Information on EC 1.14.15.1 - camphor 5-monooxygenase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
1.14.15.1
-
RECOMMENDED NAME
GeneOntology No.
camphor 5-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
(+)-camphor degradation
-
-
(-)-camphor degradation
-
-
SYSTEMATIC NAME
IUBMB Comments
(+)-camphor,reduced putidaredoxin:oxygen oxidoreductase (5-hydroxylating)
A heme-thiolate protein (P-450). Also acts on (-)-camphor and 1,2-campholide, forming 5-exo-hydroxy-1,2-campholide.
CAS REGISTRY NUMBER
COMMENTARY hide
9030-82-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Pseudomonas putida C1 / ATCC 17453
-
-
-
Manually annotated by BRENDA team
soil isolate, strain C5
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(+)-alpha-pinene + putidaredoxin + O2
(+)-cis-verbenol + (+)-myrtenol + (+)-verbenone + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
-
(+)-alpha-pinene + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
(+)-camphor + O2 + reduced putidaredoxin
exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
show the reaction diagram
(+)-camphor + reduced putidaredoxin + NADH + H+ + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + NAD+ + H2O
show the reaction diagram
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
(+)-camphor + reduced putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(+)-camphor + reduced putidaredoxin + O2
borneol + oxidized putidaredoxin + H2O
show the reaction diagram
(+)-exo-5-hydroxycamphor + reduced putidaredoxin + O2
5-oxocamphor + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(1R)-(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(1R)-(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
(1R)-5,5-difluorocamphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
(1R)-5-exo-methoxycamphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
(1R)-5-methylenylcamphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
(1R)-camphor + putidaredoxin + O2
5-exo-(1R)-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
(1R)-camphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
(1R)-camphor enol ether + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(1R)-camphor N-methyl imine + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(1R)-camphor oxime + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(1R)-endo-borneol allyl ether + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(1R)-endo-borneol methyl ether + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(1R)-endo-borneol propyl ether + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(1R)-iso-borneol methyl ether + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(1R)-norcamphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
(1S)-camphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
(4S)-limonene + putidaredoxin + O2
?
show the reaction diagram
-
the 7-position is the major site of hydroxylation by P450cam
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-
?
(R)-3-ethylhexanol + putidaredoxin + O2
2-ethylhexanoic acid + 2-ethyl-1,2-hexanediol + 2-ethyl-1,3-hexanediol + 2-ethyl-1,4-hexanediol + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
ratio: 50:13:15:8
?
(R)-exo-5-hydroxycamphor + O2 + reduced putidaredoxin
2,5-diketocamphane + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
(S)-3-ethylhexanol + putidaredoxin + O2
2-ethylhexanoic aicd + 2-ethyl-1,2-hexanediol + 2-ethyl-1,3-hexanediol + 2-ethyl-1,4-hexanediol + oxidized putidaredoxin + H2O
show the reaction diagram
-
the (S)-isomer is turned over 1.4times faster than the (R)-isomer
ratio: 15:53:28:10
?
1,2,4,5-tetrachlorobenzene + putidaredoxin + O2
2,3,5,6-tetrachlorophenol + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
1,2-campholide + putidaredoxin + O2
5-exo-hydroxy-1,2-campholide + oxidized putidaredoxin + H2O
show the reaction diagram
1,2-dibromo-3-chloropropane + O2 + reduced putidaredoxin
1-bromo-3-chloroacetone + allyl chloride + H2O + putidaredoxin + Br-
show the reaction diagram
-
dehalogenation, bromochloroacetone is the major conversion product when the incubation medium is saturated with oxygen, while allyl chloride is the sole product in the absence of oxygen
a number of bromochloropropene are also formed to a minor extent by an elimination mechanism, product determination
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-
1,2-dichlorobenzene + putidaredoxin + O2
2,3-dichlorophenol + 3,4-dichlorophenol + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
1,3,5-trichlorobenzene + putidaredoxin + O2
2,4,6-trichlorophenol + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
1,3,5-trichlorobenzene + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
1,3-dichlorobenzene + putidaredoxin + O2
2,6-dichlorophenol + 2,4-dichlorophenol + 2,5-dichlorophenol + 2,3-dichlorophenol + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
1,4-dichlorobenzene + putidaredoxin + O2
2,5-dichlorophenol + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
1-dehydrocamphor + putidaredoxin + O2
exo-5,6-epoxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
1-ethyl-2-methylbenzene + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
the S-enantiomer is preferred by the wild-type enzyme
-
-
?
