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Literature summary extracted from

  • Vandemeulebroucke, A.; Aldag, C.; Stiebritz, M.; Reiher, M.; Hilvert, D.
    Kinetic consequences of introducing a proximal selenocysteine ligand into cytochrome P450cam (2015), Biochemistry, 54, 6692-6703 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.15.1 recombinant expression of wild-type and mutant enzymes in Escherichia coli, the selenoenzyme mutant SeP450cam C357U/R365L/E366Q is expressed in Escherichia coli strain XL1 blue cotransformed with plasmid pSUABC, whereas mutant R365L/E366Q P450cam and wild-type P450cam are expressed in a Escherichia coli strain BL21 Gold, which enhances the yields of the latter proteins but not of SeP450cam Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
1.14.15.1 C357U site-directed mutagenesis, selenocysteine increases the affinity for oxygen 3-4-fold and accelerates the formation of superoxide 50fold, but the net effect of the C357U mutation on substrate hydroxylation is minimal because the second electron transfer step is much faster than superoxide formation under normal turnover conditions. As a consequence, selenocysteine is an excellent surrogate for the proximal cysteine in P450cam, maintaining both high monooxygenase activity and coupling efficiency Pseudomonas putida
1.14.15.1 C357U/R365L/E366Q site-directed mutagenesis, structural, electronic, and catalytic properties of cytochrome P450cam are subtly altered when the cysteine that coordinates to the heme iron is replaced with a selenocysteine, mapping of the effects of the sulfur-to-selenium substitution on the individual steps of the catalytic cycle. The more electron-donating selenolate ligand has only negligible effects on substrate, product, and oxygen binding, electron transfer, catalytic turnover, and coupling efficiency. Off-pathway reduction of oxygen to give superoxide is the only step significantly affected by the mutation. Incorporation of selenium accelerates this uncoupling reaction approximately 50fold compared to sulfur, but because the second electron transfer step is much faster, the impact on overall catalytic turnover is minimal. Quantum mechanical calculations, overview. Steady-state kinetic analysis revealed that the selenocysteine substitution has essentially no effect on the specific catalytic activity or the binding interaction with the electron donor Pdx, as both kcat and KM,Pdx are very similar for wild-type and mutant enzymes Pseudomonas putida
1.14.15.1 R365L/E366Q site-directed mutagenesis Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.15.1 additional information
-
 additional information detailed comparative pre-steady-state kinetic and steady-state analysis of enzyme mutant SeP450cam C357U/R365L/E366Q and its the cysteine-containing wild-type, overview Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.15.1 Fe2+ in heme 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
-
 ?
1.14.15.1 (+)-camphor + reduced putidaredoxin + O2 Pseudomonas putida ATCC 12633
-
 (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.15.1 Pseudomonas putida P00183
-
-
1.14.15.1 Pseudomonas putida ATCC 12633 P00183
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.15.1 recombinant wild-type and mutant enzymes from Escherichia coli by anion exchange chromatography and hydrophobic interaction chromatography Pseudomonas putida

Reaction

EC Number Reaction Comment Organism Reaction ID
1.14.15.1 (+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O proposed catalytic cycle for cytochrome P450, overview Pseudomonas putida
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.15.1 (+)-camphor + reduced putidaredoxin + O2
-
 Pseudomonas putida (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?
1.14.15.1 (+)-camphor + reduced putidaredoxin + O2
-
 Pseudomonas putida ATCC 12633 (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?
1.14.15.1 (1R)-(+)-camphor + reduced putidaredoxin + O2
-
 Pseudomonas putida (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?
1.14.15.1 (1R)-(+)-camphor + reduced putidaredoxin + O2
-
 Pseudomonas putida ATCC 12633 (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
 ?
1.14.15.1 additional information putidaredoxin (Pdx) and putidaredoxin reductase (PdR) are expressed in Escherichia coli strain BL21 from a pMG211 plasmid as C-terminally His6-tagged proteins, and purified by nickel affinity chromatography and gel filtration Pseudomonas putida ?
-
 ?
1.14.15.1 additional information putidaredoxin (Pdx) and putidaredoxin reductase (PdR) are expressed in Escherichia coli strain BL21 from a pMG211 plasmid as C-terminally His6-tagged proteins, and purified by nickel affinity chromatography and gel filtration Pseudomonas putida ATCC 12633 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.14.15.1 cytochrome p450cam
-
 Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.15.1 25
-
 assay at 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 heme structural, electronic, and catalytic properties of cytochrome P450cam are subtly altered when the cysteine that coordinates to the heme iron is replaced with a selenocysteine Pseudomonas putida
1.14.15.1 putidaredoxin
-
 Pseudomonas putida

General Information

EC Number General Information Comment Organism
1.14.15.1 malfunction structural, electronic, and catalytic properties of cytochrome P450cam are subtly altered when the cysteine that coordinates to the heme iron is replaced with a selenocysteine, mapping of the effects of the sulfur-to-selenium substitution on the individual steps of the catalytic cycle. The more electron-donating selenolate ligand has only negligible effects on substrate, product, and oxygen binding, electron transfer, catalytic turnover, and coupling efficiency. Off-pathway reduction of oxygen to give superoxide is the only step significantly affected by the mutation. Incorporation of selenium accelerates this uncoupling reaction approximately 50fold compared to sulfur, but because the second electron transfer step is much faster, the impact on overall catalytic turnover is minimal. Quantum mechanical calculations, Lewis structure of our P450cam active site model and quantum mechanically optimized structures (with TPSS/SVP) of the oxygen-bound forms with S and Se in the triplet spin state, overview Pseudomonas putida