Plant Biotransformation

ANAND C. R.
S2 BIOTECHNOLOGY
Biotransformation
Chemical conversion of a substance
mediated by living organisms or enzyme
p re p a r a t i o n s d e r i v e d t h e re f ro m .

In plants
• Biotransformation of Pesticides and heavy
metals
• Biotransformation using plant cultured cells

12/8/21 2
Pesticide biotransformation

• may occur via multistep processes

known as cometabolism.
–b i o t r a n s f o r ma t i o n o f a n o r g a n i c
c o m po u n d no t us e d a s a n e n e r g y
s o urc e o r a s a c o n s t i t u t i v e e l e m e n t
o f t h e o r g a n i s m.

12/8/21 3
 Individual reactions of degradation–detoxification pathways

> oxidation
> reduction
> hydrolysis
> conjugation

12/8/21 4
 Diverse Metabolic Pathways

depends on
the chemical structure of the xenobiotic
compound
the organism
environmental conditions
metabolic factors
the regulating expression of these
biochemical pathways

12/8/21 5
 A three-phase process

12/8/21 6
 • Generally, Phase II
metabolites have little or
no phytotoxicity and
may be stored in
cellular organelles.

12/8/21 7
 Conjugation and secondary conjugation of
picloram in leafy spurge (Euphorbia esula L.)
as proposed by Frear et al. (1989).

12/8/21 8
 Primary Metabolism
 Oxidative Transformations
– mediated by oxidative enzymes,
– e.g., cytochrome P450s- The most extensively
studied oxidative enzymes
– peroxidases
– polyphenol oxidases.

12/8/21 9
Cytochrome P450s
• Hemethiolate proteins
• Produce many secondary metabolites
– plant growth regulators
– isoprenoids
– alkaloids.
• CYP superfamily of genes
– highly conserved residues around the heme
portion of the protein
– occur in clusters in the genome (Frey et al. 1997).
12/8/21 10
 Cytochrome P450s
• monooxygenase reaction (hydroxylation)
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
12/8/21 11
 P450-mediated herbicide metabolism

• Studied using the phenylurea herbicides,

particularly chlortoluron.
• chlortoluron- metabolized to two metabolites
by at least two different P450 enzymes
(Mougin et al. 1990)
• Cytochrome P450 inhibitors
– piperonyl butoxide or 1-aminobenzotriazole
– tetcyclacis

12/8/21 12
 Cytochrome
P450
Inhibitors
+ chlortoluron

12/8/21 13
Herbicide resistance mediated by
P450s
Demonstrated in blackgrass (Alopecurus
myosuroides) (Menendez and De Prado 1997) and
rigid ryegrass (Lolium rigidum) (Preston et al. 1996)

12/8/21 blackgrass ryegrass 14

 Herbicide resistance mediated by P450s

• May arise via two scenarios:

– (1) mutation of an existing P450, allowing
increased binding and metabolism of the
herbicide
– (2) increased activity of existing P450s (Barrett
2000).

12/8/21 15
 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

12/8/21 16
 Peroxidases, Phenoloxidases, and Related
Oxidoreductases

Other reactions

12/8/21 17
 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.

• l
12/8/21 18
 Hydrolysis

12/8/21 19
 Ester hydrolysis
• commonly carried out by esterases
• lesser extent by lipases and proteases.
• Microbial and plant esterases have a
characteristic GLY-X-SER-X-GLY motif
(Brenner 1988).
• The SER acts as a nucleophile, enabling
ester bond cleavage (Cygler et al. 1995).

12/8/21 20
 Ester hydrolysis
• Herbicides esterified to increase absorption
and selectivity
– fenoxaprop-ethyl, diclofop-methyl, and 2,4-DB
• In plants, the ester bond is metabolized,
forming the acid (more phytotoxic)
• Immediate herbicide detoxification
– Deesterification, as with methyl-thifensulfuron

12/8/21 21
atrazine and other s-triazines
• metabolized in plants via
– N-dealkylation by cytochrome P450s
– Hydrolytic dehalogenation or
– displacement of chlorine with
glutathione(GSH) (Lamoureux et al. 1998)

12/8/21 22
amide hydrolysis

 Propanil resistance
Due to enhanced hydrolysis by aryl acylamidase
 resistant barnyardgrass (Carey et al. 1995a, 1997)
 resistant jungle-rice (Echinochloa colona) biotypes
(Leah et al. 1994
Due to high levels of aryl acylamidase
 Rice (Oryza sativa L.)