1-ethyl-3-methylbenzene + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
the S-enantiomer is preferred by the wild-type enzyme
-
-
?
1-ethyl-4-methylbenzene + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
the S-enantiomer is preferred by the wild-type enzyme
-
-
?
1-methylimidazole + O2 + reduced putidaredoxin
?
show the reaction diagram
-
-
-
-
?
2-adamantanone + O2 + reduced putidaredoxin
5-hydroxy-2-adamantanone + oxidized putidaredoxin + H2O
show the reaction diagram
3 2-methylpentane + 3 reduced ferreredoxin + O2
2-methyl-pentan-2-ol + 2-methyl-pentan-3-ol + 2-methyl-pentan-4-ol + 3 oxidized ferredoxin
show the reaction diagram
-
52.5% 2-methyl-pentan-2-ol + 13% 2-methyl-pentan-3-ol, and 3% 2-methyl-pentan-4-ol for the wild-type enzyme, 5% + 12% + 30% for the mutant Y96A
-
?
3-chloroindole + O2 + reduced putidaredoxin
isatin + H2O + Cl- + oxidized putidaredoxin + ?
show the reaction diagram
-
no substrate of wild-type, substrate of mutants E156G/V247F/V253G/F256S, T56A/N116H/D297N and G60S/Y75H
-
-
?
3-chloroperbenzoic acid + O2 + reduced putidaredoxin
?
show the reaction diagram
5,5-difluorocamphor + O2 + reduced putidaredoxin
?
show the reaction diagram
-
-
-
-
?
5,5-difluorocamphor + putidaredoxin + O2
5,5-difluoro-9-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
5-exo-bromocamphor + putidaredoxin + O2
5-ketocamphor + oxidized putidaredoxin + Br- + H2O
show the reaction diagram
-
(+)- and (-)-enantiomer
-
?
5-methylenyl-camphor + O2 + reduced putidaredoxin
?
show the reaction diagram
adamantane + reduced ferreredoxin + O2
1-adamantol + 2-adamantol + oxidized ferredoxin + H2O
show the reaction diagram
-
98% 1-adamantol + 2% 2-adamantol for the wild-type enzyme, 97% + 3% for the mutant Y96A
-
?
adamantanone + O2 + reduced putidaredoxin
?
show the reaction diagram
-
-
-
-
?
adamantanone + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
?
adamantenone + reduced putidaredoxin + O2
?
show the reaction diagram
-
-
-
-
?
benzo[a]pyrene + putidaredoxin + O2
3-hydroxybenzo[a]pyrene + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
beta-ionone + O2 + reduced putidaredoxin
4-hydroxy-beta-ionone + oxidized putidaredoxin + H2O
show the reaction diagram
camphane + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
cyclooctane + reduced ferreredoxin + O2
cyclooctanol + cyclooctanone + oxidized ferredoxin + H2O
show the reaction diagram
-
99% cyclooctanol + 1% cyclooctanone for the wild-type enzyme, 97% + 3% for the mutant Y96A
-
?
DL-camphor + reduced putidaredoxin + NADH + H+ + O2
exo-5-hydroxycamphor + oxidized putidaredoxin + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
ethylbenzene + putidaredoxin + O2
1-phenylethanol + oxidized putidaredoxin + H2O
show the reaction diagram
-
at 5% of the reaction with (+)-camphor
ratio of (R)- to (S)-1-phenylethanol produced depends on mutant form
?
fluoranthene + putidaredoxin + O2
3-fluoranthol + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
hexane + reduced ferreredoxin + O2
hexan-2-ol + hexan-3-ol + oxidized ferredoxin + H2O
show the reaction diagram
-
56% hexan-2-ol + 44% hexan-3-ol for the wild-type enzyme and mutant Y96A
-
?
imidazole + O2 + reduced putidaredoxin
?
show the reaction diagram
-
-
-
-
?
indole + O2 + reduced putidaredoxin
3-hydroxyindole + oxidized putidaredoxin + H2O
show the reaction diagram
-
no substrate of the wild-type enzyme, but a good substrate for Y96 mutants, mutant screening, overview
3-hydroxyindole undergoes spontaneous air oxidation to produce the insoluble dye indigo
-
?
