cytochrome p450 (cyp) a biocatalyst for many applicationsusers.unimi.it/tberdid/cyp450_a.pdf ·...
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CytochromeCytochrome P450 (CYP)P450 (CYP)
a biocatalyst for many applicationsa biocatalyst for many applications
• Introduction to CYP450s• Structure • Activity• Mechanism• Bacterial and human CYPs• Human CYPs and xenobiotics
• Applications CYPs in biotechnologyCYPs in bioremediation CYPs in medicineCYPs as biosensors
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• superfamily of ubiquitous monoxygenases• > 7000 known sequences• 57 human (450 rice)• many isoforms
• bacterial CYPs are soluble with substrate specificity• mammalian CYPs are membrane bound, with loose
substrate specificity• a metallo-enzyme ……
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Fe-heme (heme b)
Fe-protoporphirin IX
Soluble P450cam from Pseudomonas putida
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Cys357 binds as thiolate
Conserved fifth axial ligand: a cysteine
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Human CYP3A4 (soluble fraction)
Pseudomonas putidaP450cam
(CYP450cam /CYP101)
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Mixed function monooxygenase
RH + O2+ 2 e- + 2 H+ → ROH + H2O
Insertion of an oxygen atom into a C-H bond
of non-activated substrates…..
NADH and NADPH are primary electron donors
RH + O2+ NAD(P)H + H+ → ROH + NAD(P)+ + H2O
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Fatty acid hydroxylation (n=1-16; m=0-7)
Epoxidation
Hydroxylation of aromatic rings
O-dealkylation
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Regio- and stereoselective reactions
5-exo derivative
…….through changes of
• coordination
• spin state
• redox potential
of the metal center
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The distal cavity is hydrophobic but…..
…..Tyr96 with the polar OH group exposed in distal cavity
1phc
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hexacoordinated low spin Fe(III)
Low reduction potential E° = -330 mV
a water molecule is coordinated to Fe(III)Resting state
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Camphor is the substrate
hydrogen-bond between camphorcarbonyl group and Tyr-OH
2cpp
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low spin Fe(III) E° = -330 mV
high spin Fe (III) E° = -170 mV
high spin Fe (II)
1st electron fromputidaredoxin (E°=-239 mV)
oxygen bindingFe3+-O2-
ferric superoxide
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Fe3+-O22-
ferric peroxide
H
H+
H+
H2O
2nd electronrate determining step
We were here
heterolytic cleavage ofO-O bond
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1dz8
Proton pathway
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H
H+
H2Ohighly reactive
oxo-Ferrilic Fe(IV) + porphyrin radicalIntermediate
O-O bond cleavage
possible resonance structures
the heterolytic O-O bond cleavage leavesa 6 electron O
stabilized as Fe(V)=O or as…
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• high intrinsic kinetic isotopic effect kH/kD = 10-14
radicalic process
high rate of oxygen rebound kOH>>109 s-1
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radical clocks
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Porphyrin π− π∗ transitionsSoret band
Q bands
resting state
after substrate binding
UV-vis spectroscopy
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Enzyme titration with substrate
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∆ε
UV-vis difference spectra upon interaction withbinding molecules
(nm)
• type I
not bound to iron
• type II
bound to iron
BLUE SHIFT
RED SHIFT
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High spin Fe (II)
1st electron fromputidaredoxin (-239 mV)
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Origin of the P450 name = pigment at 450nm
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Strong absorption at 450 nm
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PeroxidesPeroxides or or oxygenoxygenatomatom donorsdonors
peroxide shunt
H
HOOH
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H
Uncoupling : escape of O2- and H2O2
HH22OO22
OO22--
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Mixed function monooxygenase
RH + O2+ 2 e- + 2 H+ → ROH + H2O
NAD(P)Hdoes not transfer electrons directly to CYP active site
NADH and NADPH are primary electron donors
RH + O2+ NAD(P)H + H+ → ROH + NAD(P)+ + H2O
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1st electron fromputidaredoxin (E° =-239 mV)
2nd electronrate determining step
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P450
ER membrane Substrate and O 2
Cytochrome P450 membrane topology
cytosol
NH2
bacterial CYPs are soluble
hydrophobic tail
mammalian CYPs are membrane bound
heme
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P450
FAD
FeS
NADPH2 e-
Ferredoxin
Ferredoxin Reductase
Mitochondrial inner membrane
Substrate and O 2
Class I P450 enzymes
(CYP450cam)
matrix
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Class II P450 enzymes
P450
FAD
FMN
NADPH2 e-
NADPH Cytochrome P450Reductase (NCR)
ER membrane Substrate and O 2
cytosol
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Substrate and O 2
P450
FAD
FMN
NADPH2 e-
BM3 from Bacillum Megaterium
☺ a single protein
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Bacterial CYP
