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In plants Biotransformation of Pesticides and heavy metals Biotransformation using plant cultured cells Tuesday, March 01, 2011 2Pesticide biotransformation: 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. Tuesday, March 01, 2011 3 Pesticide biotransformationIndividual reactions of degradation–detoxification pathways > oxidation > reduction > hydrolysis > conjugation : Individual reactions of degradation–detoxification pathways > oxidation > reduction > hydrolysis > conjugation Tuesday, March 01, 2011 4Diverse Metabolic Pathways : 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 Tuesday, March 01, 2011 5A three-phase process: A three-phase process Tuesday, March 01, 2011 6Slide 7: Generally, Phase II metabolites have little or no phytotoxicity and may be stored in cellular organelles. Tuesday, March 01, 2011 7Slide 8: Conjugation and secondary conjugation of picloram in leafy spurge ( Euphorbia esula L.) as proposed by Frear et al. (1989). Tuesday, March 01, 2011 8Primary Metabolism: Primary Metabolism Oxidative Transformations mediated by oxidative enzymes, e.g., cytochrome P 450 s- The most extensively studied oxidative enzymes peroxidases polyphenol oxidases . Tuesday, March 01, 2011 9Cytochrome P450s: 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). Tuesday, March 01, 2011 10Cytochrome P450s: Cytochrome P450s monooxygenase reaction (hydroxylation) RH + O 2 + NAD(P)H + H + ROH + H 2 O + NAD(P) + Other P450-mediated reactions Dehydration Dimerization Deamination Dehydrogenation Heteroatom dealkylation Epoxidation Reduction C–C or C=N cleavage Tuesday, March 01, 2011 11P450-mediated herbicide metabolism: 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 Tuesday, March 01, 2011 12Slide 13: Cytochrome P450 Inhibitors + chlortoluron Tuesday, March 01, 2011 13Herbicide resistance mediated by P450s Demonstrated in blackgrass (Alopecurus myosuroides) (Menendez and De Prado 1997) and rigid ryegrass (Lolium rigidum) (Preston et al. 1996): 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 Tuesday, March 01, 2011 14Herbicide resistance mediated by P450s: 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). Tuesday, March 01, 2011 15Peroxidases, Phenoloxidases, and Related Oxidoreductases: 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 Tuesday, March 01, 2011 16Peroxidases, Phenoloxidases, and Related Oxidoreductases: Other reactions Peroxidases , Phenoloxidases , and Related Oxidoreductases Tuesday, March 01, 2011 17Hydrolytic Transformations: 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 Tuesday, March 01, 2011 18Slide 19: Hydrolysis Tuesday, March 01, 2011 19Ester 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). Tuesday, March 01, 2011 20Ester hydrolysis: 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 Tuesday, March 01, 2011 21Slide 22: 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) Tuesday, March 01, 2011 22Slide 23: 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.) Tuesday, March 01, 2011 23Aromatic Nitroreductive Processes: Aromatic Nitroreductive Processes N- dealkylation of trifluralin observed in peanut ( Arachis hypogaea L.) and aryl nitroreduction observed in sweet potato ( Ipomoea batatas L.) Tuesday, March 01, 2011 24Aromatic Nitroreductive Processes: Aromatic Nitroreductive Processes Observed metabolism of pentachloronitrobenzene in peanut ( Arachis hypogaea L.) via aryl nitroreduction , and glutathione S- transferase – mediated dechlorination or nitrite release. Tuesday, March 01, 2011 25Carbon–Phosphorus Bond Cleavage Reactions: 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 Tuesday, March 01, 2011 26Genetically 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 : 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 . G lyphosate tolerance in several transgenic crops is but due to a herbicide-insensitive target site, namely CP4 5-enolpyruvylshikimate-3-phosphate synthase Tuesday, March 01, 2011 27 Biotransformation of glyphosate, highlighting C–P lyase and glyphosate oxidoreductase (GOX) enzymatic reactions. : Biotransformation of glyphosate , highlighting C–P lyase and glyphosate oxidoreductase (GOX) enzymatic reactions. Tuesday, March 01, 2011 28Pesticide Conjugation 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. Tuesday, March 01, 2011 29Carbohydrate and Amino Acid Conjugation: 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 Tuesday, March 01, 2011 30Slide 31: The most common glucose conjugates are O- glucosides Tuesday, March 01, 2011 31Differential conjugation of 2,4-D : 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 Tuesday, March 01, 2011 32Slide 33: Tuesday, March 01, 2011 33Slide 34: 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). Tuesday, March 01, 2011 34Slide 35: Tuesday, March 01, 