Biotransformation Presented By PreetHi. G. U i sem msC BioteCHnoloGy
Biotransformati on Chemical alteration of a substance within the body, as by the action of enzymes Vital to survival Key in defense mechanism….
Uptake and excretion of hydrophilic and lipophilic compounds UPTAKE UPTAKE UPTAKE BIOTRANS- ORGAN ORGAN FORMATION EXCRETION EXCRETION EXCRETION Primarily biotransformation makes lipophilic compounds more hydrophilic
REACTIONS PHASE I : modification PHASE II : conjugation PHASE III : transport
A small polar group is either exposed on the toxicant or added to the toxicant… Oxidation Reduction Hydrolysis Acetylation
PHASE I REACTION OXIDATION substrate loses electrons addition of oxygen, dehydrogenation, or simply transfer of electrons…
Aliphatic hydroxylation Sulphur oxidation R - CH2 – CH2 – CH3 R – CH2 – CHOH – CH3 R - S - R’ R - S - R’Aromatic hydroxylation De-sulphurnation S O R R OH R1R2P - X R 1R2P - X + SEpoxidation Oxidative dehalogenation O X X O R - CH CH - R’ R - CH - CH - R’ R-C-H R - C - OH R - C - H + HXN-, O-, or S-dealkylation H H H R - (N, O, S) - CH3 R – (NH2, OH, SH) + CH2O Deamination OR – CH2 – NH2 R - C - H + NH3N - hydroxylation O O R - NH - C – CH3 R - NOH - C – CH3
PHASE I REACTION REDUCTION Substrate gains electrons Occurs when oxygen content is low Common reaction ○ azo reduction ○ dehalogenation ○ disulfide reduction ○ nitro reduction ○ N-oxide reduction ○ sulfoxide reduction
PHASE I REACTION HYDROLYSIS Addition of water splits the molecule into two fragments or smaller molecules -OH gp to one fragment and –H to other Eg : Larger chemicals such as esters, amines, hydrazines, and carbamates
Conjugation Endogenous substance is added to the reactive site of the Phase I metabolite more water-soluble
t tyPe ii Methylation Peptide conjugation Glucuronidation Glutathione conjugation Sulfation Glycosylation Acetylation
GLUCURONIDE CONJUGATION glucuronic acid from glucose Sites involve substrates having O2, N2 or S bonds Includes xenobiotics as well as endogenous substances Reduces toxicity..(sometimes produce carcinogenic substances) Excreted: kidney or bile depending on conjugate size
GlUCUronide ConJUGation COOH COOH O O GlucuronylR – OH + O UDP transferase O R + UDP OH OH HO HO OH OH
SULPHATE CONJUGATION Decreases toxicity readily excreted by urine Sulphotransferase PAPS limits the pathway
glucuronidation or sulfation can conjugate the same xenobiotics Primary, secondary, phenols, catechols, N- oxides, amines undergo this…
GLUTATHIONE CONJUGATION Conjugate loses glutamic acid and glycine Cysteine is N-acetylated to give stable mercapturic acid derivatives
H H NGlutamic H O H Hacid N O O O H O H O H H N + O H H S O N H HCysteine S O N O O H O + H H H N GlutathioneGlycine O O H
ACETYLATION the water solubility of parent molecule and their excretion Masks the functional group of parent from participating in conjugations Acetyl transferases Aromatic amines or hydrazine group to amides or hydrazides
Methylation Makes slightly less soluble Masks available functional groups Types O- methylation N- methylation S- methylation
ENZYMESENZYMES microsomal…. Phase I and glucuronidation enzymes Cytosolic enzymes….phase II and oxidation and reduction Mitochondrial, nuclei and lysosomes contain a little transforming activity….
MICROSOMAL NONMICROSOMALPhase I reactions Phase I reactions – Most oxidation and – Most hydrolysis reduction – Some oxidation and – Some hydrolysis reduction Phase II reactionsPhase II reactions ALL except Glucuronide – ONLY Glucuronide conjugation conjugation • Not inducible• Inducible CP, MT etc – Drugs, diet, etc. SER
CYTOCHROME P450 ENZYME SYSTEM Mixed function oxidase Commonly in microsomes Important in plant terpenoid biosynthesis In phase I reactions Contains 2 enz NADPH CYP reductase and cyp 450
CYTOCHROME P450 ENZYME SYSTEM superfamily of heme-dependent proteinsexpressed in mammals mainly in the liver, with lower levels of expression in the small intestine, lungs, kidneys, brain and placentaIn man, to date 57 different P450 isoforms have been identified, which were assigned to 18 families and 43 subfamilies based on their protein sequences
TYPES Microsomal P450 systems: electrons are transferred from NADPH via cytochrome P450 reductase. Mitochondrial P450 systems: employ adrenodoxin reductase and adrenodoxin to transfer electrons from NADPH to P450.
