functional characterization of the first prenyltransferase involved in the biosynthesis of coumarins, important molecules in the defense system of higher plants

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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
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What are coumarin?

• 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)

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Biosynthetic pathway of phenylpropanoid
4CL / HCT / C3H
Lignin
C4H/4CL
PAL
Flavonoid
Chlorogenic Acid.
COCoA
Phenylalanine
Cinnamic acid
p-coumaroyl coA
C2H
Introduction
Umbelliferone
Prenyltransferase
Prenyltransferase
Osthenol
DMS
Linear form
Angular form
Furanocoumarins
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What is prenylation?
The addition of an isoprene unit
Prenyltransferase (PT) enzyme that catalyze the
prenylation step
polyphenol
Isoprene group
Prenylated polyphenol
Prenylated phenols in plants
Introduction
Euphorbiaceae
Rutaceae
Leguminosae
Anti-poliferative activity
Drug enhancement effects
Anti-tyrosinase
Moraceae
Umbelliferae
Anti-glycosidase
Anti-cancer
Bactericide
Anti-oxidant
Phytoalexin
(Park et al., 2003Stevens et al., 2000 Riberio
et al., 2008)
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Prenylated phenols in plants
Introduction
Leguminosae
Umbelliferae
Rutaceae
Celery, parsley
Lime, lemon, Ruta
SfN8DT
Angelica
EST Sequencing
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1) Identification of new candidate genes
Flavonoids
Vitamin E
Experimental strategy
Flavonoids or others?
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Experimental strategy
Done by Prof Yazaki (collaboration)
Identification of new candidate genes
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
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2) Heterologous expression system
The principle of the approach
Promotor
N-Term
ORF
Agrobacterium
Plasmid with concerned gene
Experimental strategy
Protein extraction(4 days post inoculation)
(Voinnet et al., 2003 Kapila et al., 1997)
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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
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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
N-Term
SfN8DT
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
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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

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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-Lime
PT-Ruta
PT-Angelic
PT-Lemon
PT-Celery
PT-Parsley
Results
Without enzyme
With enzyme
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Biochemical parameters?
Results
Optimal pH8.2
Km24 /-3 µM
In agreement with already characterized
prenyltransferase (Sfn8DT, SfG6DT)
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Biochemical parameters?
Phenylalanine
Inhibition of the reaction with psoralen
Umbelliferone
Prenyltransferase
Demetylsuberosin
Osthenol
Results
Quantity Of DMS in nM
Angelicin
Psoralen
Linear furanocoumarins
Angular furanocoumarins
Conc. Of Psoralen in µM
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Conclusions

• 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
Angular furanocoumarins
Linear furanocoumarins
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Project 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
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Thank you for your attention!
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PT- parsley is bifunctional?
Umbelliferone
Prenyltransferase
Demetylsuberosin
Osthenol
Psoralen
Linear furanocoumarins
Without enzyme
With enzyme