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Enzyme Nomenclature
Information on EC 1.14.15.1 - camphor 5-monooxygenase
for references in articles please use BRENDA:EC1.14.15.1
Word Map on EC 1.14.15.1
- 1.14.15.1
- heme
-
spin
-
substrate-free
-
ferrous
-
high-spin
-
substrate-bound
- dioxygen
-
low-spin
-
camphor-bound
-
p450terp
-
soret
- chloroperoxidase
-
adrenodoxin
- novosphingobium
- aromaticivorans
-
ferryl
-
monooxygenation
-
monoxygenase
-
self-sufficient
-
thiolate-ligated
- p450eryf
- biotechnology
- synthesis
- analysis
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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
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EC NUMBER 
COMMENTARY 
1.14.15.1
-
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RECOMMENDED NAME
GeneOntology No.
camphor 5-monooxygenase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
multi-component mixed function oxidase, consisting of putidaredoxin reductase, putidaredoxin and cytochrome P450cam, heme-thiolate protein
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
model for putidaredoxin activity, primary role of putidaredoxin is to prevent uncoupling by enforcing conformations of enzyme that prevent loss of substrate and to enforce conformations that permit efficient proton transfer
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
putidaredoxin acts as a shuttle for transport of electrons from putidaredoxin reductase to enzyme
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
binding of putidaredoxin forces selection of the active conformation of enzyme
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
the acvtive conformation of enzyme is stabilized by binding of the substrate at the active site
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
oxygen-transfer reaction via a monooxygenation mechanism
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
reaction mechanism, and second reductive step of the mechanism of interaction and electron transfer, overview
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
K+ plays an important role in substrate binding and structural and conformational stability of the enzyme
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
mechanism of O2 activation: binding of O2 to ferrous P450cam to yield the ferric-superoxo form, oxyP450cam, followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam, camphor is bound to all enzyme forms, overview
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
the active site structure changes due to putidaredoxin-enzyme interaction involving salt bridge and hydrogen bonding network between Arg112, His355, Leu356, and the heme ligand Cys357
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
active binding of substrate camphor, analysis by density functional theory calculations, residue Tyr96 is important forming a strong hydrogen bond, catalytic cycle of cytochrome P450, the strong hydrogen bonding is not affected by the enzyme's environment, reaction mechanism, overview
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
via H2O2 and a nucleophilic intermediate, the substrate can modulate the properties of both the monoxygenase active-oxygen intermediates and the proton-delivery network that encompasses them, the catalytic cycle, overview
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
rebound mechanism of C-H hydroxylation by P450, molecular dynamics
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
reaction mechanism
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
reaction mechanism and cycle of the enzyme including postulated intermediates, detailed overview
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
reaction mechanism involving a heme ring and residue Asp297, modelling of the hydroxylation of camphor and the hydrogen abstraction from heme using combined quantum mechanical/molecular mechanical method
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
proposed catalytic cycle for cytochrome P450, overview
-
(+)-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
-
(+)-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; proposed catalytic cycle for cytochrome P450, overview
-
-
(+)-camphor + reduced putidaredoxin + O2 = (+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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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.
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CAS REGISTRY NUMBER
COMMENTARY
9030-82-4
-
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
657684, 658115, 658971, 658988, 671612, 672144, 672413, 673054, 674197, 675233, 676941, 701533, 701902, 702698, 704181, 711234, 711268, 744192, 744343, 744365, 744368, 744434, 744637, 744698, 744704, 745164, 745613, 745614, 746282, 746306, 746482
UniProt
-
657898, 657999, 658056, 659396, 659528, 659635, 659825, 671535, 672070, 672115, 672742, 672760, 673037, 674152, 674438, 674474, 674516, 674959, 675234, 676280, 702301, 702799, 702820, 704713, 705331, 706783, 724338, 724762, 725602, 725873, 744714
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-
Accession Number PC00183
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-
Accession Number PC00183; PpG786, mutant derived from PpG1
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-
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
physiological function
additional information
evolution
-
the enzyme belongs to the superfamily of cytochrome P450 monooxygenases
evolution
-
cytochrome P450cam (CYP101) from Pseudomonas putida is the model enzyme for the P450 superfamily
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
malfunction
-
alanine substitutions of the residues involved in putidaredoxin-enzyme interactions do not influence the rates of electron transfer, interaction analysis of enzyme wild-type and mutant (D125A, H352A, and H361A) proteins with putidaredoxxin wild-type and mutant (Y33A, S42A, and S44A) proteins, kinetics, overview
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
-
physiological function
-
Pseudomonas putida is capable of detoxification of camphor and borneol, overview
physiological function
-
cytochrome P450cam carries out the conversion of camphor to 5-exohydroxycamphor under conditions in which the bacterium uses camphor as the primary carbon source for growth
physiological function
-
Pseudomonas putida is capable of detoxification of camphor and borneol, overview
-
additional information
-
CYP101A1 is a soluble monomeric heme-containing camphor monooxygenase
additional information
-
both the apoenzyme and the camphor-bound enzyme of CYP101D2 have open conformations with an access channel. In the active site of the camphor-bound form, the camphor carbonyl interacts with the heme-iron-bound water. The observed open structures may be conformers of the CYP101D2 enzyme that enable the substrate to enter the buried active site via a conformational selection mechanism. Two other potential camphor-binding sites exist: one located in the access channel, flanked by the B/C and F/G loops and the I helix, and the other in a cavity on the surface of the enzyme near the F helix side of the F/G loop. The second and third binding sites may be intermediate locations of substrate entry and translocation into the active site, substrate binding structure and multi-step substrate-binding mechanism, overview
additional information
-
NMR structure analysis of the enzyme CYP101A1 with substrate (+)-camphor bound the active site and substrate-free enzyme after removal of (+)-camphor, molecular dynamics simulations and modeling, overview. Portions of a beta-rich region adjacent to the active site shift so as to partially occupy the vacancy left by removal of substrate. The accessible volume of the active site is reduced in the substrate-free enzyme relative to the substrate-bound structure
additional information
-
P450cam from Pseudomonas putida oxidizes (1R)-(+)-camphor to 5-exo-hydroxy camphor and further to 5-oxo-camphor
additional information
-
substrate recognition and selectivity, enzyme-substrate interactions, NMR structrue analysis, Analysis of 1H,15N-TROSY-HSQC spectra of CYP-(+)-camphor-CO, CYP-adamantenone-CO and CYP-norcamphor-CO. overview. Replacing the native substrate camphor with adamantanone or norcamphor causes perturbations in NMR-detected NH correlations assigned to the network,which includes portions of a beta-sheet and an adjacent helix that is remote from the active site
additional information
-
double electron-electron resonance measurements of intermolecular distances in the Pdx/P450cam complex show that the geometry of the complex is nearly identical for the open and closed states of P450cam
additional information
-
structures of the Pdx-P450cam complex, potential electron transfer pathways and interactions between Pdx Asp38 and P450cam Arg112, as well as hydrophobic contacts between the Pdx Trp106 and P450cam residues, favorable interactions exist between Pdx Tyr33 and P450cam Asp125, as well as between Pdx Ser42 and P450cam His352, overview
additional information
-
cofactor putidaredxin-bound and substrate-bound wild-type and E195C/A199C mutant enzymes, camphor-free, cyanide-bound P450cam, and apoenzyme structure analysis, structure analysis using crystal structure, PDB IDs 4JWS and 3L63. Cyanide and camphor can bind simultaneously in the active site
additional information
-
both the apoenzyme and the camphor-bound enzyme of CYP101D2 have open conformations with an access channel. In the active site of the camphor-bound form, the camphor carbonyl interacts with the heme-iron-bound water. The observed open structures may be conformers of the CYP101D2 enzyme that enable the substrate to enter the buried active site via a conformational selection mechanism. Two other potential camphor-binding sites exist: one located in the access channel, flanked by the B/C and F/G loops and the I helix, and the other in a cavity on the surface of the enzyme near the F helix side of the F/G loop. The second and third binding sites may be intermediate locations of substrate entry and translocation into the active site, substrate binding structure and multi-step substrate-binding mechanism, overview
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additional information
-
P450cam from Pseudomonas putida oxidizes (1R)-(+)-camphor to 5-exo-hydroxy camphor and further to 5-oxo-camphor
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(+)-alpha-pinene + putidaredoxin + O2
(+)-cis-verbenol + (+)-myrtenol + (+)-verbenone + oxidized putidaredoxin + H2O
-
-
-
-
-
(+)-alpha-pinene + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
(+)-camphor + O2 + reduced putidaredoxin
exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
(+)-camphor + reduced putidaredoxin + NADH + H+ + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + NAD+ + H2O
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
(+)-camphor + reduced putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-exo-5-hydroxycamphor + reduced putidaredoxin + O2
5-oxocamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(1R)-(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(1R)-(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
(1R)-5,5-difluorocamphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
?