isoborneol + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
linalool + O2 + reduced putidaredoxin
8-hydroxy-linalool + oxidized putidaredoxin + H2O
show the reaction diagram
norcamphor + O2 + reduced putidaredoxin
?
show the reaction diagram
-
-
-
-
?
norcamphor + reduced putidaredoxin + O2
?
show the reaction diagram
-
-
-
-
?
norcamphor + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
peracetic acid + O2 + reduced putidaredoxin
?
show the reaction diagram
phenanthrene + putidaredoxin + O2
1-phenanthrol + 2-phenanthrol + 3-phenanthrol + 4-phenanthrol + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
pyrene + putidaredoxin + O2
1-pyrenol + 2-pyrenol + 1,6-pyrenequinone + 1,8-pyrenequinone + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
thiocamphor + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
show the reaction diagram
-
-
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
show the reaction diagram
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
(+)-camphor + reduced putidaredoxin + O2
borneol + oxidized putidaredoxin + H2O
show the reaction diagram
(1R)-camphor + putidaredoxin + O2
5-exo-(1R)-hydroxycamphor + oxidized putidaredoxin + H2O
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome
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state of cytochrome that is two equivalents of oxidation greater than the ferric form (Cpd I species), state of cytochrome that is one equivalent of oxidation greater than the ferric form (Cpd II species), two-electron-oxidized state of P450 or peroxidases containing both an oxoferryl center [FeIV=O] and either a tryptophanyl or tyrosyl radical, analogous to Cpd ES in cytochrome c peroxidase (Cpd ES species)
-
cytochrome b5
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a cytochrome P450 enzyme, cytochrome b5 is bound in the reduced CYP101-camphor-carbon monoxide complex, cytochrome b5 perturbs many of the same resonances in the complex as Pdx, including those for residues involved in substrate access to and orientation within the active site of CYP101, chemical shifts, overview
-
cytochrome m
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cytochrome P-450
-
-
-
cytochrome P450
-
FAD
-
increase of activity, can replace FMN
Ferredoxin
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NADPH
putidaredoxin
-
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
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe3+
-
the enzyme requires K+ to drive formation of the characteristic high-spin state of the heme Fe3+ upon substrate binding
Tl+
-
can substitute for K+ and minimize the effects of K+ absence on conformational perturbences upon putdaredoxin binding
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Metyrapone
-
enzyme P450cam bound to metyrapone is constrained in the closed conformation
putidaredoxin
-
-
-
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
bis(2-ethylhexyl) sulfosuccinate
-
improves the initial activity of P450cam in two-phase emulsions with initial camphor concentrations of 5-15 mM. P450cam is activated in the surfactant-free emulsions, and addition of bis(2-ethylhexyl) sulfosuccinate sodium salt improves the activity even further, at least over the range of camphor concentrations for which initial rates are readily measurable in all media. The largest rate enhancement is 4.5fold. Nearly 50times more product is formed in the surfactant-stabilized emulsions than is achieved in aqueous buffer
cumene hydroperoxide
-
considerable amount of the putative Cpd II species accumulates, even at pH 7.4. By contrast, reactions with meta-chloroperbenzoic acid at pH 7.4 yield very little of the ca. 420 nm species, unless methanol is included
glycerol
-
activates in vivo and in vitro
methanol
-
reaction with meta-chloroperbenzoic acid in the presence of methanol (3% or ca. 1 M after mixing) at pH 7.4 and 25C results in the accumulation of a considerable fraction of the P450cam as the putative Cpd II species
tetrahydrofuran
-
activation
additional information
-
involvement of X-proline isomerization in enzyme function
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0013 - 0.388
(+)-Camphor
0.086
(R)-2-ethylhexanol
-
-
0.068
(S)-2-ethylhexanol
-
-
0.0024 - 0.0559
1-Methylimidazole
0.0046 - 0.44
3-chloroindole
0.0075 - 0.3
imidazole
0.077 - 0.083
O2
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.9 - 2.75
(+)-Camphor
0.0014 - 0.186
3-chloroindole
0.833
5,5-difluorocamphor
-
cytochrome P-450
2.5 - 51
camphor
34
cytochrome m
-
P-450cam, constituent part of the multi-component enzyme
-
17.9 - 40.3
NADH
55 - 66
O2
2
putidaredoxin
-
constituent protein of the multi-component oxygenase
-
56.4 - 64
reduced putidaredoxin
-
additional information
additional information
-
enzymatic substrate turnover with cytochrome b5 as the effector
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.05 - 1.68
3-chloroindole
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00038 - 0.0007
putidaredoxin