12/8/21 23
Aromatic Nitroreductive Processes
N-dealkylation
of trifluralin
observed in
peanut (Arachis
hypogaea
L.) and aryl
nitroreduction
observed in
sweet potato
(Ipomoea
b12/8/21
atatas L.) 24
 Aromatic Nitroreductive Processes
Observed metabolism of
pentachloronitrobenzene
in peanut (Arachis
hypogaea L.) via aryl
n i t r o r e d u c ti o n , a n d
glutathione S-transferase–
m e d i a t e d d e c h l o r i n a ti o n
or nitrite release.

12/8/21 25
 Carbon–Phosphorus Bond Cleavage
Reactions
• Organophosphonates used as pesticides
• Plants do not possess the ability to break the C–P
bond
• Glufosinate
– Microbial mineralization occur in the environment
– Genetically engineered glyphosate-tolerant crops
• the GOX gene isolated from E.coli was fused with the
chloroplast transit peptide from the small subunit of ribulose-
1,5-bisphosphate carboxylase/oxygenase

12/8/21 26
Genetically engineered glyphosate-tolerant crops
Two genes for acetyltransferase, bar and pat, isolated from Streptomyces
hygroscopicus and Streptomyces viridochromogenes, respectively have also
been used
Once acetylated, glufosinate does not inhibit glutamine synthetase.
Glyphosate tolerance in several transgenic crops is but due to a herbicide-
insensitive target site, namely CP4 5-enolpyruvylshikimate-3-phosphate
synthase

12/8/21 27
Biotransformation of glyphosate, highlighting C–P lyase and glyphosate oxidoreductase (GOX)
enzymatic reactions.
12/8/21 28
 Pesticide Conjugation Reactions
• Metabolic processes whereby an exogenous
or endogenous natural compound is joined to
a pesticide or its metabolite(s) facilitating
detoxification, compartmentalization,
sequestration, and/or mineralization
• Often involves utilization of existing enzymatic
machinery
• Therefore a co-metabolic process.

12/8/21 29
 Carbohydrate and Amino Acid Conjugation

• Glucose conjugation to pesticides results in

several metabolites
– O-, S-, and N-glucosides
– glucose ester
– gentibioside (e.g., 6-O-b-D-glucopyranosyl-D-
glucose)
– malonyl-glucose conjugates

12/8/21 30
 The most common glucose conjugates are O-
glucosides
12/8/21 31
 Differential conjugation of 2,4-D
• Susceptible Broadleaf weeds
– produce glucose ester metabolites
– readily susceptible to hydrolysis
– yields phytotoxic 2,4-D
• 2,4-D–tolerant wheat
– produces amino acid conjugates and O-glucosides
– stable non-phytotoxic metabolites that are not
easily hydrolyzed

12/8/21 32
 Twenty amino acids have been found to conjugate
with 2,4-D

Amino acid conjugation very common in plants

12/8/21 33
• Uridine diphosphate–glucosyl (UDPG)
transferase
– an enzyme involved in cellulose
biosynthesis
– mediates pesticide–glucose conjugation
(Klambt 1961) and pesticide–glucose ester
conjugation reactions (Mine et al. 1975).

12/8/21 34
 Pesticide–sugar conjugates
can undergo further
conjugation with
malonate via reaction with
malonyl CoA
In tomato (Lycopersicon esculentum L.)
the herbicide metribuzin is conjugated
to glucose
subsequently conjugated to malonate
Forming the N-malonyl–glucose
conjugate (Frear et al. 1985)

A range of UDPG transferase

activity within various
tomato cultivars confers
differential tolerance of
these cultivars to metribuzin

12/8/21 35
 Plant Glutathione Conjugation Reactions

• Glutathione
– γ-L-glutamyl-L-cysteinyl glycine [GSH]
– ubiquitously distributed in most aerobic
organisms
– Phloem mobile
– degraded by carboxypeptidases and
transpeptidases in the cytoplasm and vacuoles
• synthesis limited by
– availability of cysteine and hence by the
concentration of sulfate ions.
12/8/21 36
 GSH conjugation
Non enzymati c
not common
plants with low glutathione S-
transferase (GST) acti vity uses this
E.g. increased GSH concentrati ons
protect wheat from fenoxaprop
injury via Non enzymati c
conjugati on
12/8/21 37
 enzymatic conjugation
of xenobiotics with GSH
Glutathione-S-transferases
• homo- or heterodimer,
multifunctional enzymes
located in the cytosol
• Catalyze the nucleophilic
attack of the sulfur atom
of GSH by the electrophilic
center of the substrate

