudomonas putida ATCC 12633 | P00183 | - |
- |
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 | 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 |
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 | 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 | Comment | Organism |
---|---|---|
? | x * 47561.6, recombinant mutant R365L/E366Q, mass spectrometry, x * 47607.4, recombinant mutant C357U, mass spectrometry | Pseudomonas putida |
Synonyms | Comment | Organism |
---|---|---|
cytochrome p450cam | - |
Pseudomonas putida |
P450cam | - |
Pseudomonas putida |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Pseudomonas putida |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.4 | - |
assay at | Pseudomonas putida |
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 |