Biotransformation refers to the chemical alteration of substances within living organisms, typically involving enzymatic reactions. These reactions make compounds more water-soluble so they can be more easily excreted from the body. Biotransformation occurs in three phases - Phase I involves oxidation, reduction, and hydrolysis reactions; Phase II involves conjugating reactions like glucuronidation and sulfation; Phase III involves transport of conjugated compounds out of cells and organs. The liver is a major site of biotransformation, with cytochrome P450 enzymes and conjugating enzymes playing important roles in Phase I and Phase II reactions. Biotransformation is vital for the metabolism of drugs and other xenobiotics in the body.

1. Biotransformation
Presented By
PreetHi. G. U
i sem msC BioteCHnoloGy

2. Biotransformati
on
 Chemical alteration of a substance within the
body, as by the action of enzymes
 Vital to survival
 Key in defense mechanism….

5. 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

6. REACTIONS
 PHASE I : modification
 PHASE II : conjugation
 PHASE III : transport

8.  A small polar group is either exposed on
the toxicant or added to the toxicant…
 Oxidation
 Reduction
 Hydrolysis
 Acetylation

9. PHASE I REACTION
OXIDATION
 substrate loses electrons
 addition of oxygen, dehydrogenation, or
simply transfer of electrons…

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

11. 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 + S
Epoxidation Oxidative dehalogenation
O
X X O
R - CH CH - R’ R - CH - CH - R’
R-C-H R - C - OH R - C - H + HX
N-, O-, or S-dealkylation H H
H
R - (N, O, S) - CH3 R – (NH2, OH, SH) + CH2O
Deamination O
R – CH2 – NH2 R - C - H + NH3
N - hydroxylation
O O
R - NH - C – CH3 R - NOH - C – CH3

12. 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

14. 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

16.  Conjugation
 Endogenous substance is added to the
reactive site of the Phase I metabolite
 more water-soluble

18. t
tyPe ii
 Methylation  Peptide conjugation
 Glucuronidation  Glutathione conjugation
 Sulfation  Glycosylation
 Acetylation

19.  glucuronide conjugation
 sulfate conjugation
 acetylation
 amino acid conjugation
 glutathione conjugation
 methylation

20. COFACTORS
 tyPe
1- reaCtive/ aCtivated
CofaCtor
a)UDP- Glucuronic acid
b)PAPS
c)Acetyl CoA
d)SAM

21.  tyPe 2- reaCtive XenoBiotiC
a)Glutathione
b)Aminoacids(glycine,glutamine,
taurine)

22.  Glucuronosyltransferase
 Sulfotransferase
 Glutathione-S-transferase
 Acetyltransferase

23.  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

25. GlUCUronide ConJUGation
COOH COOH
O O
Glucuronyl
R – OH + O UDP transferase O R + UDP
OH OH
HO HO
OH OH

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

27. SULFATE CONJUGATION

28.  glucuronidation or sulfation can conjugate
the same xenobiotics
 Primary, secondary, phenols, catechols, N-
oxides, amines undergo this…

29. GLUTATHIONE CONJUGATION
 Conjugate loses glutamic acid and glycine
 Cysteine is N-acetylated to give stable
mercapturic acid derivatives

30. H H
N
Glutamic H O H H
acid N
O O
O H
O H
O H
H N
+ O
H H S O
N H
H
Cysteine S O N
O
O
H O
+ H
H H
N Glutathione
Glycine O
O
H

33. 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

34. Methylation
 Makes slightly less soluble
 Masks available functional groups
 Types
O- methylation
N- methylation
S- methylation

36. PHASE II REACTIONS
 Aminoacid conjugation

37. GENETICS
Nfr2- nuclear factor erythroid derived
Inactive oxidative stress active
CP nucleus

38.  Additional conjugation reaction
 ABC family (MDR proteins)
 Conjugates and their metabolites can be
excreted from cells

39.  Anionic transporter :
OATP1B1/SLCO1B1
 Cationic transporters :
OATP1B3/SLCO1B3
 ABC transporters: P glycoprotein

41. ENZYMES
ENZYMES
 microsomal…. Phase I and glucuronidation enzymes
 Cytosolic enzymes….phase II and oxidation and
reduction
 Mitochondrial, nuclei and lysosomes contain a little
transforming activity….