(1R)-5-exo-methoxycamphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
?
(1R)-5-methylenylcamphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
?
(1R)-camphor + putidaredoxin + O2
5-exo-(1R)-hydroxycamphor + oxidized putidaredoxin + H2O
(1R)-camphor enol ether + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
(1R)-camphor N-methyl imine + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
(1R)-camphor oxime + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
(1R)-endo-borneol allyl ether + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
(1R)-endo-borneol methyl ether + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
(1R)-endo-borneol propyl ether + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
(1R)-iso-borneol methyl ether + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
(1R)-norcamphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
?
(1S)-camphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
?
(4S)-limonene + putidaredoxin + O2
?
-
the 7-position is the major site of hydroxylation by P450cam
-
-
?
(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
-
-
ratio: 50:13:15:8
?
(R)-exo-5-hydroxycamphor + O2 + reduced putidaredoxin
2,5-diketocamphane + oxidized putidaredoxin + H2O
-
-
-
-
?
(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
-
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
-
-
-
?
1,2-campholide + putidaredoxin + O2
5-exo-hydroxy-1,2-campholide + oxidized putidaredoxin + H2O
1,2-dibromo-3-chloropropane + O2 + reduced putidaredoxin
1-bromo-3-chloroacetone + allyl chloride + H2O + putidaredoxin + Br-
-
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
-
-
1,2-dichlorobenzene + putidaredoxin + O2
2,3-dichlorophenol + 3,4-dichlorophenol + oxidized putidaredoxin + H2O
-
-
-
?
1,3,5-trichlorobenzene + putidaredoxin + O2
2,4,6-trichlorophenol + oxidized putidaredoxin + H2O
-
-
-
?
1,3,5-trichlorobenzene + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
1,3-dichlorobenzene + putidaredoxin + O2
2,6-dichlorophenol + 2,4-dichlorophenol + 2,5-dichlorophenol + 2,3-dichlorophenol + oxidized putidaredoxin + H2O
-
-
-
?
1,4-dichlorobenzene + putidaredoxin + O2
2,5-dichlorophenol + oxidized putidaredoxin + H2O
-
-
-
?
1-dehydrocamphor + putidaredoxin + O2
exo-5,6-epoxycamphor + oxidized putidaredoxin + H2O
-
-
-
?
1-ethyl-2-methylbenzene + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
the S-enantiomer is preferred by the wild-type enzyme
-
-
?
1-ethyl-3-methylbenzene + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
the S-enantiomer is preferred by the wild-type enzyme
-
-
?
1-ethyl-4-methylbenzene + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
the S-enantiomer is preferred by the wild-type enzyme
-
-
?
2-adamantanone + O2 + reduced putidaredoxin
5-hydroxy-2-adamantanone + oxidized putidaredoxin + H2O
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
-
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 + ?
-
no substrate of wild-type, substrate of mutants E156G/V247F/V253G/F256S, T56A/N116H/D297N and G60S/Y75H
-
-
?
5,5-difluorocamphor + putidaredoxin + O2
5,5-difluoro-9-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
?
5-exo-bromocamphor + putidaredoxin + O2
5-ketocamphor + oxidized putidaredoxin + Br- + H2O
-
(+)- and (-)-enantiomer
-
?
adamantane + reduced ferreredoxin + O2
1-adamantol + 2-adamantol + oxidized ferredoxin + H2O
-
98% 1-adamantol + 2% 2-adamantol for the wild-type enzyme, 97% + 3% for the mutant Y96A
-
?
adamantanone + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
?
benzo[a]pyrene + putidaredoxin + O2
3-hydroxybenzo[a]pyrene + oxidized putidaredoxin + H2O
-
-
-
-
?
beta-ionone + O2 + reduced putidaredoxin
4-hydroxy-beta-ionone + oxidized putidaredoxin + H2O
camphane + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
cyclooctane + reduced ferreredoxin + O2
cyclooctanol + cyclooctanone + oxidized ferredoxin + H2O
-
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
-
-
-
-
?
ethylbenzene + putidaredoxin + O2
1-phenylethanol + oxidized putidaredoxin + H2O
-
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
-
-
-
-
?
hexane + reduced ferreredoxin + O2
hexan-2-ol + hexan-3-ol + oxidized ferredoxin + H2O
-
56% hexan-2-ol + 44% hexan-3-ol for the wild-type enzyme and mutant Y96A
-
?
indole + O2 + reduced putidaredoxin
3-hydroxyindole + oxidized putidaredoxin + H2O
-
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
-
-
-
-
?
norcamphor + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
phenanthrene + putidaredoxin + O2
1-phenanthrol + 2-phenanthrol + 3-phenanthrol + 4-phenanthrol + oxidized putidaredoxin + H2O
-
-
-
-
?
pyrene + putidaredoxin + O2
1-pyrenol + 2-pyrenol + 1,6-pyrenequinone + 1,8-pyrenequinone + oxidized putidaredoxin + H2O
-
-
-
-
?