Bacterial P450 systems: employ a ferredoxin reductase and a ferredoxinCYB5R/cyb5/P450 systems: both electrons required by the CYP come from cytochrome b5.FMN/Fd/P450 systems: originally found in Rhodococcus sp. in which a FMN-domain-containing reductase is fused to the CYP.P450 only systems, which do not require external reducing power. Notable ones include CYP5 (thromboxane synthase), CYP8(prostacyclin synthase), and CYP74A ( allene oxide synthase).
NOMENCLATURE 40% seq homology Cyto proteins Families Coloured Designated by numerals 450nm>55% seq homology CYP2D6 40-55% aa seq homol Subfamilies Iso enzymes Designated by capital letters Designated by numerals
Ah receptor-hsp90 Cell HC HC(inducer) Nucleus HC-AhR HC-AhR P450 gen hsp90 XRE P450 protein P450 mRNA • Bioactivation Toxicity • Detoxification HC: Hydrocarbon (inducer) Elimination XRC: Regulator gene (stimulates transcription of P-450 gene)
P450 family FunctionCYP1, CYP2, CYP3 Metabolism of drugs and xenobiotics Fatty acids hydroxylation, biosynthesis of prostaglandins, prostacyclins and thromboxanesCYP4, CYP5, CYP8CYP7, CYP11, CYP17, CYP19(=steroid aromatase), CYP21,CYP24, CYP27, CYP39, CYP46,CYP51 Biosynthesis and metabolism of cholesterol, steroid hormones and bile acidsCYP26 Retinoic acid hydroxylationCYP20 Unknown
FLAVIN MONO OXYGENASE Microsomal enzyme mixed function amine oxidase Cofactors: NADPH, molecular O₂ Do not contain heme Broad specificity Nicotine detoxification
OTHER ENZYMES Monoamine oxidases- breakdown of neurotransmitters and antidepressant drugs Alcohol and aldehyde dehydrogenases
BIOTRANSFORMATION IN MICROORGANISMS elimination of wide range of pollutant and waste removal of contaminants by degrade/convert such compounds. adapt and become quite rapidly selected to xenobiotic compounds introduced into the environment, mainly via the usage of the compound as carbon, energy or nitrogen source.
CYP IN MICROORGANISMS Cyt P450cam (CYP101): first cytP450 3D protein structure solved by X-ray crystallography part of a camphor-hydroxylating catalytic cycle consisting of two electron transfer steps from putidaredoxin, a 2Fe-2S cluster- containing protein cofactor.
Cytochrome P450 eryF (CYP107A1) originally from the actinomycete bacterium Saccharopolyspora erythraea is responsible for the biosynthesis of the antibiotic erythromycin by C6-hydroxylation of the macrolide 6-deoxyerythronolide B.
Cyt P450 BM3 (CYP102A1) from the soil bacterium Bacillus megaterium catalyzes the NADPH-dependent hydroxylation of several long-chain fatty acids at the ω–1 through ω– 3 positions..
CytP450 119 (CYP119) isolated from the thermophillic archea Sulfolobus acidocaldarius has been used in a variety of mechanistic studies function at high temperatures, they tend to function more slowly at room temperature (if at all) and are therefore excellent mechanistic models.
IN FUNGI The commonly used azole class antifungal drugs work by inhibition of the fungal CYP 14α-demethylase. This interrupts the conversion of lanosterol to ergosterol, a component of the fungal cell membrane. Cunninghamella elegans is a candidate for use as a model for mammalian drug metabolism Significant research is going on…
BIOTRANSFORMATION IN PLANTSo large amounts of peroxidases in plantso small amounts of CYP in plant tissueso a low substrate specificity of plant peroxidases as compared to the high specificity of the plant CYP
o a wide range of action of plant peroxidaseso the similarity of in vivo metabolites of several xenobiotics in plants to those formed in vitro by peroxidases rather than to those resulting from cytochrome P-450-dependent in vitro reactionso high affinities of peroxidases to exogenous substrates
o peroxidases are located in all parts of plant cells, the plant CYP are located in the microsomal fraction only.
