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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

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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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