Biotransformation Presented by

PREETHI. G. UI sem MSC biotechnology

BIOTRANSFORMATION

Chemical alteration of a substance within the body, as by the action of enzymes

Vital to survival

Key in defense mechanism….

UPTAKE

ORGAN

EXCRETION

ORGAN

UPTAKE

EXCRETION

Primarily biotransformation makeslipophilic compounds more hydrophilic

UPTAKE

EXCRETION

BIOTRANS-FORMATION

Uptake and excretion of hydrophilic and lipophilic compounds

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…

alcohol dehydrogenation aldehyde dehydrogenation alkyl/acyclic hydroxylation aromatic hydroxylation deamination desulfuration N-dealkylation N-hydroxylation N-oxidation O-dealkylation sulphoxidation

Aliphatic hydroxylation

Aromatic hydroxylation

R R OH

R - CH CH - R’ R - CH - CH - R’

Epoxidation

O

N-, O-, or S-dealkylation

R - (N, O, S) - CH3

H

N - hydroxylation

Deamination

R - C - H + NH3

O

OO

R - NH - C – CH3 R - NOH - C – CH3

R - C - H R - C - OH R - C - H + HX

OX X

H H

R - S - R’ R - S - R’

Sulphur oxidation

S

R1R2P - X R1R2P - X + S

O

De-sulphurnation

Oxidative dehalogenation

R - CH2 – CH2 – CH3 R – CH2 – CHOH – CH3

R – (NH2, OH, SH) + CH2O

R – CH2 – NH2

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

TYPE I Methylation

Glucuronidation

Sulfation

Acetylation

TYPE II Peptide conjugation

Glutathione conjugation

Glycosylation

glucuronide conjugation

sulfate conjugation

acetylation

amino acid conjugation

glutathione conjugation

methylation

COFACTORSCOFACTORSType 1- Reactive/ Activated

Cofactor a)UDP- Glucuronic acid

b)PAPS

c)Acetyl CoA

d)SAM

Type 2- Reactive Xenobiotic a)Glutathione

b)Aminoacids(glycine,glutamine,

taurine)

Glucuronosyltransferase

Sulfotransferase

Glutathione-S-transferase

Acetyltransferase

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

R – OH + UDPO

HO

O

OH

OH

COOH

Glucuronyltransferase O

HO

O

OH

OH

COOH

R + UDP

SULPHATE CONJUGATION Decreases toxicity readily excreted by urine Sulphotransferase PAPS limits the pathway

SULFATE CONJUGATION

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

N

O

H

O

H

N

S

H

O

N

O

O

H H

OH

H

H+

+

N

OO

O

H H

O

H

H

N

O

S

H H

O

H

N

O

HH

O

H

Glutamicacid

Cysteine

GlycineGlutathione

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

MethylationMakes slightly less solubleMasks available functional groupsTypes

O- methylation

N- methylation

S- methylation

PHASE II REACTIONS

Aminoacid conjugation

GENETICSNfr2- nuclear factor erythroid derived

Inactive oxidative stress active

CP nucleus

Additional conjugation reaction

ABC family (MDR proteins)

Conjugates and their metabolites can be excreted from cells

Anionic transporter : OATP1B1/SLCO1B1

Cationic transporters : OATP1B3/SLCO1B3

ABC transporters: P glycoprotein

ENZYMES

ENZYMESmicrosomal…. Phase I and glucuronidation

enzymesCytosolic enzymes….phase II and oxidation

and reductionMitochondrial, nuclei and lysosomes contain

a little transforming activity….

MICROSOMALPhase I reactions

– Most oxidation and reduction

– Some hydrolysis

Phase II reactions– ONLY Glucuronide

conjugation• Inducible

– Drugs, diet, etc. SER

NONMICROSOMALPhase I reactions

– Most hydrolysis– Some oxidation and

reductionPhase II reactions

ALL except Glucuronide conjugation

• Not inducible CP, MT etc

ENZYMES

High molecular weight proteins..

CYP

FLAVIN MONOOXYGENASES

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 SYSTEMCYTOCHROME P450 ENZYME SYSTEM

superfamily of heme-dependent proteins

expressed in mammals mainly in the liver, with lower levels of expression in the small intestine, lungs, kidneys, brain and placenta

In man, to date 57 different P450 isoforms have been identified, which were assigned to 18 families and 43 subfamilies based on their protein sequences

REDUCTASE

P-450P-450

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 ferredoxin

CYB5R/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).