42. MICROSOMAL NONMICROSOMAL
Phase I reactions Phase I reactions
– Most oxidation and – Most hydrolysis
reduction – Some oxidation and
– Some hydrolysis reduction
Phase II reactions
Phase II reactions ALL except Glucuronide
– ONLY Glucuronide conjugation
conjugation • Not inducible
• Inducible  CP, MT etc
– Drugs, diet, etc.
 SER

43. ENZYMES
 High molecular weight proteins..

44. CYP
FLAVIN MONOOXYGENASES

45. 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

46. CYTOCHROME 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

47. REDUCTASE
P-450 P-450

48. 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.

49.  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).

50. 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

51. CYTOCHROME P 450 ENZYME ACTION

52. 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)

53. P450 family Function
CYP1, CYP2, CYP3 Metabolism of drugs and xenobiotics
Fatty acids hydroxylation, biosynthesis of
prostaglandins, prostacyclins and thromboxanes
CYP4, CYP5, CYP8
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

54. FLAVIN MONO
OXYGENASE
 Microsomal enzyme
 mixed function amine oxidase
 Cofactors: NADPH, molecular O₂
 Do not contain heme
 Broad specificity
 Nicotine detoxification

55. OTHER ENZYMES
 Monoamine oxidases- breakdown of
neurotransmitters and antidepressant
drugs
 Alcohol and aldehyde dehydrogenases

57. BIOTRANSFORMATION SITES
 Liver
 Lung
 Kidney
 Intestine
 Gut
 Skin
 Gonads

58. 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
Seminiferous tubules,
 Testes
sertoli cells

59. MICROSOMAL
FRACTION

60. PHASES OF BIOTRANSFORMATION

61. IMPORTANCE
 Drug metabolism
 Factor in multidrug resistance
 Cancer chemo therapy
 Environmental science- bioremediation or
persistence in environment

62. BIOTRANSFORMATION OF ALCOHOL

63. INTESTINAL FLORA AND
BIOTRANSFORMATION

64. DRUG METABOLISM

65. REACTION OCCURING….

69. 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.

70. 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.

71.  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.

72.  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..

73.  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.

74. 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…

76. 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

77. 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

78. o peroxidases are located in all parts of plant
cells, the plant CYP are located in the
microsomal fraction only.

79.  In plants….
 Transformation occurs in pesticide and heavy
metals
 Using plant cell cultures

80.  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.

81. REACTIONS INVOLVE…
OXIDATION
REDUCTION
HYDROLYSIS
CONJUGATION

82. OTHER ENZYMES
INVOLVED
 Peroxidases
 Phenolases
 Other oxidoreductases
 Hydrolytic enzymes
 Polymerisationof various anilines and phenols
 Usually decreases toxicity

83. HYDROLYTIC ENZYMES
 Metabolise substrates containing amide,
carbamate or ester functional group
 Extracellular
 Anaerobic or aerobic

85. 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

87. Amide hydrolysis

88. 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

89. PHASE III
 Additional conjugation

90. NON SPECIFIC
REACTIONS
 Nitroreduction
 Hydroxylations
 Glucosylation
 Oxido-reductions between alcohols and ketones
 Hydrolysis
 Epoxidation
 Reductions of carbonyl groups
 Reduction of C–C double bond

91. 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)

92.  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)

93. 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

94. IN HUMAN
 Mainly haemoglobin biotransformation
 Detoxification
 Drug metabolism
 Transformation of endogenous molecules
 hormone synthesis and breakdown
 cholesterol synthesis
 vitamin D metabolism..

95. CYP IN HUMANS
 The Human Genome Project has identified 57
human genes coding for the various cytochrome
P450 enzymes.

96. PROCESSING OF PROCARCINOGEN BY
BIOTRANSFORMING ENZYMES

97.  AGE
 GENDER
 GENETIC VARIABILITY
 POOR NUTRITION AND DISEASES
 DOSE LEVELS

98. SYNDROMES ASSOCIATED…
 GILBERTS SYNDROME
Reduced activity of glucuronyl transferase
Hyper bilirubinemia
Develops jaundice
 CRIGLER-NAJJAR SYNDROME
Autosomal recessive disorder
No UDP glucuronosyltransferase

99.  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

100. 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

101. 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