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30.26
-
putidaredoxin reductase
additional information
-
activities of wild-type and mutant enzymes
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2 - 8
-
assay at pH 6.2, pH 7.0, pH 7.4, and pH 8.0
7 - 7.4
-
assay at
7.5
-
assay at
8
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.4
-
assay at 3.4C and 25C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 47
-
tertiary structure of enzyme undergoes conformational changes in this temperature range in the absence of substrate
20 - 60
-
tertiary structure of enzyme undergoes conformational changes in this temperature range in the presence of substrate
additional information
-
temperature profile
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
Pseudomonas putida strains can use (1R)-(+) camphor as sole carbon and energy source
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
46000
-
1 * 46000
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
-
1 * 46000
tetramer
-
crystallization data
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
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, 18C, 1 week, for substrate-bound form soaking of crystals in camphor-containing solution, 18C, 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, 18C, X-ray diffraction structure determination and analysis at 2.0 A resolution
-
; Pseudomonas sp., ternary complex
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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
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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, 6C, 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 systems 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
-
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 -5C 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
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
thermal unfolding data, thermal multistep unfolding modell, Thr101 is important for thermal stability, equilibrium unfolding of wild-type C334A and mutant enzymes
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
2-mercaptoethanol retards loss of FeS-chromophore from putidaredoxin during purification
-
dialysis, loss of activity in crude extract of Pseudomonas sp. C5, restorable by NADPH-addition
-
freeze-thawing, less than 5% loss of activity of cytochrome P450cam in the presence of camphor
-
glycerol, minimizes loss of FMN during purification of putidaredoxin reductase
-
multiple freezing and thawing, at -20C, cytochrome m accumulates an equally active, heme-containing component of higher molecular weight, DTT reconverts it to native cytochrome m at 25C
-
putidaredoxin suffers degradation by repeated cycles of freezing and thawing
-
Thr101 is required for the enzyme reaction intermediate
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexane
-
P450cam is stable and retains 80% of its initial activity after 1 h in a hexane/water emulsion at agitation speeds of less than 250 rpm
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
under aerobic conditions, cytochrome P450cam decays at 25C with t1/2 of 180 min to cytochrome P420, not rapidly in the presence of camphor
-
347691
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-196C, no loss of activity of putidaredoxin reductase after repeated freeze-thaw cycles
-
-20C, 50 mM Tris buffer, pH 7.4, 50% v/v glycerol
-
-80C, 50 mM potassium phosphate buffer, pH 7.4, 50 mM KCl, 1 mM (+)-camphor
-
0-4C, after 24-96 h, the resuspended pellet of 140000*g sedimentation of Pseudomonas sp. C5 loses hydroxylation capacity, restorable with NADPH and THF
-
0C, putidaredoxin slowly loses its prosthetic group, 2-mercaptoethanol retards apoprotein formation
-
4C, monomeric cytochrome P450cam can be stored at low protein concentrations for several days without appreciable accumulation of the dimer
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by anion exchange, hydrophobic interaction and gel filtration
-
by gel filtration
-
native C334A enzyme mutant from Pseudomonas putida strain ATCC 17453 by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration
-
recombinant C334A enzyme mutant from Escherichia coli strain NCM533 by ammonium sulfate fractionation, dialysis, anion exchange chromatography, and gel filtration
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recombinant enzyme from Escherichia coli strain BL21(DE3)
-
recombinant enzyme to 90% purity from Escherichia coli strain DH5alpha
-
recombinant His-tagged CYP101D2 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, gel filtration, and anion exchange chromatography to over 95% purity
-
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain XL-1 Blue by nickel affinity chromatography and repeated anion exchange chromatography
-
recombinant N--terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)
-
recombinant P450cam C334A from Escherichia coli NCM533. Protein purification includes a protamine sulfate cut to precipitate nucleic acids, and an ammonium sulfate cut to isolate CYP101A1
