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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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Vancomicyn an antibiotic

Phenol coupling of the

ABAB, CDCD and DEDE rings

relays on threeindependent CYP450 enzymes

CYP74C

fragrance

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Rhizopus (fungus) Patent Upjohn 1952 (Pfizer)

JACS 1952 74 5933

Biotransformation of steroids

11α-hydroxylation

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6β-hydroxylation

Anti-cholesterolemic drug : inhibitor of HMG-CoA reductase

Sankyo Pharma, Bristol-Myers Squibb Company

1000mg/L

……. a statin: Pravastatin

Eur J Biochem 1989 184 707

Streptomices ca.

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• Saccharopolyspora erythraea CYP107 (Cyt P450eryF)

10 asymmetric carbons

Woodward R. performed its total synthesis

……. an antibiotic: Erythromycin A

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• purified enzyme:

NAD(P)H

Reductase Protein (CRP, NPR)

RH + O2+ NAD(P)H + H+ → ROH + NAD(P)+ + H2O

• whole cell approach

Problems:

stability

limited substrate uptake

low conversion rates

toxicity of the substrate or of the products

thermal stability

heterologous expression

engineering enzymes

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Taxol, a diterpenoid used as antineoplastic drug

8 out 19 steps in the taxol biosynthesis depend on CYP450 enzymes

• two CYP450 have been expressed in yeast (Saccaromices cerevisiae) toghether with their NPR from Taxus cuspidata ….

Taxus brevifolia

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Taxol

Biotechnol. Bioeng. 2005 89 588

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the right enzyme CYP153 in a different organism

(-)-perillyl alcohol

Terpene derivative with anticarginogenic properties (Phase II for breast, colorectal, pancreatic cancer )

(-) limonene

• Bacillus stearothermophilus BR388 not regiospecific

• Mycobacterium sp. strain HXN-1500 regio and stereospecificCYP153 + ferridoxin + ferridoxin reductase

• expressed in Pseudomonas p.Appl. Environ. Microbiol. 2005 71 1737

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Biotransformation of steroids

Cortisol11-Deoxycortisol

Curvularia sp.

• Immobilization of fungal spores

• cosolvent and cyclodextrines to improve steroid solubility in aqueous medium

100 tons per year

Schering / now Bayer HealthCare

11β-hydroxylation

Problems:

substrate solubility in aqueous medium

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Human CYPs expressed in E.coli

membranes

cells

Drug primary metabolites are often bioactive molecules

Synthesis of drug metabolites with human CYPs →heterologous expression

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Potato transfected with CYP1A1 become resistant to chlortoluron (CT)

Human CYPs are able to transform pollutants, PAH

Plant transfection to afford resistance to herbicides

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Rice plants transfected with human CYP2A1, 2B6, 2C19

Tolerance to atrazine, metolachlor, norflurazon

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Bioremediation?

Purified plant enzyme (+ NPR) immobylization in polyaphrons (oil-in-water emulsions)

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Enzyme engineeringto improve the enzyme performance

directed evolution

Nature review Drug Discovery 2002 1 359

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Enzyme engineeringto improve the enzyme performance

fusion protein (CPR+CYP450)

rational approach (conserved sequence motif, substrate recognition sites)

2006 24 324

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to avoid costly reductants

NADH vs NADPH

peroxide (H2O2 or cumene hydroperoxide)

electrodes

in a single polypeptide chain heme and flavine binding domains [1048 aa]

Enzyme engineering

Substrate and O2

BM3 from Bacillum MegateriumCYP102 (CYPBM3)

P450

2 e-FAD

FMN

NADPH

heme

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CYP102 (CYPBM3)

native activity →subterminal hydroxylation of C12-C20fatty acids

Enhanced tolerance to H2O2

Enhanced tolerance to organic solvents

Improved thermal stability

Enzyme engineering afforded mutants with

BM3 from Bacillum Megaterium

2UWHBM3-PALM Heme domain

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Enantioselective epoxidation of terminal alkenes

Drug metabolite production

Enzyme engineering afforded mutants able to perform

NADPH regeneration via ADH

BM3 from Bacillum Megaterium

E.Coli lysate

Frances H. Arnold

ADH alcool deidrogenasi

da Thermoanaerobium Brockii stabile e specifica per piccoli alcoli secondari

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25 mutations

Frances H. Arnold

Short-chain alkane hydroxylation

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CYP101 (CYPcam) mutants vs CYP102 (CYPBM3) mutants

Short chain alkane hydroxylation (ethane → ethanol)

Styrene, ethylbenzene oxidation

Enzyme engineering afforded mutants able to perform

F87W/Y96H mutant showed enhanced activity towards PAH

F87W/Y96H/V247L mutant showed enhanced activity pentachlorobenzene

CYP101 (CYPcam)native activity → regio and stereo-selective camphor 5-exo-hydroxylation

(+)-valencene (+)-nootkatone

CYP101 mutants slower rate high selectivity

CYP102 mutants higher rate poor selctivity

aroma di pompelmo, ma anche repellente e insetticida ecologico!

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Direct electron supply from electrodes

unmodified electrodes → no E shift upon substrate binding, no products observed

Problems:

correct enzyme conformation ( P420)

coupling of electron flow to substrate conversion

reductase binding domain substrate binding domain

positive patch negative patch cysteine gold spacefill

E shift

current

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modified electrodes → clay→ surfactants (DDAB, PEI, PSS, PEO), gold NPs& chitosan→ covalent linkage (protein-spacer-electrode)→ covalent linkage of engineered protein→ engineered electrode (microsome)

cisteamina

GMBS

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coupling current to substrate conversion

→ high uncoupling percentage

Metoprolol current atengineered electrode (microsome)

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Hydrophobic electrode surfaces

CPR/CYP in microsomes

Scepticism: CYP3A4 poor selectivity

better CYP7A (cholesterol) or CYP51(lanosterol)

Good responses using

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Towards sensor chip for ligand binding

Ligand binding affects spin state and SQUID response

NHS = N-idrossisuccinimide

EDC = Derivato di carbodiimide

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Gene Directed Enzyme Pro-drug TherapyGDEPT

Cyclophosphamide (CPA) a pro-drug

4- hydroxycyclophosphamide(4OH-CPA): the drugTumor population

with tranducedtumor cell

Pro-drug activatingP450-enzyme

CPA 4OH-CPA

PRO-DRUG ACTIVATION BASED CHEMOTHERAPY

Amplification of responsevia bystander cytotoxicity

Mol Cancer Ther 2007;6(11)

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