CYP2D6

Cyto proteinsColoured

450nm

FamiliesDesignated by numerals

SubfamiliesDesignated by capital letters

Iso enzymesDesignated by numerals

40-55% aa seq homology>55% seq homology

40% seq homologyNOMENCLATURE

CYTOCHROME P 450 ENZYME ACTION

HC(inducer)

Ah receptor-hsp90

HC

P450 mRNAP450 protein

• Bioactivation• Detoxification

Toxicity

Elimination

Cell

HC-AhR

hsp90

HC-AhR

XRE

P450 gen

Nucleus

HC: Hydrocarbon (inducer)XRC: Regulator gene (stimulates transcription of P-450 gene)

P450 family Function

CYP1, CYP2, CYP3 Metabolism of drugs and xenobiotics

CYP4, CYP5, CYP8

Fatty acids hydroxylation, biosynthesis of prostaglandins, prostacyclins and thromboxanes

CYP7, CYP11, CYP17, CYP19 (=steroid aromatase), CYP21, CYP24, CYP27, CYP39, CYP46, CYP51 Biosynthesis and metabolism of cholesterol, steroid

hormones and bile acids

CYP26 Retinoic acid hydroxylation

CYP20 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

http://www.sciencedirect.com/science/article/pii/S1357272511003098

BIOTRANSFORMATION SITES

Liver Lung Kidney Intestine Gut Skin Gonads

ENZYME CONTAINING CELLS IN VARIOUS ORGANS

Liver Kidney Lung

Intestine

Skin Testes

Parenchymal cells

Proximal tubular cells

Clara cells, type II alveolar cells

Mucosa lining cells, enterocytes

Epithelial cells

Seminiferous tubules, sertoli cells

MICROSOMAL FRACTION

PHASES OF BIOTRANSFORMATION

IMPORTANCE

Drug metabolism Factor in multidrug resistance Cancer chemo therapy Environmental science- bioremediation or

persistence in environment

BIOTRANSFORMATION OF ALCOHOL

INTESTINAL FLORA AND BIOTRANSFORMATION

DRUG METABOLISM

REACTION OCCURING….

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 PLANTS

o large amounts of peroxidases in plants

o small amounts of CYP in plant tissues

o 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 peroxidases o 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 reactions

o 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 Multistep process 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.

REACTIONS INVOLVE…

OXIDATIONREDUCTION HYDROLYSISCONJUGATION

OTHER ENZYMES INVOLVED

Peroxidases Phenolases Other oxidoreductases Hydrolytic enzymes

Polymerisation of various anilines and phenolsUsually 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

Amide hydrolysis

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

PHASE III

Additional conjugation

NON SPECIFIC REACTIONS

NitroreductionNitroreduction HydroxylationsHydroxylations GlucosylationGlucosylation Oxido-reductions between alcohols Oxido-reductions between alcohols

and ketonesand ketones HydrolysisHydrolysis EpoxidationEpoxidation Reductions of carbonyl groupsReductions of carbonyl groups Reduction of C–C double bondReduction of C–C double bond

REACTION Hydroxylation

Nitroreduction Glucosylation

Oxido reductions Hydrolysis

EXAMPLE Warfarin to alcohol(C.roseus)

TNT to ADNT(D.inoxia) Butyric acid to 6- o butyryl-

glucose(N.plumbaginifolia

Alcohols to ketones(N.tabaccum) 1-phenyl ethyl acetate to R

alcohols(Spirodela oligorrhiza)

Epoxidation

Reduction of carbonyl group

Reduction of C=CC=C

(−)-(4R)-isopiperitinone to (−)-7-hydroxyisopiperitonone(Menthapiperita)

Ketones and aldehydes to alcohols(N.sylvestris)

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 BY BIOTRANSFORMING ENZYMES

AGE

GENDER

GENETIC VARIABILITY

POOR NUTRITION AND DISEASES

DOSE LEVELS

SYNDROMES ASSOCIATED…

GILBERTS SYNDROMEReduced activity of glucuronyl transferase

Hyper bilirubinemia

Develops jaundice CRIGLER-NAJJAR SYNDROME

Autosomal recessive disorder

No UDP glucuronosyltransferase

CROHN’S DISEASEAn 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