thiocamphor + reduced putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
CYP101D1, CYP101D2
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
CYP101D1, CYP101D2
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
672100, 672115, 672144, 672413, 672742, 672760, 673037, 673054, 673071, 674152, 674197, 674438, 674474, 674516, 674959, 675234, 676280, 676941, 701902, 701533
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
terminal monooxygenase in a three-component camphor-hydroxylating system from Pseudomonas putida, the reaction cycle requires two distinct electron transfer processes from the [2Fe-2S] containing putidaredoxin to P450cam
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
analysis of protein-protein interactions between enzyme and cofactor, overview
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
binding of camphor is strongly dependent on the concentration of alcohols, alcohol expels camphor out of the heme cavity of the enzyme by affecting tertiary structure of Cyt P450cam as well as by modifying the solubility properties of camphor in aqueous medium, overview
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
determination of appearance of transient intermediates at 3°C by double mixing rapid scanning stopped-flow spectroscopy, electron transfer from reduced putidaredoxin gives high spin deoxyferrous P-450, substrate-free enzyme also binds the cofactor, but the cofactor does not deliver the electrons to the substrate-free oxyferrous enzyme, binding structure of camphor-bound oxyferrous P450-CAM with reduced putidaredoxin, functional implications of the formation of the perturbed oxyferrous intermediate, overview
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
mechanism of camphor hydroxylation incorporating an NADH-regeneration system, overview
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
regio- and stereoselective C-H bond hydroxylation, rebound mechanism, overview
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
regio- and stereospecific hydroxylation
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
second reductive step of the mechanism of interaction and electron transfer, overview
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
wild-type dioxygen complex structure: high occupancy and a ordered structure of the iron-linked dioxygen and two 'catalytic' water molecules that form part of a proton relay system to the iron-linked dioxygen, Thr252 accepts a hydrogen bond from the hydroperoxy (Fe(III)-OOH) intermediate that promotes the second protonation on the distal oxygen atom, leading to O-O bond cleavage and compound I formation
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
wild-type enzyme, but not mutant T252A
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
the reduced enzyme exhibits lower-amplitude motions of secondary structural features than the oxidized enzyme on all of the time scales accessible, and these differences are more pronounced in regions of the enzyme involved in substrate access to the active site (B' helix and beta3 and beta5 sheets) and binding of putidaredoxin (C and L helices), the iron-sulfur protein that acts as the effector and reductant of CYP101 in vivo
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
though the active site of the enzyme resides deep inside the protein matrix, the substrate is recognized at the surface of the enzyme and directed towards the active site through the access channel. The threonine 192 that resides on the F-G loop and directed towards the putative substrate access channel of the enzyme, plays an important role in recognition of the substrate at the surface of the enzyme
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
conformational change in CYP101 upon binding of putidaredoxin that re-orients bound camphor appropriately for hydroxylation
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
ferric hydroperoxo complex, elusive reactive species of cytochrome P450cam, and the hydroxo intermediate (formed during camphor hydroxylation) in the catalytic cycle of cytochrome P450cam all have a doublet ground state which have a pronounced multiconfigurational character in the case of compound I and the hydroxo intermediate
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
the 7-propionate side chain plays a role in maintaining the high affinity of cytochrome P450cam for its substrate, the Asp297 and Gln322 residues are capable of undergoing a 7-propionate-associated conformational change in the protein interior
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
the Cpd II-like species is ineffective at hydroxylating camphor, but can be readily reduced by ascorbate to ferric P450cam, which can then bind camphor to form the high-spin heme
-
-
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
the (+)- and (-)-enantiomers serve as substrates
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
when deuterated at either 5-exo- or 5-endo-position, only 5-exo-hydroxycamphor is the product
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
Pseudomonas putida C1 / ATCC 17453
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
?
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
-
-
-
?
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
-
-
-
-
?
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
-
99% 5-exo-hydroxycamphor by wild-type enzyme and 92% by Y96A mutant
-
?
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
-
substrate of CYP101D1 and CYP101D2
-
-
?
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
-
-
-
?
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
substrate of CYP101D1 and CYP101D2
-
-
?
(+)-camphor + reduced putidaredoxin + NADH + H+ + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + NAD+ + H2O
-
-
-
-
?
(+)-camphor + reduced putidaredoxin + NADH + H+ + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + NAD+ + H2O
-
the catalytic cycle of P450cam requires two electrons, both of which are donated by putidaredoxin (Pdx), a ferredoxin containing a [2Fe-2S] cluster, structures of the Pdx-P450cam complex, overview
-
-
?
(+)-camphor + reduced putidaredoxin + NADH + H+ + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + NAD+ + H2O
-
-
-
-
?
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
724338, 724762, 725602, 725873, 744192, 744343, 744365, 744637, 744698, 744704, 745613, 746282, 746306
-
-
?
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
the enzyme shows regio- and stereospecific hydroxylation. Activation and cleavage of the oxygen molecule in the P450cam catalytic cycle is accompanied by two electron transfers from putidaredoxin. Ferric P450cam can accept the first electron from diverse chemical reductants and putidaredoxin homologues, but the second requires putidaredoxin as donor
-
-
?
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
?
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-camphor + reduced putidaredoxin + O2
borneol + oxidized putidaredoxin + H2O
-
under low oxygen conditions borneol is formed instead of 5-ketocamphor
-
-
?
(+)-camphor + reduced putidaredoxin + O2
borneol + oxidized putidaredoxin + H2O
-
the hydroxylation reaction proceeds via a catalytic cycle in which the reduction of dioxygen is coupled to the oxidation of the substrate. A key intermediate in the catalytic cycle is the iron-oxo species (Fe(IV)=O)
-
-
?
(+)-camphor + reduced putidaredoxin + O2
borneol + oxidized putidaredoxin + H2O
-
under low oxygen conditions
-
-
?
(+)-camphor + reduced putidaredoxin + O2
borneol + oxidized putidaredoxin + H2O
-
under low oxygen conditions borneol is formed instead of 5-ketocamphor
-
-
?
(+)-camphor + reduced putidaredoxin + O2
borneol + oxidized putidaredoxin + H2O
-
the hydroxylation reaction proceeds via a catalytic cycle in which the reduction of dioxygen is coupled to the oxidation of the substrate. A key intermediate in the catalytic cycle is the iron-oxo species (Fe(IV)=O)
-
-
?
(+)-camphor + reduced putidaredoxin + O2
borneol + oxidized putidaredoxin + H2O
-
under low oxygen conditions
-
-
?
(1R)-(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(1R)-(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
?
(1R)-camphor + putidaredoxin + O2
5-exo-(1R)-hydroxycamphor + oxidized putidaredoxin + H2O
-
putidaredoxin transfers electrons from NADH to P450cam in a coupled assay method
-
-
?
(1R)-camphor + putidaredoxin + O2
5-exo-(1R)-hydroxycamphor + oxidized putidaredoxin + H2O
-
putidaredoxin transfers electrons from NADH to P450cam
-
-
?
(1R)-camphor + putidaredoxin + O2
5-exo-(1R)-hydroxycamphor + oxidized putidaredoxin + H2O
-
putidaredoxin transfers electrons from NADH to P450cam in a coupled assay method
-
-
?
(1R)-camphor + putidaredoxin + O2
5-exo-(1R)-hydroxycamphor + oxidized putidaredoxin + H2O
-
putidaredoxin transfers electrons from NADH to P450cam
-
-
?
(1R)-camphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
?
(1R)-camphor + putidaredoxin + O2
? + oxidized putidaredoxin + H2O
-
-
-
-
?
1,2-campholide + putidaredoxin + O2
5-exo-hydroxy-1,2-campholide + oxidized putidaredoxin + H2O
-
-
-
?
1,2-campholide + putidaredoxin + O2
5-exo-hydroxy-1,2-campholide + oxidized putidaredoxin + H2O
-
-
-
?
1,2-campholide + putidaredoxin + O2
5-exo-hydroxy-1,2-campholide + oxidized putidaredoxin + H2O
Pseudomonas putida C1 / ATCC 17453
-
-
-
?
1,2-campholide + putidaredoxin + O2
5-exo-hydroxy-1,2-campholide + oxidized putidaredoxin + H2O
-
-
-
?
2-adamantanone + O2 + reduced putidaredoxin
5-hydroxy-2-adamantanone + oxidized putidaredoxin + H2O
-
CYP101D1 and CYP101D2
-
-
?
2-adamantanone + O2 + reduced putidaredoxin
5-hydroxy-2-adamantanone + oxidized putidaredoxin + H2O
-
CYP101D1 and CYP101D2
-
-
?
3-chloroperbenzoic acid + O2 + reduced putidaredoxin
?
-
compound I, ferryl iron plus a porphyrin pi-cation radical (Fe(IV)=O/Por(+)), and compound ES, Fe(IV)=O/Tyr(), in reactions of substrate-free ferric enzyme with 3-chloroperbenzoic acid, compound ES arises by intramolecular electron transfer from nearby tyrosines to the porphyrin pi-cation radical of compound I, active site changes influence electron transfer from nearby tyrosines and affect formation of intermediates, the tyrosyl radical is assigned to Tyr96 for wild type or to Tyr75 for the Y96F variant, overview
-
-
?
3-chloroperbenzoic acid + O2 + reduced putidaredoxin
?
