Chemical conversion of a substance mediated by living organisms or enzymes Can result in DETOXIFICATION and BIOACTIVATION Vital to survive Key in defense mechanism

1. MBB – 609 – Plant tissue culture and genetic transformation(1+2)
Topic – Biotransformation
Department of Agricultural
Biotechnology
Presented By -
Jyoti Prakash Sahoo
01ABT/PHD/17
Dept. of Agril. Biotech.
OUAT, BBSR
Course Instructor –
Dr. Gyana Ranjan Rout
Professor and Head
Dept. of Agril. Biotech.
OUAT, BBSR
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Biotransformation
 C h e m i c a l c o n ve r s i o n o f a s u b s t a n c e m e d i a t e d b y l i vi n g
o r g a n i s m s o r e n z y m e s
 C a n r e s u l t i n D E TO X I F I C AT I O N a n d B I O AC T I VAT I O N
 Vi t a l t o s u r vi ve
 K e y i n d e f e n s e m e c h a n i s m
R T
The modern field of xenobiotic
metabolism grew from the convictions of
a Welshman named R. Tecwyn
Williams

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PHASE I : modification
PHASE I I : conjugation
PHASE I I I : transport
Biotransformation
Reactions
 Oxidation
 Reduction
 Hydrolysis
 Acetylation
o Glucuronide conjugation
o Sulfate conjugation
o Acetylation
o Amino acid conjugation
o Glutathione conjugation
o Methylation

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Phase I - Modification
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
Reaction
Involved

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REDUCTION –
 Substrate gains electrons
 Occurs when oxygen content is low
azo reduction
dehalogenation
disulfide
reduction
nitro reduction
N-oxide reduction
sulfoxide
reduction
Reaction
Involved
HYDROLYSIS –
 Addition of water splits the molecule into two fragments or smaller
molecules
 -OH to one fragment and –H to other
 Eg : Larger chemicals such as esters, amines, hydrazines, and

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Phase II - Conjugation
 Endogenous substance is added to the reactive site of the Phase I
metabolite
 more water-soluble
 Type I reactive/Activated Cofactor
a) UDP- Glucuronic acid b) PAPS (3'-
Phosphoadenosine-5'-phosphosulfate (PAPS ) c)
Acetyl CoA d) SAM (S-Adenosyl methionine)
 Type II reactive Xenobiotics
a) Glutathion b) Aminoacids (Glycine, Glutamine,
Taurine)
Cofactors
 Glucuronosyl
transferase
Sulfotransferas
Acetyltransferase
Glutathione – S -
Enzymes

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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)

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SULPHATE
CONJUGATION
Decreases toxicity readily excreted by urine
 Sulphotransferase
 PAPS limits the pathway
GLUTATHION
CONJUGATION
•Conjugate loses glutamic acid and
glycine
•Cysteine is N-acetylated to give
stable mercapturic acid derivatives

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 The water solubility of parent molecule and their excretion
 Masks the functional group of parent from participating in
conjugations
 Enzyme involved - Acetyl transferases
 Aromatic amines or hydrazine group to amides or hydrazides
ACETYLATIO
N
METHYLATIO
N
Makes slightly less soluble
Masks available functional
groups

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 Additional conjugation reaction
 Conjugates and their metabolites can be excreted from cells
Phase III - Transport
Anionic transporter :
OATP1B1/SLCO1B1
Cationic transporters :
OATP1B3/SLCO1B3
ABC transporters: P glycoprotein
Transporter
s
Additional
Conjugation

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ENZYMES
ENZYMES – High Molecular Weight Protein
 microsomal…. Phase I and glucuronidation enzymes
 Cytosolic enzymes….phase II and oxidation and
reduction
 Mitochondrial, nuclei and lysosomes contain a little
transforming activity….
CYTOCHROME P450 ENZYME
SYSTEM
 Mixed function oxidase
 Commonly in microsomes
 Important in plant terpenoid biosynthesis
 Contains 2 enzymes NADPH CYP reductase and cyp 450

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FLAVIN MONO OXYGENASE
 Microsomal enzyme
 Mixed function amine oxidase
 Cofactors: NADPH, molecular O₂
 Do not contain heme group
 Broad specificity
 Nicotine detoxification
 Monoamine oxidases- breakdown of
neurotransmitter and antidepressant drugs
 Alcohol and aldehyde dehydrogenases

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SITES FOR BIOTRANSFORMATION IN ANIMALS - ENZYME
CONTAINING CELLS IN VARIOUS ORGANS
 Liver - Parenchymal cells
 Kidney - Proximal tubular cells
 Lung - Clara cells, type II alveolar cells
 Intestine - Mucosa lining cells,
enterocytes
 Skin - Epithelial cells
 Testes - Seminiferous tubules, sertoli
cells
Phase of
Biotransformation

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DRUG METABOLISM

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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
i. Indirect transformation:- In controlled biosynthesis,
altered antibiotics are produced in the presence of
inhibitors or modified precursors in the medium.
ii. Direct transformation: Hydrolysis of the functional
groups led to inactivation of the antibiotics.
Transformation of Antibiotics

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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
In Plants
 Transformation occurs in pesticide and heavy
metals
 Using plant cell cultures

