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FUNCTIONAL CHARACTERIZATION OF THE FIRST PRENYLTRANSFERASE INVOLVED IN THE BIOSYNTHESIS OF COUMARINS, IMPORTANT MOLECULES IN THE DEFENSE SYSTEM OF HIGHER.

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Presentation on theme: "FUNCTIONAL CHARACTERIZATION OF THE FIRST PRENYLTRANSFERASE INVOLVED IN THE BIOSYNTHESIS OF COUMARINS, IMPORTANT MOLECULES IN THE DEFENSE SYSTEM OF HIGHER."— Presentation transcript:

1 FUNCTIONAL CHARACTERIZATION OF THE FIRST PRENYLTRANSFERASE INVOLVED IN THE BIOSYNTHESIS OF COUMARINS, IMPORTANT MOLECULES IN THE DEFENSE SYSTEM OF HIGHER PLANTS Fazeelat Karamat Laboratoire dAgronomie et Environnement Director of thesis : Hehn Alain Co-Director of thesis: Bourgaud Frédéric / Olry Alexandre Séminaire RP2E - 19/01/2012 – F. Karamat

2 Secondary metabolites PhenylpropanoidsCoumarins Implication of coumarins Self defense system of plants (Insecticides, Bactericide and Allelochemicals) Introduction What are coumarin ? Séminaire RP2E - 19/01/2012 – F. Karamat Other implications in different industries Pharmaceutical industry (Anti coagulant, Antitumoral, Anti-HIV, Antifungic) Cosmetic industry ( Dye, Fragrances) Food industry ( Flavors)

3 PhenylalanineCinnamic acidp-coumaroyl coA Lignin Flavonoid Linear form PAL C4H/4CL 4CL / HCT / C3H… C2H Angular form Umbelliferone COCoA Furanocoumarins Prenyltransferase Chlorogenic Acid……. Biosynthetic pathway of phenylpropanoid Introduction Séminaire RP2E - 19/01/2012 – F. Karamat DMS Osthenol

4 The addition of an isoprene unit Prenylated phenols in plants Leguminosae Euphorbiaceae Rutaceae Moraceae Umbelliferae (Park et al., 2003;Stevens et al., 2000; Riberio et al., 2008) Prenyltransferase (PT) : enzyme that catalyze the prenylation step Isoprene group polyphenol Prenylated polyphenol What is prenylation ? Séminaire RP2E - 19/01/2012 – F. Karamat Introduction Anti-cancer Anti-tyrosinase Anti-glycosidase Phytoalexin Anti-poliferative activity Drug enhancement effects Anti-oxidant Bactericide

5 EST Sequencing Introduction Séminaire RP2E - 19/01/2012 – F. Karamat Prenylated phenols in plants Leguminosae Rutaceae Umbelliferae SfN8DT Celery, parsley Lime, lemon, Ruta Angelica

6 1) Identification of new candidate genes Experimental strategy Séminaire RP2E - 19/01/2012 – F. Karamat Flavonoids Vitamin E Flavonoids or others?

7 Done by Prof Yazaki (collaboration) Plant Heterologous expression system Nicotiana benthamiana. Experimental strategy Identification of new candidate genes Cloning of the genes in plasmidic expression vectors Heterologous expression of the corresponding proteins Séminaire RP2E - 19/01/2012 – F. Karamat Functional characterization of proteins

8 N-Term ORF Promotor 2) Heterologous expression system The principle of the approach (Voinnet et al., 2003; Kapila et al., 1997) Séminaire RP2E - 19/01/2012 – F. Karamat Protein extraction(4 days post inoculation) Plasmid with concerned gene Experimental strategy Agrobacterium

9 Validation of the system in two steps 1) Test with the Jellyfish Green Fluorescent Protein (GFP) -GFP +GFP Visible spectra Fluorescence excitation/emission spectra 2) Heterologous expression system Experimental strategy GFP Promotor N-Term Séminaire RP2E - 19/01/2012 – F. Karamat

10 Validation of the system in two steps 2) Test with a membrane-bound-enzyme : the Sophora flavescens prenyltransferase ( a flavonoid specific prenyltransferase that has been characterized already) N-TermSfN8DT Promotor Séminaire RP2E - 19/01/2012 – F. Karamat Infiltration of tobacco leaves Crude membrane extracts Ultra-turrax was used to prepare a homogeniezed mixture of fresh plant material. Centrifugation and ultracentrifugation was done to get membrane pellets 4 days post inoculation 2) Heterologous expression system Experimental strategy

11 The heterologous expression system is functional Reaction Without enzyme With enzyme (presence of 8dN) naringenin Validation of the system in two steps 2) Test with an enzyme : the Sophora flavescens prenyltransferase Results SfN8DT Promotor N-Term Séminaire RP2E - 19/01/2012 – F. Karamat Primary Results

12 Test of the 6 candidate genes DMAPPGPPFPP Xanthotoxol --- Bergaptol --- Herniarine --- Umbelliferone methoxy coumarin methoxy coumarin --- 6,7- methoxy coumarin --- Coumarin ethoxy coumarin methoxypsoralen methoxypsoralen --- psoralen --- Isoprenic moeity PT-Lime Putative PT ORF Promotor N-Term Without enzyme With enzyme Séminaire RP2E - 19/01/2012 – F. Karamat Results Expression of 6 candidate PTs genes PT-Ruta PT-Lemon PT-Angelic PT-Parsley PT-Celery

13 Optimal pH=8.2 Km=24 +/-3 µM Biochemical parameters ? Results Séminaire RP2E - 19/01/2012 – F. Karamat In agreement with already characterized prenyltransferase (Sfn8DT, SfG6DT)

14 Inhibition of the reaction with psoralen Biochemical parameters ? Results Séminaire RP2E - 19/01/2012 – F. Karamat Umbelliferone Demetylsuberosin Osthenol Linear furanocoumarins Angular furanocoumarins Psoralen Angelicin Prenyltransferase Phenylalanine Quantity Of DMS in nM Conc. Of Psoralen in µM

15 In this work we developed another way to screen the activity of PT enzymes using a plant transient expression system (previously it was done in yeast) We showed a potential way to regulate the production of furanocoumarins in plants thanks to the negative feedback of psoralen Umbelliferone Demetylsuberosin Osthenol Linear furanocoumarins Angular furanocoumarins Psoralen Angelicin Prenyltransferase Conclusion and perspective Conclusions Séminaire RP2E - 19/01/2012 – F. Karamat - We characterized a new enzyme involved in the biosynthetic pathway of coumarins. It is the first prenyltransferase identified in parsley, a furanocoumarin producing plant. Phenylalanine

16 1)Parsley PT In parsley - Tissue specific expression pattern of gene ( leaves, stem, roots and flower) - Subcellular localization ( mitochondria, endoplasmic reticulum) In Ruta graveolens : -Stable overexpression of the gene in order to do metabolic engineering Conclusion and perspective Séminaire RP2E - 19/01/2012 – F. Karamat Project perspective 2) The five other PT genes ? -Enlarged metabolic screening -Stable expression in Ruta graveolens (under way) / metabolic characterization

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18 Umbelliferone Demetylsuberosin Osthenol Prenyltransferase Without enzyme With enzyme Linear furanocoumarins Psoralen PT- parsley is bifunctional ?


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