12/8/21 38
 Corn (Zea mays L.) GST gene enzyme
 N-terminus of this dimeric
enzyme is highly conserved and
binds GSH at the G-site
 The less conserved C-terminal is
an α-helix that binds substrates,
including herbicides, at the H-
site
 These two binding domains are
kinetically independent
12/8/21 39
The role of GSTs and GSH in plants
1. metabolism of secondary products, including
cinnamic acid (Edwards and Dixon 1991) and
anthocyanins (Marrs et al. 1995)
2. regulation and transport of both endogenous
and exogenous compounds; for
compartmentalization in the vacuole or cell wall
3. Protection against oxidative stress from
herbicides, air pollutants, pathogen attack and
heavy metal exposure

12/8/21 40
• Glutathione conjugates and their terminal
metabolites are stored in the vacuole or
bound to the cell wall
• Glutathione conjugate pumps in the
tonoplast membrane carry GSH conjugates
across the membrane
• In the vacuole, peptidases release the
glutathionyl moiety

12/8/21 41
 Secondary conjugation (phase III)

12/8/21 42
 In Vitro Methods for Studying
Pesticide
Metabolism in Plants
• cell and tissues cultures
• cell extracts
• purified enzymes, or
• Subcellular fractions
• very powerful tools to help
elucidate microbial, plant, and
mammalian pesticide metabolism.
12/8/21 43
 Applications
(1) prediction of metabolites
that are likely present
before initiation of an in
vivo study
(2) generation of metabolites
in sufficient quantities for
12/8/21
identification 44
 Applications
(4) characterization of nonextractable
residues
(5) ‘‘metabolic profiling’’ to determine
the rate and pattern of metabolism
between species,
(6) determination of genetics and
enzymology of the metabolic
pathway.
12/8/21 45
 Detoxification of heavy metal ions in plant
cells
• By phytochelatines (mainly) and
metallothionins
• PCs are synthesized from GSH
• MTs are small gene-encoded, Cys-rich
polypeptides
• Both are peptide ligands

12/8/21 46
 PCs HAVE THE GENERAL STRUCTURE
(γ-Glu-Cys)n-Gly

PCs form a family of structures

with increasing repetitions of the
γ-Glu-Cys dipeptide followed by
a terminal Gly; (γ-Glu-Cys)n-Gly

n- as high as 11,
general range of 2
to 5.

They are structurally

related to glutathione
(GSH; γ-Glu-Cys-Gly)

structural variants
identified in some
plant species

12/8/21 47
Detoxification of heavy metal ions in plant cells by phytochelatines and
sequestration as well as of organic pollutants by glutathione S-transferases
and degradation of the reaction products including sequestration

(1) Glutamylcysteine synthetase; (2) glutathione synthetase; (3)

-

12/8/21 phytochelatine synthase; (4) glutathione S-transferase (GST). 48

 Myriophyllum quitense reacts to the pollution stress increasing the activity of
glutathione-S-transferases (CDNB and Fluorodifen), glutathione reductase (GR)
and peroxidase (POD).

12/8/21 49
B i o t r a n s f o r m a t i o n u s i n g p l a n t c u l t u re d c e l l s
Biotransformations have great potential to
generate novel products or to produce known
products more efficiently.
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.
12/8/21 50
 The plant cultured cells
have abilities of the regio-
and stereoselective
hydroxylation, oxido-
reduction, hydrogenation,
glycosylation, and
hydrolysis for various
organic compounds

Reaction
types
include:
12/8/21 51
 • Some precursors are either insoluble or
very poorly soluble in the aqueous
phase, resulting in very low
bioconversion rates.
• Cyclodextrins- cyclic oligosaccharides
able to form inclusion complexes with a
variety of apolar ligands
• Since tolerance of plant cell cultures to
organic phases is low, cyclodextrin-
complexed precursors could be used to
facilitate bioconversion of water-
12/8/21 52
 Pathway biotransformation
• Exploit a characteristic biosynthetic pathway of
the plant or use a natural intermediate of the
normal biosynthetic pathway
• Biotransformation of digitoxin and
digitoxigenin in cultures of Digitalis purpurea.
– Digitalis sp. produces digitoxin and its
12-hydroxy derivative digoxin, both of
which are important cardiovascular drugs

12/8/21 53
• Digitoxin 12β-hydroxylase, a cytochrome
P450 monoxygenase, plays a vital role in this
biotransformation by Digitalis.
• Digitoxigenin was converted to digitoxigen-3-
one, 3-epidigitoxigenin and digoxigenin by D.
lantana shoot cultures

12/8/21 54
 Rauwolfia serpentina
• In cell suspension cultures, borohydride
reduction of ajmaline to
dihydrochanoajmaline followed by a
flavin-mediated photooxidation to
raumcline

