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1.14.15.1: camphor 5-monooxygenase

This is an abbreviated version!
For detailed information about camphor 5-monooxygenase, go to the full flat file.

Word Map on EC 1.14.15.1

Reaction

(+)-Camphor
+
reduced putidaredoxin
 +
O2
=
(+)-exo-5-hydroxycamphor
 +
oxidized putidaredoxin
 +
H2O

Synonyms

2-bornanone 5-exo-hydroxylase, bornanone 5-exo-hydroxylase, CamC, camphor 5-exo-hydroxylase, camphor 5-exo-methylene hydroxylase, camphor 5-exohydroxylase, camphor 5-hydroxylase, Camphor 5-monooxygenase, camphor hydroxylase, camphor hydroxylase cytochrome P450cam, camphor methylene hydroxylase, camphor monooxygenase, class I cytochrome P450, CYP101, CYP101A1, CYP101B1, CYP101C1, CYP101D1, CYP101D2, CYP111A2, Cyt P450cam, cytochrome P-450-CAM, cytochrome P450 cam, cytochrome P450(cam), cytochrome p450cam, cytochrome P450cam monooxygenase, d-camphor monooxygenase, D-camphor-exo-hydroxylase, haem mono-oxygenase CYP101, methylene hydroxylase, methylene monooxygenase, moe, oxygenase, camphor 5-mono-, P450cam, P450cam monooxygenase, P450tcu

ECTree

     1 Oxidoreductases
         1.14 Acting on paired donors, with incorporation or reduction of molecular oxygen
             1.14.15 With reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen into the other donor
                1.14.15.1 camphor 5-monooxygenase

Crystallization

Crystallization on EC 1.14.15.1 - camphor 5-monooxygenase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant apoenzyme and camphor-bound enzyme of CYP101D2, hanging drop vapour diffusion method, mixing of 0.001 ml of 50 mg/ml protein in 20 mM Tris-HCl, pH 8.0, and 150 mM KCl, with 0.001 ml of reservoir solution containing 0.1 M Tris/HCl, pH 8.3, 2.1 M ammonium sulfate, and 4% v/v PEG 400, equilibration against 0.2 ml reservoir solution, 18°C, 1 week, for substrate-bound form soaking of crystals in camphor-containing solution, 18°C, 1 week-1 month, X-ray diffraction structure determination and analysis at 2.4 A and 2.2. A resolution, respectively, molecular replacement and structure modeling
purified recombinant His-tagged wild-type and mutant enzymes, hanging drop vapour diffusion method, mixing of 0.001 ml of 50 mg/ml protein in crystallisation buffer containing 20 mM Tris, pH 8.0, and 150 mM KCl, with 0.001 ml of reservoir solution containing 0.1 M Tris, pH 8.3, 5% v/v PEG 400, and 1.9 M ammonium sulfate, equilivration against 0.1 ml of reservoir solution, 18°C, X-ray diffraction structure determination and analysis at 2.0 A resolution
atomistic simulations study. The diffusion of camphor along the pathway near the substrate recognition site is thermodynamically preferred. The diffusion near the substrate recognition site is triggered by a transition from a heterogeneous collection of closed ligand-bound conformers to the basin comprising the open conformations of cytochrome P450cam. The accompanying conformational change includesthe retraction of the F and G helices and the disorder of the B' helix
camphor-free or camphor-bound P450cam mutant C334A in the absence of substrate and at high and low K+ concentration, protein in 50 mM Tris, pH 7.4, with or without 2-4 mM camphor, mixed with crystallization solution containing 50 mM Tris, pH 7.4, and 12-22% PEG 8000 with and without 200 mM K+, sitting drop vapour diffusion method, 6°C, X-ray diffraction structure determination and analysis at 1.50-1.79 A resolution
docking simulations for binding of 3-chloroindole, the wild-type does not accommodate 3-chloroindole in the active site, whereas all the mutants do. The mutants did not differ significantly in the Fe-N, Fe-C-2 or Fe-C-3 distances for 3-chloroindole
double electron-electron resonance studies. The geometry of the complex is nearly identical for the open and closed states of P450cam. Putaredoxin makes a single distinct interaction with its binding site on the enzyme and triggers the conformational change through very subtle structural interactions
fusion of putidaredoxin reductase PdR to the carboxy-terminus of camphor monooxygenase CYP101A1 (P450cam) via a linker peptide and reconstitution of camphor hydroxylase activity with free putidaredoxin gives a functional system with comparable in vivo camphor oxidation activity as the native system. In vitro, the fused system’s steady state NADH oxidation rate is 2fold faster than that of the native system. In contrast to the native system, NADH oxidation rates for the fusion enzyme show nonhyperbolic dependence on putidaredoxin concentration
mutant F87W/Y96F/V247L in complex with 1,3,5-trichlorobenzene or (+)-alpha-pinene
-
mutant Y96F/F87W/V247L, binding of substrate (+)-alpha-pinene in two orientations related by rotation of the molecule
Pseudomonas putida PpG786, cytochrome m
-
Pseudomonas sp., ternary complex
-
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
purified recombinant wild-type and D251N and T252A mutant enzymes in complex with O2, usage of a high pressure oxygen cell, a single crystal first is transferred into cryobuffer containing 50 mM Tris-HCl, pH 7.4, 0.4-0.6 M KCl, 1 mM D-camphor, 30% polyethylene glycol 4000, and 20% glycerol followed by reduction with 10 mM sodium dithionite for 10 min under anaerobic condition, soaking in the oxygen-saturated cryobuffer at -5°C for 5 min, X-ray diffraction structure determination and analysis at 1.55-2.10 A resolution
-
quantum mechanics and molecular mechanics study on the hydrogen abstraction reaction during hydroxylation of camphor in the quartet state. An energy barrier of 21.3 kcal/mol and a standard free energy of activation of 16.8 kcal/mol are obtained
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
space group P212121, structure at 2 A resolution
-
structure of the ferrous dioxygen adduct at 0.91 A resolution
-
the reconstituted P450cam at 1.8 A resolution reveals that the asymmetric one-legged heme is incorporated into the heme pocket in the same plane and in essentially the same conformation as the heme of the wild-type. A unique array of water molecules extending from the Tyr96 residue to the outside of the protein are present in the crystal structure
two ferric P450cam structures partially complexed with (+)-camphor, by sitting-drop vapour-diffusion method, at 1.3 A (soaked crystals) or 1.35 A (unsoaked crystals) resolution. Belongs to space group P43212, unsoaked crystals have unit-cell parameters of a = b = 63.38, c = 247.30, whereas soaked crystals have unit-cell parameters of a = b = 63.61 and c = 250.39. Structure of the unsoaked P450cam shows an active site that is partially occupied by (+)-camphor and a water molecule liganded to the haem iron and rotamers of Thr101. (+)-Camphor-bound form is the major component and the water-bound form is the minor component. In the soaked P450cam, the population of the major component increases, while the minor component decreases. (+)-Camphor binding induces rotation of Thr101 to form a hydrogen bond that acts as a hydrogen donor to a peripheral haem propionate. This bonding contributes to redox-potential change