-
recombinant P450cam mutant C334A from Escherichia coli strain BL21(DE3) by anion exchange chromatography and gel filtration
-
recombinant wild-type and mutant enzymes from Escherichia coli by anion exchange chromatography and gel filtration to homogeneity
-
recombinant wild-type and mutant enzymes from Escherichia coli by anion exchange chromatography and hydrophobic interaction chromatography
-
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by a process including anion exchange chromatography and gel filtration
-
recombinant wild-type enzyme and chimeric enzyme fusion protein P450cam-PdR from Escherichia coli strain BL21 (DE3)
-
soluble proteins separated by ultracentrifugation and purified using Ni2+-nitrilotriacetate column and gel filtration
-
to homogeneity by SDSPAGE
-
wild-type and mutants
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ArR/Arx electron-transport chain co-expressed with the CYP enzymes in Escherichia coli. Amplified genes incorporated into the pRSFDuet-Arx vector using the Nde I and Kpn I/EcoR V restriction sites, plasmids pETDuet-Arx-ArR and one of the P450 expressing plasmids pRSFDuet-Arx-CYP transformed into Escherichia coli competent BL21(DE3) cells
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expressed in Escherichia coli strain JM109
-
expression in Escherichia coli
-
expression of C334A enzyme mutant in Escherichia coli strain NCM533
-
expression of enzyme and cofactor, separately, in Escherichia coli strain DH5alpha
-
expression of enzyme and mutant cofactor in Escherichia coli strain BL21(DE3)
-
expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
-
expression of P450cam C334A in Escherichia coli NCM533
-
expression of wild-type and mutant enzymes in Escherichia coli
-
expression of wild-type enzyme, encoded by gene camC, with gene camB, encoding the putidaredoxin, in Escherichia coli strains BL21(DE3), expression of mutant enzymes in Escherichia coli strain DH10B
-
exxpression of N-terminally His-tagged CYP101D2 in Escherichia coli strain BL21(DE3)
-
functional co-expression with glycerol dehydrogenase in Escherichia coli strain BL21(DE3)
-
functional expression in Escherichia coli DH5alpha of tricistronic constructs consisting of P450cam encoded by the first cistron and the auxiliary proteins, putidaredoxin and putidaredoxin reductase by the second and the third
-
gene camC, expression of mutant enzymes in Escherichia coli strain BL21(DE3)
gene camC, recombinant expression of wild-type enzyme and chimeric enzyme fusion protein P450cam-PdR in Escherichia coli strain BL21 (DE3)
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individual expression of enzyme and cofactor putidaredoxin in Escherichia coli
-
mutant C334A transformed into chemically competent Escherichia coli NCM533 cells
-
mutants overexpressed from Escherichia coli strain NCM533 harboring modified pDNC334A plasmids that encode the appropriate mutant of CYP101 under control of the lac promoter
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open reading frames for P450cam mutants including a 6 x His carboxyl-terminal tag cloned into the pCW(Ori+) expression vector using the NdeI and XbaI restriction sites, expressed in Escherichia coli
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overexpression in Escherichia coli
-
overexpression in Escherichia coli strain BL21(DE3)
-
overexpression of P450cam mutant C334A in Escherichia coli strain BL21(DE3)
-
overexpression of wild-type and mutant enzyme sin Escherichia coli
-
pCHC1 plasmid, encoding the wild type cytochrome P450cam (C334A mutant of the native enzyme). PCR products transformed into Escherichia coli XL1-blue super-competent cells. Wild-type and mutants expressed in Escherichia coli BL21 (DE3) cells
-
plasmids transformed into Escherichia coli Bl21(DE3) competent cells
-
Pseudomonas putida, cam operon has been isolated, cloned and expressed in Escherichia coli, review
-
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
-
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
-
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
-
recombinant expression of wild-type enzyme, mutant P450cam[Tyr96Phe]-RhFRed, and other enzyme mutants in Escherichia coli strain BL21(DE3)
-
the enzyme is CAM plasmid encoded
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L253V
-
site-directed mutagenesis, the mutant shows 95% reduced activity compared to the wild-type enzyme, crystal structure analysis of mutant enzyme with bound substrate
M98F
-
site-directed mutagensis, the mutant shows 85% reduced activity compared to the wild-type enzyme, crystal structure analysis of mutant enzyme with bound substrate
Y96A
-
site-directed mutagensis, the mutant shows increased affinity for hydrocarbon substrates including adamantane, cyclooctane, hexane and 2-methylpentane, the monooxygenase activity of the mutant towards alkane substrates is enhanced compared to the wild-type enzyme, crystal structure analysis of mutant enzyme with bound substrate
