functional characterization of the first prenyltransferase involved in the biosynthesis of coumarins, important molecules in the defense system of higher plants Fazeelat Karamat Laboratoire d’Agronomie et Environnement Director of thesis : Hehn Alain Co-Director of thesis: Bourgaud Frédéric / Olry Alexandre Séminaire RP2E - 19/01/2012 – F. Karamat

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

Biosynthetic pathway of phenylpropanoid 4CL / HCT / C3’H… Lignin PAL C4H/4CL Flavonoid Chlorogenic Acid……. COCoA Phenylalanine Cinnamic acid p-coumaroyl coA C2’H Introduction Umbelliferone Prenyltransferase Prenyltransferase Osthenol DMS Furanocoumarins Linear form Angular form Séminaire RP2E - 19/01/2012 – F. Karamat

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

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

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

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

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

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

2) Heterologous expression system 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) Promotor SfN8DT N-Term Infiltration of tobacco leaves 4 days post inoculation Ultra-turrax was used to prepare a homogeniezed mixture of fresh plant material. Experimental strategy Centrifugation and ultracentrifugation was done to get membrane pellets Crude membrane extracts Séminaire RP2E - 19/01/2012 – F. Karamat

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

Results Expression of 6 candidate PTs genes Promotor Putative PT ORF N-Term Isoprenic moeity Test of the 6 candidate genes DMAPP GPP FPP Xanthotoxol - Bergaptol Herniarine Umbelliferone 6- methoxy coumarin 7- methoxy coumarin 6,7- methoxy coumarin Coumarin 7-ethoxy coumarin 5-methoxypsoralen 8-methoxypsoralen psoralen PT-Ruta PT-Lime PT-Lemon PT-Angelic PT-Parsley PT-Celery Results Without enzyme With enzyme Séminaire RP2E - 19/01/2012 – F. Karamat

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

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

- Conclusions Conclusion and perspective 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 characterized a new enzyme involved in the biosynthetic pathway of coumarins. It is the first prenyltransferase identified in parsley, a furanocoumarin producing plant. Phenylalanine Umbelliferone Conclusion and perspective Prenyltransferase Prenyltransferase We showed a potential way to regulate the production of furanocoumarins in plants thanks to the negative feedback of psoralen - Demetylsuberosin Osthenol Angelicin Psoralen Linear furanocoumarins Angular furanocoumarins Séminaire RP2E - 19/01/2012 – F. Karamat

Project perspective Conclusion and perspective 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 2) The five other PT genes ? -Enlarged metabolic screening -Stable expression in Ruta graveolens (under way) / metabolic characterization Séminaire RP2E - 19/01/2012 – F. Karamat

Thank you for your attention!

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