12/8/21 55
 Nonspecific biotransformations
• N i t ro re d u c t i o n
• H y d ro x y l a t i o n s
• Glucosylation
• O x i d o - re d u c t i o n s b e t w e e n a l c o h o l s
and ketones
• H y d ro l y s i s
• Epoxidation
• R e d u c t i o n s o f c a r b o n y l g ro u p s
• Reduction of C–C double bond
12/8/21 56
 C. roseus
• Hydroxylation of warfarin to the
corresponding alcohol (Hamada et al., 1993).
• Cell suspension cultures hydroxylated
geraniol, nerol, (+) and (−) carvone to 5β-
hydroxyneodihydroxycarveol

Catharanthus roseus

12/8/21 57
 Nitroreduction
• Biotransformation of TNT into 2,4,6-
aminodinitrotoluene (ADNT) has been
investigated in plant cell cultures of
Datura innoxia, C. roseus and
Myrophyllum plants
Catharanthus roseus

12/8/21 58
Datura innoxia Myrophyllum
 Glucosylation
• facilitate the conversion of water-insoluble
compounds to water-soluble compounds.
• it is difficult to perform by microorganisms
or by chemical synthesis
• Plant cell cultures capable of glucosylation
of a variety of exogenously added
compounds
• phenols
• phenylpropanoic acid
• their analogues.
12/8/21 59
 Glucosylation
• Butyric acid- to obtain 6-O-butyryl-D-glucose,
which extends its half-life and prolongs its
bioactivity -Nicotiana plumbaginifolia
• Phenylcarboxylic acids Aconitum japonicum

– Glycyrrhiza echinata
– Aconitum japonicum Glycyrrhiza
echinata
– Dioscoreophyllum cumminsii Dioscoreophyllum
– N. tabacum cumminsii
N. tabacum

12/8/21 60
 Oxido-reductions between alcohols and
ketones
• Callus cultures of Myrtillocactus
geometrizans and N. tabacum
– Biotransformed Δ2-carene into diastereomeric
alcohols
– Myrtillocactus oxidized these alcohols to the
corresponding ketones.
Myrtillocactus

12/8/21 61
 Hydrolysis
• Enantioselective hydrolysis
– useful for the optical resolution of racemic
acetates
– biotransformation of (RS)-1-phenylethyl acetate
and its derivatives
• cultured cells of Spirodela oligorrhiza
• gave (R)-alcohols

12/8/21 62
 Epoxidation
• useful for the modification of cytotoxic
sesquiterpenes
• biotransformation of (−)-(4R)-isopiperitinone
by Mentha piperita yielded
– three hydroxylated derivatives
– two epoxidized derivatives
• (−)-7-hydroxyisopiperitonone
• its glucosides

12/8/21 63
 Reductions of C=O & C=C
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

12/8/21 64
N. sylvestris
 Biotransformations using plant enzymes

• most suitable for economical

ymes
production of pharmaceuticals
Papain • enzyme applicability when
nitrilases compared with cell systems
depends upon the balance
yclases
between
oloxidases – activity losses during the
isolation procedure
peroxidases
– superiority of the bioconversion
xygenases efficiency of the resulting
hrome P450
preparation
oxygenase
12/8/21 65
 Biotransformations
• The reaction types and stereochemistry
depends on the functional group in the
substrates and the structural moieties
in the vicinity of the functional group.
• Therefore, the biotransformations by
plant cultured cells are considered to
serve as important tools for the
structural modification of molecules to
give compounds possessing useful
12/8/21 66
SUMMING
UP…..
•Understanding the plant enzymatic systems
involved in metabolic processes provides a basis for
developing novel,
more effective, and environmentally benign
herbicides and
safeners.
•The most significant recent advances in our
understanding of PC biosynthesis and function have
come from molecular genetic studies using a variety
of model systems. There is considerable potential for
the application of that understanding to optimize
the process of phytoremediation
•Fundamental information, such as the reaction
12/8/21 67
types, stereospecificity and regioselectivity in the
references…..
Pesticide metabolism in plants and microorganisms
Laura L. Van Eerd
Robert E. Hoagland
Robert M. Zablotowicz
J. Christopher Hall
Biotransformation using plant cultured cells
Kohji Ishihara
Hiroki Hamada
Toshifumi Hirata
Nobuyoshi Nakajima
Biotransformations using plant cells, organ cultures and
enzyme systems: current trends and future prospects
Archana Giri, Vikas Dhingra, C. C. Giri, Ajay Singh,
Owen P. Ward and M. Lakshmi Narasu

12/8/21 68
12/8/21 69