C136A
-
altered NADH turnover rate
C136S
-
site-directed mutagenesis, putidaredoxin binding compared to wild-type
C148A
-
altered NADH turnover rate
C285S
-
site-directed mutagenesis, putidaredoxin binding compared to wild-type
C357H
-
no activity
C357M
-
site-directed mutagenesis, comparison of the mutant structure to the wild-type one
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
C58A
-
altered NADH turnover rate
C58S
-
site-directed mutagenesis, putidaredoxin binding compared to wild-type
C85A
-
altered NADH turnover rate
C85S
-
site-directed mutagenesis, putidaredoxin binding compared to wild-type
D125A
-
site-directed mutagenesis
D251N
-
site-directed mutagenesis, the mutant shows altered conformation of the I helix groove and misses the catalytically important water molecules in the dioxygen complex leading to lower catalytic activity and slower proton transfer to the dioxygen ligand compared to the wild-type enzyme
D38A
-
site-directed mutagenesis, the mutant shows altered electron transfer activity with higher Kd values for ferric P450cam and about 20% of the first electron transferring ability compared to the wild-type enzyme, the mutant forms a complex with 1,3-dimethoxy-5-methyl-1,4-benzoquinone
D38N
-
site-directed mutagenesis, the mutant shows altered electron transfer activity with higher Kd values for ferric P450cam and about 20% of the first electron transferring ability compared to the wild-type enzyme
D97F/P122L/Q183L/L244Q
-
mutant isolated by Sequence Saturation Mutagenesis, converts 3-chloroindole to isatin
E14C/S29C/C85S/C73S
-
site-directed mutagenesis, putidaredoxin binding compared to wild-type
E156G/V247F/V253G/F256S
-
mutant isolated by Sequence Saturation Mutagenesis, shows the highest maximal velocity in the conversion of 3-chloroindole to isatin
E195C/A199C/C334A
-
site-directed mutagenesis, substrate and cofactor binding of the mutant compared to the wild-type, overview
E366Q
-
site-directed mutagenesis
F87A/Y96F
F87L/Y96F
F87W/Y96F
-
enhanced binding and oxidation of (+)-alpha-pinene
F87W/Y96F/L244A
-
enhanced binding and oxidation of (+)-alpha-pinene, production of 86% (+)-cis-verbenol + 5% (+)-verbenone
F87W/Y96F/L244A/V247L
-
enhanced binding and oxidation of (+)-alpha-pinene
F87W/Y96F/V247L
G120A/Y179H/G248S/D297H
-
mutant isolated by Sequence Saturation Mutagenesis, converts 3-chloroindole to isatin
G248D
-
low catalytic activity
G248E
-
low catalytic activity, incubation with camphor, putidaredoxin reductase, and NADH results in partial covalent binding of heme to protein, pronase digestion of heme-bound protein releases 5-hydroxyheme
G326A
-
site-directed mutagenesis in order to decrease the flexibility of the polypeptide at that point, spin state fractions with different substrates and compared to the wild-type enzyme. The mutant shows 40% reduced activity compared to the wild-type enzyme
G60S/Y75H
-
mutant isolated by Sequence Saturation Mutagenesis, shows highest Km/kcat values for the conversion of 3-chloroindole to isatin
G93C/K314R/L319M
-
mutant isolated by Sequence Saturation Mutagenesis, converts 3-chloroindole to isatin
H352A
-
site-directed mutagenesis
H361A
-
site-directed mutagenesis
I396A
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
I396G
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
I396V
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
K344C
-
altered NADH turnover rate
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
L244F/V247L
-
site-directed mutagenesis, the mutant exhibits moderate to high R-selectivity toward ethylmethylbenzene substrates and shows a narrow width of the binding pocket
L244N/V247L
-
site-directed mutagenesis, the mutant displays the highest S-selectivity toward substrates 1-ethyl-2-methylbenzene and 1-ethyl-3-methylbenzene, and low R-selectivity toward 1-ethyl-4-methylbenzene and shows a narrow width of the binding pocket
M184V/T185F
-
site-directed mutagenesis, the mutation introduces changes above the heme plane, prefers S-orientation of 1-ethyl-4-methylbenzene in the binding pocket of mutant, enantioselectivities of 1-ethyl-2-methylbenzene and 1-ethyl-3-methylbenzene are similar to the wild-type enzyme
M395I
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
M96Y
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
N244L
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
P89I
-
yields a mixture of both bound camphor orientations, that seen in putidaredoxin-free and that seen in putidaredoxin-bound CYP101. A mutation in CYP101 that destabilizes the cis conformer of the Ile-88-Pro-89 amide bond results in weaker binding of putidaredoxin
R112C
-
altered NADH turnover rate
R364C
-
altered NADH turnover rate
R365L
-
site-directed mutagenesis
R365L/E366Q
R66A
-
site-directed mutagenesis, reduced mutant electron transfer activity and increased Kd values for ferric P450cam compared to the wild-type enzyme