-
reaction mechanism of substrate-free ferric cytochrome P450cam, via FeIV-O plus porphyrin Pi-cation radical, overview
-
-
?
5-methylenyl-camphor + O2 + reduced putidaredoxin
?
-
wild-type enzyme and mutant T252A, in absence of the primary oxidant species of P450, the precursor species FeOOH can effect double bond activation of 5-methylenyl-camphor initiated by a homolytic cleavage of the O-O-bond and formation of an OH radical bound to the secondary oxidant by hydrogen bonding interaction, overview
-
-
?
beta-ionone + O2 + reduced putidaredoxin
4-hydroxy-beta-ionone + oxidized putidaredoxin + H2O
-
CYP101C1 and CYP101B1. CYP101C1 oxidizes beta-ionone to 4-hydroxy-beta-ionone (75%) with one other, unidentified product. This latter compound is the major product of beta-ionone oxidation by CYP101B1 (90%) where 4-hydroxy-beta-ionone is the minor product (10%)
-
-
?
beta-ionone + O2 + reduced putidaredoxin
4-hydroxy-beta-ionone + oxidized putidaredoxin + H2O
-
CYP101C1 and CYP101B1. CYP101C1 oxidizes beta-ionone to 4-hydroxy-beta-ionone (75%) with one other, unidentified product. This latter compound is the major product of beta-ionone oxidation by CYP101B1 (90%) where 4-hydroxy-beta-ionone is the minor product (10%)
-
-
?
linalool + O2 + reduced putidaredoxin
8-hydroxy-linalool + oxidized putidaredoxin + H2O
-
CYP111A2 and CYP111A1
-
-
?
linalool + O2 + reduced putidaredoxin
8-hydroxy-linalool + oxidized putidaredoxin + H2O
-
CYP111A2 and CYP111A1
-
-
?
peracetic acid + O2 + reduced putidaredoxin
?
-
reaction mechanism of substrate-free ferric cytochrome P450cam, via FeIV-O plus tyrosyl radical, overview
-
-
?
additional information
?
-
-
substrate binding and activity data for wild-type CYP101D2 and variants with different substrates, gas chromatography analysis of products, overview
-
-
-
additional information
?
-
substrate binding and activity data for wild-type CYP101D2 and variants with different substrates, gas chromatography analysis of products, overview
-
-
-
additional information
?
-
-
effects of heme environment on the hydrogen abstraction reaction of camphor in catalysis, overview
-
-
-
additional information
?
-
-
modelling of the hydroxylation of camphor using crystal structure, PDB code 1DZ9, and combined quantum mechanical/molecular mechanical method, heme propionate side chains are not involved in catalysis, Asp297 is important for the reaction mechanism, overview
-
-
-
additional information
?
-
-
relative stability of dibromochloropropane and products, overview
-
-
-
additional information
?
-
-
release of the substrate is caused both due to increased solubility of the substrate in solution in presence of alcohol and due to change in the tertiary structure of the active site of the enzyme, addition of alcohols to cytochrome P450cam causes a small change in the secondary structural elements but a significant change in the tertiary structural organization of the enzyme
-
-
-
additional information
?
-
-
the properties and reactivity of the oxyheme and of both the primary and the annealed intermediates are modulated by a bound substrate, including alterations in the properties of the heme center, the presence of any alternative substrate increases the lifetime of hydroperoxoferri-P450cam no less than about 20fold, especially 5-methylenyl-camphor
-
-
-
additional information
?
-
-
substrate specificities of wild-type and mutant enzymes, overview
-
-
-
additional information
?
-
-
under conditions of low oxygen, Pseudomonas putida cells and the isolated P450cam reduce camphor to borneol, product analysis by GC-MS
-
-
-
additional information
?
-
-
an additional coupling pathway transpires during H2O2 shunting of the cycle of wild-type P450cam and in mutant T252A. The reaction starts with the FeIII(O2H2) intermediate, which transforms to Cpd I via a O-O homolysis/H-abstraction mechanism. The substrate 5-methylenylcamphor prevents H2O2 release, while the protein controls the FeIII(O2H2) conversion to Cpd I by nailing through hydrogen-bonding interactionsthe conformation of the HO radical produced during O-O homolysis. This conformation prevents HO readical attack on the porphyrin’s meso position, as in heme oxygenase, and prefers H-abstraction from FeIVOH thereby generating H2O + Cpd I. Cpd I then performs substrate oxidations
-
-
-
additional information
?
-
-
analysis of the molecular substrate recognition of wild-type and mutant P450cams by infrared (IR) spectroscopy, differing conformational heterogeneity in the active site of the P450cam variants and changes in heterogeneity upon binding of different substrates likely contribute to their variable affinities via a conformational selection mechanism, overview. Although P450cam is relatively specific for camphor, it also recognizes a number of small, camphor-like substrates, albeit with variable affinity. Substrate binding and active site structure, overview
-
-
-
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
-
-
-
additional information
?
-
-
comparison of substrate binding and catalytic ability of wild-type enzyme with putidareductase and putidaredoxin (ternary complex), and recombinant fusion enzyme P450cam-putidareductase with putidaredoxin, overview. The wild-type and mutant systems show comparable activity with camphor. Further oxidation of 5-exo-hydroxycamphor to 5-oxo-camphor by the fusion enzyme is 39% lower than for the native system
-
-
-
additional information
?
-
-
enzyme 450cam binds camphor and converts from the closed to open conformation upon binding putidaredoxin, the binding thermodynamics of Pdx differ when the conformation of P450cam is held in different states, thermodynamic analysis, overview
-
-
-
additional information
?
-
-
enzyme P450cam catalyzes the regioselective hydroxylation of d-camphor to 5-exo-hydroxycamphor. P450cam hydroxylates thiocamphor with a regioselectivity lower than camphor but higher than norcamphor. The high regioselectivity of camphor hydroxylation, the hydrogen bond (H-bond) between the camphor ketone and the side chain of active site residue Y96 influences the local electrostatics of the active site but has little effect on either the relative populations of conformational states or the nature of the energy landscapes within the conformations. Infrared spectrometric analysis of enzyme-substrate intercations, overview
-
-
-
additional information
?
-
-
enzyme P450cam produces benzyl alcohols with only moderate enantioselectivities, enantioselective benzylic hydroxylation catalysed by P450 monooxygenases, enantioselectivity of a library of active-site mutants of chimeric P450cam-RhFRed towards the benzylic hydroxylation of structurally related regioisomers of ethylmethylbenzene, molecular dynamics simulations and computational molecular modelling, overview
-
-
-
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
-
-
-
additional information
?
-
-
the P450FeIII-OOH intermediate must be the oxidizing species. The mechanism of hydrogen peroxide binding to the substrate-free form of P450cam is mainly governed by the ability of H2O2 to undergo deprotonation at the hydroxo ligand coordinated to the iron(III) center under conditions of pH > pKP450
-
-
-
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
-
-
-
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
-
-
-
additional information
?
-
-
under conditions of low oxygen, Pseudomonas putida cells and the isolated P450cam reduce camphor to borneol, product analysis by GC-MS
-
-
-
additional information
?
-
-
substrate specificities of wild-type and mutant enzymes, overview
-
-
-
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
(1R)-camphor + putidaredoxin + O2
5-exo-(1R)-hydroxycamphor + oxidized putidaredoxin + H2O
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
P00183
-
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
672100, 672115, 672144, 672413, 672742, 672760, 673037, 673054, 673071, 674152, 674197, 674438, 674474, 674516, 674959, 675234, 676280, 676941
-
-
?
(+)-camphor + O2 + reduced putidaredoxin
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
terminal monooxygenase in a three-component camphor-hydroxylating system from Pseudomonas putida, the reaction cycle requires two distinct electron transfer processes from the [2Fe-2S] containing putidaredoxin to P450cam
-
-
?