2011 35Plant 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. Plant Glutathione Conjugation Reactions Tuesday, March 01, 2011 36GSH conjugation: Non enzymatic not common plants with low glutathione S- transferase (GST) activity uses this E.g. increased GSH concentrations protect wheat from fenoxaprop injury via Non enzymatic conjugation GSH conjugation Tuesday, March 01, 2011 37enzymatic conjugation of xenobiotics with GSH: 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 Tuesday, March 01, 2011 38Corn (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 Corn ( Zea mays L.) GST gene enzyme Tuesday, March 01, 2011 39The role of GSTs and GSH in plants: metabolism of secondary products, including cinnamic acid (Edwards and Dixon 1991) and anthocyanins ( Marrs et al. 1995) regulation and transport of both endogenous and exogenous compounds; for compartmentalization in the vacuole or cell wall Protection against oxidative stress from herbicides, air pollutants, pathogen attack and heavy metal exposure The role of GSTs and GSH in plants Tuesday, March 01, 2011 40Slide 41: 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 Tuesday, March 01, 2011 41Secondary conjugation (phase III): Secondary conjugation (phase III) Tuesday, March 01, 2011 42In 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. In Vitro Methods for Studying Pesticide Metabolism in Plants Tuesday, March 01, 2011 43Applications: (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 Tuesday, March 01, 2011 44Applications: Applications Tuesday, March 01, 2011 45 (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.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 Detoxification of heavy metal ions in plant cells Tuesday, March 01, 2011 46Slide 47: PCs HAVE THE GENERAL STRUCTURE ( γ - Glu-Cys )n- Gly Tuesday, March 01, 2011 47Slide 48: 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). Tuesday, March 01, 2011 48Slide 49: Tuesday, March 01, 2011 49Biotransformation using plant cultured cells : Biotransformation using plant cultured cells 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. Tuesday, March 01, 2011 50Slide 51: Tuesday, March 01, 2011 51Slide 52: Tuesday, March 01, 2011 52 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 environmentPathway biotransformation: 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 Tuesday, March 01, 2011 53Slide 54: Digitoxin 12β-hydroxylase, a cytochrome P 450 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 Tuesday, March 01, 2011 54Rauwolfia serpentina: Rauwolfia serpentina In cell suspension cultures, borohydride reduction of ajmaline to dihydrochanoajmaline followed by a flavin -mediated photooxidation to raumcline Tuesday, March 01, 2011 55Nonspecific biotransformations: Nonspecific biotransformations Nitroreduction Hydroxylations Glucosylation Oxido -reductions between alcohols and ketones Hydrolysis Epoxidation Reductions of carbonyl groups Reduction of C–C double bond Tuesday, March 01, 2011 56C. roseus: C. roseus Hydroxylation of warfarin to the corresponding alcohol ( Hamada et al., 1993 ). Cell suspension cultures hydroxylated geraniol , nerol , (+) and (−) carvone to 5β-hydroxyneodihydroxycarveol Tuesday, March 01, 2011 57 Catharanthus roseusNitroreduction : Tuesday, March 01, 2011 58 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 roseusGlucosylation: Tuesday, March 01, 2011 59 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: Tuesday, March 01, 2011 60 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: Tuesday, March 01, 2011 61 Oxido -reductions between alcohols and ketones Callus cultures of Myrtillocactus geometrizans and N. tabacum Biotransformed Δ 2 -carene into diastereomeric alcohols Myrtillocactus oxidized these alcohols to the corresponding ketones . MyrtillocactusHydrolysis: Tuesday, March 01, 2011 62 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 )-alcoholsEpoxidation: Tuesday, March 01, 2011 63 Epoxidation useful for the modification of cytotoxic sesquiterpenes biotransformation of (−)-(4 R )- isopiperitinone by Mentha piperita yielded three hydroxylated derivatives two epoxidized derivatives (−)-7-hydroxyisopiperitonone its glucosidesReductions of C=O & C=C: Tuesday, March 01, 2011 64 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 N. sylvestrisBiotransformations using plant enzymes : Tuesday, March 01, 2011 65 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 preparationSlide 66: B iotransformations 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. Tuesday, March 01, 2011 66SUMMING UP…..: 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 . Tuesday, March 01, 2011 67references…..: 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 Tuesday, March 01, 2011 68Slide 69: Tuesday, March 01, 2011 69 You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.