17.  M a y o c c u r vi a m u l t i s t e p p r o c e s s e s k n o w n a s c o -
m e t a b o l i s m
 B i o t r a n s f o r m a t i o n o f a n o r g a n i c c o m p o u n d n o t
u s e d a s a n e n e r g y s o u r c e o r a s a c o n s t i t u t i ve
e l e m e n t o f t h e o r g a n i s m .
Pesticide
Biotransformation
Individual reactions of degradation–detoxification
pathways
o Oxidation
o Reduction
o Hydrolysis
o Conjugation
Diverse Metabolic Pathways depend
on
• the chemical structure of the xenobiotic
compound
• the organism
• environmental conditions
• metabolic factors
• the regulating expression of these
biochemical pathways
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18. Pesticide Biotransformation - A three-phase
process
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 Generally, phase II metabolites have
little or no phytotoxicity and may be
stored in cellular organelles.

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 Oxidative Transformations
 mediated by oxidative enzymes,
 e.g., cytochrome P450s- The most extensively studied
oxidative enzymes
 peroxidases and polyphenol oxidases
Primary
Metabolism
 Monooxygenase reaction, e.g., insertion of
one atom of oxygen into an organic
substrate (RH) while the other oxygen atom
is reduced to water
cytochrome P450s
RH + O2 + NAD(P)H + H+ ROH + H2O +
NAD(P)+
Other P450 - mediated
reactions
• Dehydration
• Dimerization
• Deamination
• Dehydrogenation
• Heteroatom
dealkylation
• Epoxidation
• Reduction
• C–C or C=N cleavage

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Peroxidases, Phenoloxidases, and Related
Oxidoreductases
 catalyze the polymerization of various anilines and phenols
 In most instances, polymerization products have reduced
toxicity compared with the substrate
Other
reactions

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Hydrolytic
Transformations
 Hydrolytic enzymes
 capable of metabolizing a variety of substrates, particularly
those containing amide, carbamate, or ester functional
groups
 compartmentalized or extracellular
 reactions can occur under aerobic or anaerobic conditions
Hydrolysis

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Carbon–Phosphorus Bond Cleavage
Reactions
Biotransformation of glyphosate, highlighting C–P lyase and glyphosate oxidoreductase (GOX)
enzymatic reactions.

23.  All Roundup Ready crops contain an enzyme known as EPSPS (5-
enolpyruvylshikimate-3-phosphate synthase) that is resistant to
the effects of glyphosate.
 The enzyme is an important catalyst in the biochemical pathway for
synthesis of the aromatic amino acids phenylalanine, tryptophan,
and tyrosine.
EPSP
S
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Biotransformation using plant cultured
cells
Plant cell cultures exhibit a vast biochemical potential for
production of specific secondary metabolites.
 A wide variety of chemical compounds including aromatics,
steroids, alkaloids, coumarins and terpenoids can undergo
biotransformations using plant cells, organ cultures and
enzymes.
• Oxidations
• Reductions
• Hydroxylations
• Methylations
• Acetylations
• Isomerizations
• Glycosylations
• Esterfications
Types of
reactions

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Nitroreductio
n
 Biotransformation of TNT into 2,4,6-
aminodinitrotoluene (ADNT) has been
investigated in plant cell cultures of
Datura innoxia, C. roseus and
Myrophyllum plants
Datura innoxia
Myrophyllum
Catharanthus
roseus
Glucosylati
on
 Butyric acid to obtain 6-O-butyryl-D-
glucose, which extends its half-life and
prolongs its bioactivity –
Nicotiana plumbaginifolia
 Phenylcarboxylic acids
 Glycyrrhiza echinata
 Aconitum japonicum
 Dioscoreophyllum cumminsii
 N. tabacum
Glycyrrhiz
a echinata
Aconitum japonicum
Dioscoreophyllu
m cumminsii
N. tabacum

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 Callus cultures of Myrtillocactus geometrizans and
N. tabacum
 Biotransformed Δ2-carene into diastereomeric
alcohols
 Myrtillocactus oxidized these alcohols to the
corresponding ketones.
Myrtillocactus
 Enantio selective hydrolysis
 useful for the optical resolution of racemic
acetates
 biotransformation of (RS)-1-phenylethyl acetate and
its derivatives
 cultured cells of Spirodela oligorrhiza

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Carbonyl group
 reduction of ketones and aldehydes to the
corresponding alcohols
 Whole cells, cell-free extracts or culture broth from cell
suspension cultures of N. sylvestris or C. roseus
C–C double bond
 Cultured cell lines of Astasia longa produced two different
enone reductases, which reduced the C–C double bond
of carvone
N. sylvestris
 useful for the modification of cytotoxic
sesquiterpenes
 biotransformation of isopiperitinone by Mentha
piperita yielded
 three hydroxylated derivatives
 two epoxidized derivatives

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Applicatio
n
 prediction of metabolites that are likely present before initiation of an in
vivo study
 generation of metabolites in sufficient quantities for identification
 detection of intermediate metabolites, which may provide insight into the
metabolic pathway
 characterization of nonextractable residues
 ‘‘metabolic profiling’’ to determine the rate and pattern of metabolism
between species,
 determination of genetics and enzymology of the metabolic pathway
 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

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Reference
s
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

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