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
Pseudomonas putida C1 / ATCC 17453
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + putidaredoxin + O2
(R)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
i.e. (+)-exo-5-hydroxycamphor
-
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
Q2G8A2
-
-
-
?
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
-
-
-
-
?
(+)-camphor + reduced ferredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized ferredoxin + H2O
Q2G8A2
-
-
-
?
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
P00183
-
-
-
?
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-camphor + reduced putidaredoxin + O2
(+)-exo-5-hydroxycamphor + oxidized putidaredoxin + H2O
-
-
-
-
?
(+)-camphor + reduced putidaredoxin + O2
borneol + oxidized putidaredoxin + H2O
-
under low oxygen conditions borneol is formed instead of 5-ketocamphor
-
-
?
(+)-camphor + reduced putidaredoxin + O2
borneol + oxidized putidaredoxin + H2O
-
under low oxygen conditions borneol is formed instead of 5-ketocamphor
-
-
?
(1R)-camphor + putidaredoxin + O2
5-exo-(1R)-hydroxycamphor + oxidized putidaredoxin + H2O
-
putidaredoxin transfers electrons from NADH to P450cam in a coupled assay method
-
-
?
(1R)-camphor + putidaredoxin + O2
5-exo-(1R)-hydroxycamphor + oxidized putidaredoxin + H2O
-
putidaredoxin transfers electrons from NADH to P450cam in a coupled assay method
-
-
?
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
cytochrome
-
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
-
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
-
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
-
cytochrome m
-
cytochrome P-450cam, essential, b-type heme-thiolate protein of P-450-class
-
cytochrome m
-
-
-
cytochrome m
-
optimal ratio of putidaredoxin reductase, putidaredoxin, cytochrome m is 1:10:2
-
cytochrome P450
-
CYP101D2 is a cytochrome P450 monooxygenase
-
cytochrome P450
-
cytochrome P450cam, importance and role of the polar residues, e.g. Tyr33, involved in the Pdx-P450cam interaction, overview. Interaction key residues are Pdx Asp38, Arg66, and Trp106, as well as P450cam Arg109 and Arg112, crystal structure of the Pdx-P450cam complex
-
cytochrome P450
-
; the putidaredoxin reductase-camphor monooxygenase fusion enzyme shows the 450 nm Soret band characteristic of the CYP superfamily
-
cytochrome P450
-
-
-
Ferredoxin
-
the CYP101D2 likely ferredoxin-binding site on the proximal face is largely positively charged, similar to that of CYP101D1
-
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
NADH
-
requirement, reduces reductase flavoprotein and putidaredoxin, but not P450cam, in the absence of camphor
NADH
-
-
347692, 347693, 347694, 347695, 347697, 347699, 347700, 347702, 347708, 347713, 658115, 744637, 744698, 744704, 746282
NADPH
-
requirement, instead of NADH, with campholide as substrate, Pseudomonas sp. C5
putidaredoxin
-
cannot be replaced by other FeS-proteins or the phospholipid of the hepatic microsomal P450 system
-
putidaredoxin
-
essential, iron-sulfur redox protein, Fe2S2*Cys4-class, e-transfer agent to and effector of cytochrome P450cam
-
putidaredoxin
-
the putidaredoxin-binding site is only minimally affected by cytochrome b5
-
putidaredoxin
-
the reaction cycle requires two distinct electron transfer processes from the [2Fe-2S] containing putidaredoxin to P450cam, altered binding and electron transfer with the putidaredoxin mutant C73S, structure and model of oxidized and reduced forms, overview
-
putidaredoxin
-
binding causes conformational changes involving K+, in absence of K+ multiple conformations occur, overview
-
putidaredoxin
-
electron transfer from putidaredoxin to the ferric-superoxo enzyme form, overview
-
putidaredoxin
-
analysis of protein-protein interactions between enzyme and cofactor, structural changes upon complex formation, overview
-
putidaredoxin
-
wild-type and mutant C73S/C85S cofactor, the mutant shows about 2fold improved substrate conversion
-
putidaredoxin
-
the substrate-free oxyferrous enzyme also reacts readily with reduced putidaredoxin
-
putidaredoxin
-
optimal ratio of putidaredoxin reductase, putidaredoxin, cytochrome m is 1:10:2
-
putidaredoxin
-
binding of putidaredoxin converts a single X-proline amide bond in CYP101 from trans or distorted trans to cis
-
putidaredoxin
-
transferring electrons from NADH to P450cam in a coupled assay method
-
putidaredoxin
-
the physiological electron transfer partner protein contains a [2Fe-2S] cluster, energetic importance and role of the polar residues, e.g. Tyr33, involved in the Pdx-P450cam interaction, overview. Interaction key residues are Pdx Asp38, Arg66, and Trp106, as well as P450cam Arg109 and Arg112, crystal structure of the Pdx-P450cam complex
-
putidaredoxin
-
Pdx, the enzyme is highly specific for its reductase, with an absolute requirement for the native putidaredoxin (Pdx) to provide the second electron transfer, binding kinetics and thermodynamics with wild-type and mutant enzymes, overview. Upon binding ferric P450cam, Pdx is now known to trigger a conformational change in the enzyme, which may provide the trigger to coordinate enzyme turnover and protect the enzyme from oxidative damage
-
putidaredoxin
-
[2Fe-2S] putidaredoxin, Pdx. Camphor-bound ferric P450cam domain forms a complex with Pdx, modeling
-
putidaredoxin
-
the catalytic cycle of P450cam requires two electrons, both of which are donated by putidaredoxin (Pdx), a ferredoxin containing a [2Fe-2S] cluster, structures of the Pdx-P450cam complex, potential electron transfer pathways and interactions between Pdx Asp38 and P450cam Arg112, as well as hydrophobic contacts between the Pdx Trp106 and P450cam residues, favorable interactions exist between Pdx Tyr33 and P450cam Asp125, as well as between Pdx Ser42 and P450cam His352, overview
-
putidaredoxin
-
-
347695, 347697, 347698, 347699, 347700, 671535, 671612, 672083, 672760, 673037, 673054, 673071, 674152, 674197, 674438, 674474, 674959, 675233, 675234, 676280, 676941, 701533, 701902, 702301, 702698, 702820, 704181, 704713, 705331, 724338, 725602, 725873, 744343, 744365, 744704, 745613, 746282
-
putidaredoxin
-
cofactor putidaredxin-bound enzyme structure analysis using crystal structure, PDB ID 4JWS
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Fe2+
Fe3+
-
the enzyme requires K+ to drive formation of the characteristic high-spin state of the heme Fe3+ upon substrate binding
Iron
K+
Tl+
-
can substitute for K+ and minimize the effects of K+ absence on conformational perturbences upon putdaredoxin binding
Fe2+
-
requirement, enzyme complex with two FeS-protein components
Fe2+
-
the reaction cycle requires two distinct electron transfer processes from the [2Fe-2S] containing putidaredoxin to P450cam
Fe2+
heme iron, located in the active site, Thr101 is important for stability, tertiary and secondary structure of the heme active site, overview
Fe2+
-
heme iron, heme centre tertiary structure and redox potential analysis of wild-type and C357M mutant enzymes, overview
Fe2+
-
heme iron, formation of an oxyferrous cytochrome P450cam during reaction, structure of the active site of oxyferrous-P450cam
Fe2+
-
Asn252 can stabilize the ferric hydroperoxy intermediate, preventing premature release of H2O2 and enabling addition of the second proton to the distal oxygen to generate the catalytic ferryl species
Fe2+
-
local protein backbone dynamics of CYP101 depend upon the oxidation and ligation state of the heme iron
Iron
-
purified reconstituted P450cam exhibits the ferrous CO-bound P450cam spectrum with the characteristic Soret band at 446 nm, indicating that the thiolate of Cys357 is ligated to the heme iron of the one-legged heme as seen in the wild-type protein
Iron
-
the hydroperoxo iron(III) intermediate P450camFeIII-OOH, i.e. Compound 0 involved in the natural catalytic cycle of P450cam, can be transiently observed in the peroxo-shunt oxidation of the substrate-free enzyme
K+
-
increase of activity, selective effector of enhanced substrate affinity to cytochrome m
K+
-
binding of K+ dramatically increases the high spin content for wild-type and mutant complexes of enzyme and substrate or substrate analogues
K+
-
effects of potassium ion binding on camphor-bound oxidized, camphor-bound reduced, and on CO-bound reduced wild-type and L358P enzyme, detailed overview, the enzyme requires K+ to drive formation of the characteristic high-spin state of the heme Fe3+ upon substrate binding, K+ binding site, overview
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Metyrapone
-
enzyme P450cam bound to metyrapone is constrained in the closed conformation
