presentation Plant Biotransformation

Plant Biotransformation

ANAND C. R.S2 BIOTECHNOLOGY

Saturday, April 8, 2023 2

Biotransformation Chemical conversion of a

substance mediated by living organisms or enzyme preparations derived there from.

In plants• Biotransformation of Pest ic ides and heavy metals• Biotransformation using plant cultured cel ls


•may occur via multistep processes known as cometabolism. –biotransformation of an organic compound not used as an energy source or as a constitutive element of the organism.

Pesticide biotransformation


Individual reactions of degradation–detoxification pathways

> oxidation> reduction > hydrolysis > conjugation


Diverse Metabolic Pathways

depends on the chemical structure of the

xenobiotic compoundthe organismenvironmental conditionsmetabolic factorsthe regulating expression of

these biochemical pathways


A three-phase process


• Generally, Phase II metabolites have little or no phytotoxicity and may be stored in cellular organelles.


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


Primary Metabolism

Oxidative Transformations– mediated by oxidative enzymes,– e.g., cytochrome P450s- The most extensively

studied oxidative enzymes– peroxidases – polyphenol oxidases.


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).


Cytochrome P450s

• monooxygenase reaction (hydroxylation) RH + O2 + NAD(P)H + H+ ROH + H2O + NAD(P)+

Other P450-mediated reactionsDehydration

DimerizationDeaminationDehydrogenationHeteroatom dealkylationEpoxidationReduction C–C or C=N cleavage


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


Cytochrome P450

Inhibitors + chlortoluron


Herbicide resistance mediated by P450s

Demonstrated in blackgrass (Alopecurus myosuroides) (Menendez and De Prado 1997) and rigid ryegrass (Lolium rigidum) (Preston et al. 1996)

blackgrass ryegrass


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).


Peroxidases, Phenoloxidases, and Related Oxidoreductases

• catalyze the polymerization of various anilines and phenols

• In most instances, polymerization productshave reduced toxicity compared with the substrate


Other reactions

Peroxidases, Phenoloxidases, and Related Oxidoreductases


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


Hydrolysis


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).


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


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)


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. 1994Due to high levels of aryl acylamidase

Rice (Oryza sativa L.)


Aromatic Nitroreductive ProcessesN-dealkylation of tr ifl ural in observed in peanut (Arachis hypogaeaL.) and aryl nitroreduction observed in sweet potato ( Ipomoea batatas L.)


Aromatic Nitroreductive ProcessesO b s e r v e d m e t a b o l i s m o f p e n t a c h l o r o n i t r o b e n z e n e i n p e a n u t ( A r a c h i sh y p o g a e a L . ) v i a a r y l n i t r o r e d u c ti o n , a n d g l u t a t h i o n e S - t r a n s f e r a s e –m e d i a t e d d e c h l o r i n a ti o n o r n i t r i t e r e l e a s e .


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


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


Biotransformation of glyphosate, highlighting C–P lyase and glyphosate oxidoreductase (GOX) enzymatic reactions.


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.


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


The most common glucose conjugates are O-glucosides


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


Twenty amino acids have been found to conjugate with 2,4-D

Amino acid conjugation very common in plants


• 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).


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


• 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.

Plant Glutathione Conjugation Reactions


Non enzymati cnot commonplants with low glutathione S-

transferase (GST) acti vity uses thisE.g. increased GSH concentrati ons

protect wheat from fenoxaprop injury via Non enzymati c conjugati on

GSH conjugation


enzymatic conjugationof 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


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

Corn (Zea mays L.) GST gene enzyme


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

The role of GSTs and GSH in plants


• 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


Secondary conjugation (phase III)


• cell and tissues cultures• cell extracts• purified enzymes, or• Subcellular fractions• very powerful tools to help

elucidate microbial, plant, and mammalian pesticide metabolism.

In Vitro Methods for Studying Pesticide

Metabolism in Plants


(1) prediction of metabolites that are likely present before initiation of an in vivo study

(2) generation of metabolites in sufficient quantities for identification

(3) detection of intermediate metabolites, which may provide insight into the metabolic pathway

Applications


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.


• By phytochelatines (mainly) and metallothionins

• PCs are synthesized from GSH• MTs are small gene-encoded, Cys-rich

polypeptides• Both are peptide ligands

Detoxification of heavy metal ions in plant cells


PCs form a family of structures with

increasing repetitions of the

γ-Glu-Cys dipeptide

followed by a terminal Gly; (γ-

Glu-Cys)n-Glyn- 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

PCs HAVE THE GENERAL STRUCTURE(γ-Glu-Cys)n-Gly


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) phytochelatine synthase; (4) glutathione S-transferase (GST).


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


Biotransformation using plant cultured cel ls

A wide variety of chemical compounds including aromatics, steroids, alkaloids, coumarins and terpenoids can undergo biotransformations using plant cells, organ cultures and enzymes.

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.


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:


• 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-insoluble precursors in a more compatible aqueous environment


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


• 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


Rauwolfia serpentina• In cell suspension cultures, borohydride

reduction of ajmaline to dihydrochanoajmaline followed by a flavin-mediated photooxidation to raumcline


Nonspecific biotransformations

• Nitroreduction• Hydroxylations• Glucosylation• Oxido-reductions between

alcohols and ketones• Hydrolysis• Epoxidation• Reductions of carbonyl groups• Reduction of C–C double bond


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

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T4X-43G312P-2&_user=2241385&_coverDate=06/30/2001&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1650962025&_rerunOrigin=google&_acct=C000056699&_version=1&_urlVersion=0&_userid=2241385&md5=5d28e8e945dfb54ff57394f216eafd07&searchtype=a


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

Datura innoxia Myrophyllum

Catharanthus roseus


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.


Glucosylation• 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

Glycyrrhiza echinata

Aconitum japonicum

Dioscoreophyllum cumminsii

N. tabacum


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


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


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


Reductions of C=O & C=CCarbonyl 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


Biotransformations using plant enzymes

• most suitable for economical production of pharmaceuticals

• enzyme applicability when compared with cell systems depends upon the balance between– activity losses during the

isolation procedure– superiority of the

bioconversion efficiency of the resulting preparation

Enzymes

1. Papain

2. Oxynitrilases

3. Cyclases

4. Phenoloxidases

5. Haloperoxidases

6. Lipoxygenases

7. Cytochrome P450 monoxygenase


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 properties.


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 types, stereospecificity and regioselectivity in the biotransformation of exogenous compounds, is essential for the development of the biotechnology for using higher plant cells.


references…..Pesticide metabolism in plants and microorganisms

Laura L. Van EerdRobert E. HoaglandRobert M. ZablotowiczJ. Christopher Hall

Biotransformation using plant cultured cells

Kohji Ishihara Hiroki HamadaToshifumi 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


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presentation Plant Biotransformation