In plants…. Transformation occurs in pesticide and heavy metals Using plant cell cultures
CO-METABOLISM Multistepprocess Not used for energy production Not a constitutive element of organism Secondary substrate metabolism Enzyme A ----------> Enzyme B -------------> Enzyme C Substrate A ----------> Product B ------------> Product C Substrate Ax-----------> Product Bx [not metabolized by enzyme C] Substrate Ax is "sufficiently similar" to Substrate A that Enzyme A can transform it to Bx, but Bx is "sufficiently different" to B so as to prevent further metabolism by Enzyme C.
OTHER ENZYMES INVOLVED Peroxidases Phenolases Other oxidoreductases Hydrolytic enzymes Polymerisationof various anilines and phenols Usually decreases toxicity
HYDROLYTIC ENZYMES Metabolise substrates containing amide, carbamate or ester functional group Extracellular Anaerobic or aerobic
ESTER HYDROLYSIS Esterases, lipases, proteases GLY-X-SER-X-GLY The SER acts as a nucleophile, enabling ester bond cleavage Increases absorption and selectivity Ester bond metabolised to form acid (more toxic) which is desterified
ROLE OF GST AND GSH IN PLANTS Metabolism of secondary products(cinnamic acid, anthocyanins) Regulation and transport of both endo and exogenous compounds Protection against oxidative stress Involved by vacuoles
NON SPECIFIC REACTIONS Nitroreduction Hydroxylations Glucosylation Oxido-reductions between alcohols and ketones Hydrolysis Epoxidation Reductions of carbonyl groups Reduction of C–C double bond
REACTION EXAMPLE Warfarin to alcohol(C.roseus) Hydroxylation Nitroreduction TNT to ADNT(D.inoxia) Butyric acid to 6- o butyryl- Glucosylation glucose(N.plumbaginifolia Oxido reductions Alcohols to ketones(N.tabaccum) 1-phenyl ethyl acetate to R Hydrolysis alcohols(Spirodela oligorrhiza)
Epoxidation (−)-(4R)-isopiperitinone to (−)-7- hydroxyisopiperitonone(Menthapip erita) Reduction of carbonyl Ketones and aldehydes to group alcohols(N.sylvestris) Reduction of C=C Carvone reduction(Astasia longa)
Major conjugation reactions in plants and animals Glucuronide formation prevalent in vertebrates Glycoside formation prevalent in plants and insects Mercapturates animals only Cysteine conjugation plants and animals Gycine conjugation plants and animals Other aminoacid conjugation plants and animals Sulphate conjugation prevalent in animals rare in plant O and S methylation animals and plants Thiocyanate formation animals and plants N- acetylation animals and plants
IN HUMAN Mainly haemoglobin biotransformation Detoxification Drug metabolism Transformation of endogenous molecules hormone synthesis and breakdown cholesterol synthesis vitamin D metabolism..
CYP IN HUMANS The Human Genome Project has identified 57 human genes coding for the various cytochrome P450 enzymes.
PROCESSING OF PROCARCINOGEN BYBIOTRANSFORMING ENZYMES
CROHN’S DISEASE An imbalance between toxic compounds and detoxifying substances on the luminal side of the gut inflammation of the intestinal mucosa ANTLEY-BIXLER SYNDROME Abnormal production of cholesterol Mutation in POR gene
APPLICATIONS Therapeutic drug monitoring Cancer chemo therapy and drug metabolism Oil degradation in marine systems Natural attenuation and bioremediation Waste biotreatment Aerobic and anaerobic degradation of organic pollutants Transformation of specific substrates into products of interest in vitro
REFERENCE http://www.eoearth.org/article/Biotransformation?topic=58074 profiles.nlm.nih.gov/ps/access/CCAAOR.pdf www.slideshare.net/shishirkawde/biotransformation-10417087 www.eolss.net/sample-chapters/c17/e6-58-04-06.pdf www.ncbi.nlm.nih.gov/pubmed/3116933 ingentaconnect.com RK Venisetty, V Ciddi - Current pharmaceutical biotechnology, 2003 web.squ.edu.om/med-Lib/MED_CD/E_CDs/.../020160r00.HTM www.eoearth.org/article/Biotransformation