additional information
-
increasing viscosity of the assay mixture, e.g. by addition of glucose or sucrose, reduces kcat
-
additional information
-
emulsions formed by strongly agitating the mixture with a vortexer denatured protein, P450cam has a complete loss of activity when incubated under these conditions for more than 5 min prior to the initiation of the reaction
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
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
methanol
-
reaction with meta-chloroperbenzoic acid in the presence of methanol (3% or ca. 1 M after mixing) at pH 7.4 and 25°C results in the accumulation of a considerable fraction of the P450cam as the putative Cpd II species
additional information
-
involvement of X-proline isomerization in enzyme function
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0046
3-chloroindole
-
mutant G120A/Y179H/G248S/D297H, pH 7.4, temperature not specified in the publication
0.016
3-chloroindole
-
mutant T56A/N116H/D297N, pH 7.4, temperature not specified in the publication
0.026
3-chloroindole
-
mutant Y179H, pH 7.4, temperature not specified in the publication
0.041
3-chloroindole
-
mutant G60S/Y75H, pH 7.4, temperature not specified in the publication
0.09
3-chloroindole
-
mutant D97F/P122L/Q183L/L244Q, pH 7.4, temperature not specified in the publication
0.15
3-chloroindole
-
mutant G93C/K314R/L319M, pH 7.4, temperature not specified in the publication
0.44
3-chloroindole
-
mutant E156G/V247F/V253G/F256S, pH 7.4, temperature not specified in the publication
additional information
additional information
-
Km-value of putidaredoxin, 0.0038 mM, steady-state kinetics of enzyme interaction with redox partners
-
additional information
additional information
-
spectroscopic and stopped flow transient kinetic studies
-
additional information
additional information
-
stopped-flow kinetics of the reaction between putidaredoxin with 1,3-dimethoxy-5-methyl-1,4-benzoquinone, kinetics of the first and the second electron transfer to P450cam of wild-type and mutant enzymes
-
additional information
additional information
thermal unfolding kinetics, substrate binding kinetics
-
additional information
additional information
-
equilibrium isotope effect on O2 binding, isotope effects and transient-state kinetics, electron transfer kinetics, steady-state kinetics, recombinant His6-tagged enzyme, overview
-
additional information
additional information
-
development of a global data fitting kinetic model that describes the time-varying concentrations of substrate and products, Michaelis-Menten kinetics
-
additional information
additional information
-
EPR/ENDOR studies of wild-type and mutant T252A enzymes
-
additional information
additional information
-
combined quantum mechanical/molecular mechanical study, molecular dynamics, overview
-
additional information
additional information
-
stopped-flow kinetics at different pH and temperature
-
additional information
additional information
-
single turnover kinetics
-
additional information
additional information
-
stopped-flow kinetic analysis of the peracid oxidation of the wild-type enzyme and substrate-free ferric mutant enzymes overview
-
additional information
additional information
-
steady-state turnover activities of the P450cam catalytic cycle, overview
-
additional information
additional information
-
interaction thermodynamics between Pdx and various states of P450cam constrained in different conformations, overview
-
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
-
additional information
additional information
-
thermodynamics of wild-type and mutant enzymes with different substrates, overview
-
additional information
additional information
-
enzyme-cofactor interaction kinetics of wild-type and mutant enzymes, steady-state and stopped-flow reaction kinetics, overview
-
additional information
additional information
-
kinetics of wild-type and mutant enzymes
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
34
cytochrome m
-
P-450cam, constituent part of the multi-component enzyme
-
additional information
additional information
-
enzymatic substrate turnover with cytochrome b5 as the effector
-
0.0014
3-chloroindole
-
mutant Y179H, pH 7.4, temperature not specified in the publication
0.0053
3-chloroindole
-
mutant G120A/Y179H/G248S/D297H, pH 7.4, temperature not specified in the publication
0.011
3-chloroindole
-
mutant D97F/P122L/Q183L/L244Q, pH 7.4, temperature not specified in the publication
0.027
3-chloroindole
-
mutant T56A/N116H/D297N, pH 7.4, temperature not specified in the publication
0.06
3-chloroindole
-
mutant G60S/Y75H, pH 7.4, temperature not specified in the publication
0.062
3-chloroindole
-
mutant G93C/K314R/L319M, pH 7.4, temperature not specified in the publication
0.186
3-chloroindole
-
mutant E156G/V247F/V253G/F256S, pH 7.4, temperature not specified in the publication
17.9
NADH
-
pH 7.4, 22°C, recombinant mutant H352A enzyme with mutant S42A putidaredoxin
26.3
NADH
-
pH 7.4, 22°C, recombinant mutant D125A enzyme with mutant Y33A putidaredoxin
34.1
NADH
-
pH 7.4, 22°C, recombinant mutant H361A enzyme with wild-type putidaredoxin; pH 7.4, 22°C, recombinant wild-type enzyme with mutant S42A putidaredoxin
34.9
NADH
-
pH 7.4, 22°C, recombinant wild-type enzyme with mutant Y33A putidaredoxin
36.3
NADH
-
pH 7.4, 22°C, recombinant mutant H352A enzyme with wild-type putidaredoxin
37.4
NADH
-
pH 7.4, 22°C, recombinant wild-type enzyme with mutant S44Aputidaredoxin
38.2
NADH
-
pH 7.4, 22°C, recombinant wild-type enzyme with wild-type putidaredoxin
40.3
NADH
-
pH 7.4, 22°C, recombinant mutant D125A enzyme with wild-type putidaredoxin
56.4
reduced putidaredoxin
-
putidaredoxin reductase-camphor monooxygenase fusion enzyme, pH 7.4, 30°C
-
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.05
3-chloroindole
-
mutant Y179H, pH 7.4, temperature not specified in the publication
0.12
3-chloroindole
-
mutant D97F/P122L/Q183L/L244Q, pH 7.4, temperature not specified in the publication
0.42
3-chloroindole
-
mutant G93C/K314R/L319M, pH 7.4, temperature not specified in the publication
0.43
3-chloroindole
-
mutant E156G/V247F/V253G/F256S, pH 7.4, temperature not specified in the publication
1.15
3-chloroindole
-
mutant G120A/Y179H/G248S/D297H, pH 7.4, temperature not specified in the publication
1.49
3-chloroindole
-
mutant G60S/Y75H, pH 7.4, temperature not specified in the publication
1.68
3-chloroindole
-
mutant T56A/N116H/D297N, pH 7.4, temperature not specified in the publication
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.4
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
22
25
30
37
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
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
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
-
Pseudomonas putida strains can use (1R)-(+) camphor as sole carbon and energy source
additional information
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
activity sediments after 14 h centrifugation of crude extract at 140000 * g, not after 2 h at 100000 * g, Pseudomonas sp. C5
-
-
Pseudomonas putida C1 / ATCC 17453
-
activity sediments after 14 h centrifugation of crude extract at 140000 * g, not after 2 h at 100000 * g, Pseudomonas sp. C5
-
-
activity sediments after 14 h centrifugation of crude extract at 140000 * g, not after 2 h at 100000 * g, Pseudomonas sp. C5
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PDB
SCOP
CATH
UNIPROT
ORGANISM
Pseudomonas putida;
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
47000
additional information
additional information
-
putidaredoxin reductase: MW 43000 Da, putidaredoxin: MW 12500 Da, cytochrome P450cam: MW 45000 Da, Pseudomonas putida PpG786, ultracentrifugal, diffusion and amino acid analysis
additional information
-
putidaredoxin reductase, MW 43500 Da, putidaredoxin, MW 11594 Da, cytochrome m, MW 45000 Da, Pseudomonas putida PpG786, analytical data; putidaredoxin reductase: MW 45000 Da, putidaredoxin: MW 11000 Da, cytochrome m: MW 44000 Da, Pseudomonas putida PpG786, gel filtration
additional information
-
FMN-containing NADH-reductase, MW 43500 Da, putidaredoxin, MW 12500 Da, cytochrome P-450cam, MW 45000 Da, Pseudomonas putida C1, ultracentrifugal analysis, gel filtration, end group analysis and quantification of prosthetic group material
additional information
-
multi-component mixed function oxidase, consisting of cytochrome P-450, MW 40000 Da, putidaredoxin and putidaredoxin reductase, slightly smaller than cytochrome P-450, Pseudomonas C1, gel filtration
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
?