R66E
-
site-directed mutagenesis, reduced mutant electron transfer activity and increased Kd values for ferric P450cam compared to the wild-type enzyme
R72C
-
altered NADH turnover rate
S190D
-
does not show any significant change in the rate constants of the substrate association, has almost no effect on the activation energy of substrate binding to the enzyme
T101M
-
ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 89:11
T101M/T185F/V247M
-
ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 87:13
T101V
site-directed mutagenesis, the mutant shows decreased thermal stability of the heme active site and reaction intermediates in the reaction, equilibrium unfolding compared to the wild-type enzyme
T185F
-
ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 78:22
T185L
-
ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 80:20
T185V
-
ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 74:26
T192E
-
rate constants of the substrate association is much lower compared to the wild-type, activation energy for the substrate association is significantly higher in the T192E mutant compared to the S190D mutant or the wild-type enzyme
T252I
-
10% of wild-type activity
T252N
-
has comparable turnover number but higher Km value relative to the wild-type enzyme, due to a decrease in the camphor binding affinity, non-productive H2O2 generation is negligible
T252N/V253T
-
has comparable turnover number but higher Km value relative to the wild-type enzyme, due to a decrease in the camphor binding affinity, non-productive H2O2 generation is negligible
T297D
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
T56A/N116H/D297N
-
mutant isolated by Sequence Saturation Mutagenesis, shows highest Km/kcat values for the conversion of 3-chloroindole to isatin
V247A
-
ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 87:13
V247L
-
increased turnover rate for NADH
V247M
-
ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 83:17
V295I
-
ratio of (R)- to (S)-1-phenylethanol produced from ethylbenzene is 76:24
V87F
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
W106A
-
site-directed mutagenesis, the mutant shows altered electron transfer activity with higher Kd values for ferric P450cam and about 20% of the first electron transferring ability compared to the wild-type enzyme
W106F
-
site-directed mutagenesis, reduced mutant electron transfer activity and increased Kd values for ferric P450cam compared to the wild-type enzyme
Y179H
-
mutant isolated by Sequence Saturation Mutagenesis, converts 3-chloroindole to isatin
Y29F
-
the cis conformer is destabilized by the absence of the hydrogen bond between the carbonyl oxygen of Ile-88 and the Tyr-29 hydroxyl group
Y33A
-
site-directed mutagenesis, reduced mutant electron transfer activity and increased Kd values for ferric P450cam compared to the wild-type enzyme
Y33F
-
site-directed mutagenesis, reduced mutant electron transfer activity and increased Kd values for ferric P450cam compared to the wild-type enzyme
Y96A
-
site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96C
-
site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96C/C334A
-
site-directed mutagenesis, substrate recognition and binding compared to the wild-type, conformational selection mechanism
Y96F/C334A
-
site-directed mutagenesis, substrate recognition and binding compared to the wild-type, conformational selection mechanism
Y96F/L244A/V247L
-
enhanced binding and oxidation of (+)-alpha-pinene, production of 55% (+)-cis-verbenol + 32% (+)-verbenone
Y96F/V247L
-
enhanced binding and oxidation of (+)-alpha-pinene
Y96F/Y75F
Y96G
-
site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96M
-
site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96N/C334A
-
site-directed mutagenesis, substrate recognition and binding compared to the wild-type, conformational selection mechanism
Y96Q
-
site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96S
-
site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96T
-
site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96Y
-
altered product spectrum
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; 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
-
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
-
E366Q
-
site-directed mutagenesis
-
R365L
-
site-directed mutagenesis
-
R365L/E366Q
-
site-directed mutagenesis; 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
-
M96Y
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
-
N244L
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
-
T297D
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
-
V87F
-
site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
-
the enzyme is useful in whole cell biocatalyst systems
biotechnology
-
bioengineered Escherichia coli cells possess a heterologous self-sufficient P450 catalytic system that may have advantages in terms of low cost and high yield for the production of fine chemicals
synthesis
additional information