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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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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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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)