-
x * 47561.6, recombinant mutant R365L/E366Q, mass spectrometry, x * 47607.4, recombinant mutant C357U, mass spectrometry
?
-
x * 47561.6, recombinant mutant R365L/E366Q, mass spectrometry, x * 47607.4, recombinant mutant C357U, mass spectrometry
-
additional information
-
multi-component mixed function monooxygenase consisting of putidaredoxin reductase, MW 47000, putidaredoxin, MW 11700, cytochrome m, MW 50500, Pseudomonas putida PpG786, SDS-PAGE
additional information
-
cytochrome P450cam dimerizes via the formation of an intermolecular disulfide bond
additional information
-
release of the substrate is caused both due to increased solubility of the substrate in solution in presence of alcohol and due to change in the tertiary structure of the active site of the enzyme, addition of alcohols to cytochrome P450cam causes a small change in the secondary structural elements but a significant change in the tertiary structural organization of the enzyme
additional information
tertiary and secondary structure of the heme active site, overview
additional information
-
secondary structure and localization of perturbation sites of cytochrome b5 and putidaredoxin, overview
additional information
-
structural changes upon complex formation between enzyme and cofactor cause an increase in beta-sheets and alpha-helix content, a decrease in population of random coil/3 10-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bondonh of amino acid carboxylic side chains
additional information
-
secondary structure analysis of wild-type and mutant C357M enzymes, overview
additional information
-
wild-type dioxygen complex structure: high occupancy and a ordered structure of the iron-linked dioxygen and two 'catalytic' water molecules that form part of a proton relay system to the iron-linked dioxygen
additional information
-
the camphor-free structure is in an open conformation characterized by a water-filled channel created by the retraction of the F and G helices, disorder of the B' helix, and loss of the K+ binding. site. Presence of K+ alone does not alter the open conformation, while camphor alone is sufficient for closure of the channel
additional information
-
three-dimensional structure and dynamics of the open and closed states of CYP101A1, NMR analysis and molecular dynamics simulations, overview
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POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
glycoprotein
glycoprotein
-
small amount of carbohydrate in putidaredoxin and in cytochrome P-450cam
glycoprotein
Pseudomonas putida C1 / ATCC 17453
-
small amount of carbohydrate in putidaredoxin and in cytochrome P-450cam
-
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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
-
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
-
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
-
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
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
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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 -20°C, cytochrome m accumulates an equally active, heme-containing component of higher molecular weight, DTT reconverts it to native cytochrome m at 25°C
-
putidaredoxin suffers degradation by repeated cycles of freezing and thawing
-
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
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
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
under aerobic conditions, cytochrome P450cam decays at 25°C with t1/2 of 180 min to cytochrome P420, not rapidly in the presence of camphor
-
347691
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-196°C, no loss of activity of putidaredoxin reductase after repeated freeze-thaw cycles
-
-80°C, 50 mM potassium phosphate buffer, pH 7.4, 50 mM KCl, 1 mM (+)-camphor
-
0-4°C, after 24-96 h, the resuspended pellet of 140000*g sedimentation of Pseudomonas sp. C5 loses hydroxylation capacity, restorable with NADPH and THF
-
0°C, putidaredoxin slowly loses its prosthetic group, 2-mercaptoethanol retards apoprotein formation
-
4°C, monomeric cytochrome P450cam can be stored at low protein concentrations for several days without appreciable accumulation of the dimer
-
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
-
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
-
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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
-
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 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)
-
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
-
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
-
overexpression of P450cam mutant C334A in Escherichia coli strain BL21(DE3)
-
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
-
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)
-
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
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
C136S
-
site-directed mutagenesis, putidaredoxin binding compared to wild-type
C285S
-
site-directed mutagenesis, putidaredoxin binding compared to wild-type
C334A
C357M
-
site-directed mutagenesis, comparison of the mutant structure to the wild-type one
C357U
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
C58S
-
site-directed mutagenesis, putidaredoxin binding compared to wild-type
C85S
-
site-directed mutagenesis, putidaredoxin binding compared to wild-type
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
F87A/Y96F
F87L/Y96F
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
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
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
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
L358P
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
Q227C
Q272C
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
S190C
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
S48C
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
T252A
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
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
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mutant isolated by Sequence Saturation Mutagenesis, converts 3-chloroindole to isatin
Y29F
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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
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site-directed mutagenesis, reduced mutant electron transfer activity and increased Kd values for ferric P450cam compared to the wild-type enzyme
Y33F
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site-directed mutagenesis, reduced mutant electron transfer activity and increased Kd values for ferric P450cam compared to the wild-type enzyme
Y75F
Y96A
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site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96C
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site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96C/C334A
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site-directed mutagenesis, substrate recognition and binding compared to the wild-type, conformational selection mechanism
Y96F
Y96F/C334A
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site-directed mutagenesis, substrate recognition and binding compared to the wild-type, conformational selection mechanism
Y96F/L244A/V247L
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enhanced binding and oxidation of (+)-alpha-pinene, production of 55% (+)-cis-verbenol + 32% (+)-verbenone
Y96F/Y75F
Y96G
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site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96M
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site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96N/C334A
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site-directed mutagenesis, substrate recognition and binding compared to the wild-type, conformational selection mechanism
Y96Q
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site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96S
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site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
Y96T
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site-directed mutagenesis, the mutant gains the ability to hydroxylate indole to 3-hydroxyindole
C357U
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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
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C357U/R365L/E366Q
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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
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R365L/E366Q
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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
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M96Y
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site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
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N244L
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site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
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T297D
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site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
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V87F
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site-directed mutagenesis, the substrate specificity is altered compared to the wild-type enzyme
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additional information
C334A
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identical with the wild-type monomer in terms of optical spectra, camphor-binding and turnover
C334A
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spectroscopically and enzymatically identical to wild-type, but does not form dimers in solution
C334A
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is identical spectroscopically and enzymatically to wild-type but does not dimerize in solution at high concentrations
C334A
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mutant, that is spectroscopically and enzymatically identical to wild-type CYP101 enzyme, but does not form dimers in solution, and so is more suitable for solution NMR studies than wild-type enzyme
C334A
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the mutation of P450cam increases the protein stability compared to the wild-type enzyme
C334A
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site-directed mutagenesis, the C334A mutant is spectroscopically and enzymatically identical to the wild type but does not form dimers in solution, and so is more suitable for NMR structure analysis than the wild type enzyme