specificity
regio and stereo selectivity
camphor
1-Me -norcamphor
norcamphor
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Y56
V295
V247
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Y56
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V247A mutant
V295I mutant
V247A
V295I
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Human CYP450
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10-15% in the liverCYP…SUPERFAMILY
ca 60% of drugs are metabolized byCyp 3A4
57 human sequences
5%9 %
30%
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Cortisol
11-Deoxdycortisol
11-Deoxycorticosterone
Corticosterone
Aldosterone
11A1
CYP17
CYP19
11B1
CYP21
CYP21
11B1
11B2CYP17
CYP17 CYP17
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3 7
12
24
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CYP27A1
CYP27B1
active form
CYP24
inactive
24 2627
1
CYP2R1
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Metabolism and clearance of xenobioticsubstances
Drugs
Chemicals (pesticides, pollutants, solvents)
Alcaloids
Colorants
Animal, plant and fungal toxins
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Reazioni catalizzate dal citocromo P450
• Idrossilazione di aromatici• Epossidazione di aromatici• Idrossilazione di alifatici• Epossidazione di alcheni• N-dealchilazione• O-dealchilazione• S-sealchilazione• N-ossidazione• N-idrossilazione• S-ossidazione• Ossidazione di aldeidi• Aromatizzazione di androgeni
● Ossidazione dell’alotano• Riduzione dell’alotano• Ossidazione dell’arginina• Taglio della catena laterale del colesterolo• Deidrogenazione• Dealogenazione• Azoriduzione• Deaminazione• Desolforazione• Idrolisi di ammidi• Idrolisi di esteri• Perossidazione• Denitrazione
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hydroxylation reactions
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inactivation through hydroxylation
phenobarbital
hydroxylation reactions
epoxidation reactions
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Carcinogenic
epoxidation reactions
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antipsychotic
pesticide
insecticide, acaricide
oxidation reactions
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analgesicparacetamol
aminopyrine, an antidolorific
chemioterapic drug
hydroxylation & dealkylation reactions
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dealkylation reactions
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N-demethylation reactions
Ring coupling
morphine caffeine
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CYP2D6 Substrates
* Antiarrhythmics: Flecainide, Mexiletine, Propafenone
* Antidepressants: Amitriptyline, Paroxetine, Venlafaxine, Fluoxetine (Prozac), Trazadone
* Antipsychotics: Clorpromazine, Haloperidol, Thoridazine
* Beta-Blockers: Labetalol, Timolol, Propanolol, Pindolol, Metoprolol
* Analgesics: Codeine, Fentanyl, Meperidine, Oxycodone, Propoxyphene
oxycodone is oxycontin, a favorite drug of abuse.morfina
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CYP3A4 Substrates
* Acetominophen (Tylenol)* Codeine (narcotic)* Cyclosporin A (an immunosuppresant),* Diazepam (Valium)* Erythromycin (antibiotic)* Lidocaine (anaesthetic),* Lovastatin (HMGCoA reductase inhibitor,
a cholesterol lowering drug),* Taxol (cancer drug),* Warfarin (anticoagulant).
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CYP2C9SUBSTRATES INHIBITORS INDUCERS
..and also
inhibitors bind on iron
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CYP2C9 complexed with warfarin (anticoagulant)
Nature 2003 424: 464
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CYP3A4 INHIBITORS
2V0M
two moleculesin the distal cavity
PNAS 2006 103 13682
Ketonazole
bergamottina
naringina
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inhibitors bind to iron
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CYP3A4 E° -330 mV
CYP3A4/ androstendione E° -270 mV
CYP3A4/ ritonavir E° -350 mV
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Induction of Cytochrome P450 Genes
endocrine control ACTH (steroid biosynthesis P450s)
PPARα peroxisome proliferators (clofibrate) CYP4 family
PXR CYP3 family
CAR phenobarbital CYP2B 40-50X in rats
AHR (benzo-a-pyrene, cigarette smoke) CYP1 family
ethanol CYP2E1 enzyme stabilization by substrate
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MC (methylcholantrene)
2,3,7,8-Tetrachlorodibenzo-p-dioxin
INDUCERSPolycyclic Aromatic Hydrocarbonsinduce biosynthesis of CYP1A
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CYP1A2 is induced also by
cigarette smoke,
charred food,
cruciferous vegatables(broccoli)
CYP3A4 is induced by IPERICO
CYP2D6 : no inducers
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Drug-drug, drug-xenobiotics, drug-food, etcinteractions
CYP substrates
CYP inhibitors
CYP substrates
CYP inducers
[substrates]
possible toxicity
[substrates]
poor effect
CYP substrateA
CYP substrateB
sustrate competition
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CYP2D6 has
no inducers
its expression ishighly variable
ranging from
no expression(lack of the genes)
high expressiondue to gene duplications
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nortr
ipty
line
plas
mat
icco
ncen
tratio
nnM
nortriptyline: antidepressant drug
Pharmacokinetics
hours
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