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.
2. Biotransformati
on
Chemical alteration of a substance within the
body, as by the action of enzymes
Vital to survival
Key in defense mechanism….
3.
4.
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
7.
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…
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
13.
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
15.
16. Conjugation
Endogenous substance is added to the
reactive site of the Phase I metabolite
more water-soluble
17.
18. t
tyPe ii
Methylation Peptide conjugation
Glucuronidation Glutathione conjugation
Sulfation Glycosylation
Acetylation
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
24.
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
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
31.
32.
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
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
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
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
61. IMPORTANCE
Drug metabolism
Factor in multidrug resistance
Cancer chemo therapy
Environmental science- bioremediation or
persistence in environment
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…
75.
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.
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
84.
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
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
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.
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