C334A
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site-directed mutagenesis, the mutation reduces protein aggregation, but has no effect on catalytic activity
C357U
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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
C357U
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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
F87W/Y96F/V247L
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enhanced activity for oxidation of 1,3,5-trichlorobenzene or (+)-alpha-pinene, compared to wild-type, analysis of active-site structure, crystallization
L358P
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stereo- and regioselectivity for d-camphor hydroxylation unchanged
L358P
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in absence of putidaredoxin, mutant shows ring-current signals typical for wild-type enzyme in presence of putidaredoxin, heme-environment of mutant mimics that of the putidaredoxin-bound wild-type, mutant accepts nonphysiological electron donors dithionite and ascorbic acid
L358P
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site-directed mutagenesis, spin-state equilibrium in the L358P mutant is more sensitive to K+ than the wild-type enzyme
Q227C
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site-directed mutagenesis, putidaredoxin binding compared to wild-type
Q272C
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site-directed mutagenesis, putidaredoxin binding compared to wild-type
R365L/E366Q
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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
S190C
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site-directed mutagenesis, putidaredoxin binding compared to wild-type
S190C
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mutation used for double electron-electron resonance studies. Residues S48C and S190C are at opposite ends of the substrate access channel to provide a longer distance measurement
S48C
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site-directed mutagenesis, putidaredoxin binding compared to wild-type
S48C
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mutation used for double electron-electron resonance studies. Residues S48C and S190C are at opposite ends of the substrate access channel to provide a longer distance measurement
T252A
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about 5% of wild-type activity, similar spectra for oxyferrous mutant and wild-type except for Soret band position blue shifts. Epoxidation substrate 5-methylenylcamphor has a anomalous binding mode for the mutant
T252A
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site-sirected mutagenesis, comparison of substrate binding properties to the wild-type enzyme, overview
T252A
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site-directed mutagenesis, the mutant does not show altered conformation of the I helix groove and the catalytically important water molecules in the dioxygen complex
T252A
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the mutant can epoxidize olefins like 5-methylenyl-camphor, but is ineffective in camphor hydroxylation
T252A
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non-productive H2O2 generation is dominant, does not oxidize camphor, substrate binding affinity is similar to that of the wild-type enzyme
T252A
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mutant displays an additional coupling pathway responsible for the epoxidation of 5-methylenylcamphor. During the reaction, camphor cannot prevent H2O2 release and hence the T252A mutant does not oxidize camphor
Y75F
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the mutant shows an altered active site structure influencing catalysis
Y75F
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reaction with meta-chloroperbenzoic acid at 25°C, pH 8.0, is similar to that with the Y96F variant, although slightly more Cpd I (and possibly some Cpd ES) is present
Y96F
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the mutant shows an altered active site structure influencing catalysis
Y96F
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reaction with peracetic acid at pH 8.0, 25°C, is similar to that with meta-chloroperbenzoic acid, except that even with 2.4 mM peracetic acid, all steps are slower than those with 0.150 mM meta-chloroperbenzoic acid
Y96F
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site-directed mutagenesis, population and dynamics of the conformational states are largely unaltered by the Y96F mutation compared to the wild-type enzyme
Y96F/Y75F
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the mutant shows an altered active site structure influencing catalysis
Y96F/Y75F
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mutants produce changes in hydrogen bonding patterns and increase hydrophobicity that affect the ratio of heterolytic to homolytic pathways in reactions with cumene hydroperoxide, resulting in a shift of this ratio from 84/16 for wild-type to 72/28 for the Y96F/Y75F double mutant
additional information
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substitutions at Tyr96, Met98 and Leu253 in CYP101D2 analogously to closely related CYP101A1 from Pseudomonas putida, reduce both the spin state shift on camphor binding and the camphor oxidation activity
additional information
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a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase in Escherichia coli whole cell biocatalysts, production of (+)-exo-5-hydroxycamphor and 5-keto-camphor, overview
additional information
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construction of the deletion mutant DELTA106, the mutant shows reduced electron transfer activity and increased Kd values for ferric P450cam compared to the wild-type enzyme
additional information
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construction of a catalytically active recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system from Pseudomonas putida coupled with enzymatic cofactor putidaredoxin mutant C73S/C85S regeneration, the cofactor mutant is 2fold more effective than the wild-type putidaredoxin
additional information
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establishment of a functional CYP101-system in a cell-like aqueous compartment build by a stable water-oil emulsion with the non-ioninc surfactant tetraethylene glycol dodecyl ether in micro-scale, the enzyme is not active in an organic-aqueous biphasic system, e.g. with etahnol or glycol, incorporating an NADH-regeneration system using recombinant His-tagged bacterial glycerol dehydrogenase, method optimization, overview
additional information
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removal of heme-7-propionate decreases the (+)-camphor affinity by approximately 3fold, but exerts less of an influence on the other steps of the catalytic cycle of the monooxygenation reaction catalyzed by P450cam, does not exert an influence on the structure and electronic properties of the heme
additional information
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in contrast to the wild-type, the more hydrophobic sites of the Tyr-to-Phe variants, compared to that of wild-type, favor homolysis more strongly and lead to considerable fractions of the Cpd II like species in reactions with peracids, especially at higher pH
additional information
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establishment of an in vitro screening system for P450cam selecting variants by the activity on NADH
additional information
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generation of a G327 insertion mutant in order to determine whether the length of the helix played a role in the sensitivity of the K' helix to substrate, the insertion mutant fails to express
additional information
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construction of a fusion enzyme composed of enzyme P450cam and putidaroxin reductase, i.e. P450cam-PdR, kinetic model based on two-site binding of putidaredoxin by P450cam-PdR and inactive dimer formation of the fusion protein. Fusion co-expression with putidaredoxin results in a functional system with in vivo camphor oxidation activity comparable to the wild-type system , but P450cam-PdR is a class I P450 fusion protein that exhibits significantly more favorable catalytic behavior than that of the wild-type system. Further oxidation of 5-exo-hydroxycamphor to 5-oxo-camphor by the fusion enzyme is 39% lower than for the native system
additional information
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interaction analysis of enzyme wild-type and mutant (D125A, H352A, and H361A) proteins with putidaredoxxin wild-type and mutant (Y33A, S42A, and S44A) proteins, kinetics, overview
additional information
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enantioselectivity of a library of active-site mutants of chimeric P450cam-RhFRed towards the benzylic hydroxylation of structurally related regioisomers of ethylmethylbenzene, computational molecular modeling, overview
additional information
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establishment of an in vitro screening system for P450cam selecting variants by the activity on NADH
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biotechnology
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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
synthesis
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selective enzymatic oxidation of (+)-alpha-pinene to verbenol, verbenone, or myrtenol by enzyme mutants
synthesis
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camphor hydroxylation in reverse micelles depends on coexistence of enzyme with putidaredoxin, putidaredoxin reductase, and NADH, but not on H2O2
synthesis
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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 enables the production of the almost fully heme-incorporated CYP-FdR fusion that is catalytically active in vivo and in vitro
additional information
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P450cam is not an antifungal target, but is an excellent model in which to study the phenomenon of drug resistance
additional information
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knowledge of the conformational landscape is central to understanding P450 activity, which has important practical ramifications for the design of therapeutics with optimized pharmacokinetics, and the manipulation of P450s, and possibly other enzymes, for biotechnological applications
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