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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 d’Agronomie et Environnement
Director of thesis : Hehn Alain Co-Director of thesis: Bourgaud Frédéric /
Olry Alexandre
Séminaire RP2E - 19/01/2012 – F. Karamat
Implication of coumarins
Self defense system of plants (Insecticides, Bactericide and Allelochemicals)
Intr
oduct
ion
O OR7
R6
R5
R8
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)
NH2HOOC HOOC HOOC
OH
Phenylalanine Cinnamic acid p-coumaroyl coA
Lignin
Flavonoid
Linear form
PAL C4H/4CL4CL / HCT / C3’H…
C2’H
O OOHO OOH
O OOH
Angular form
Umbelliferone
COCoA
Furanocoumarins
Prenyltransferase Prenyltransferase
Chlorogenic Acid…….
O OOO OO
Biosynthetic pathway of phenylpropanoid
Intr
oduct
ion
Séminaire RP2E - 19/01/2012 – F. Karamat
DMS Osthenol
The addition of an isoprene unit
Prenylated phenols in plants
Leguminosae EuphorbiaceaeRutaceae
MoraceaeUmbelliferae
(Park et al., 2003;Stevens et al., 2000; Riberio et al., 2008)
Prenyltransferase (PT) : enzyme that catalyze the prenylation step
PPi O ORO OR
Isoprene group polyphenol Prenylated polyphenol
What is prenylation?
Séminaire RP2E - 19/01/2012 – F. Karamat
Intr
oduct
ion
Anti-cancer
Anti-tyrosinase
Anti-glycosidase
Phytoalexin
Anti-poliferative activityDrug enhancement effects
Anti-oxidantBactericide
EST Sequencing
Intr
oduct
ion
Séminaire RP2E - 19/01/2012 – F. Karamat
Prenylated phenols in plants
Leguminosae RutaceaeUmbelliferae
SfN8DT Celery, parsley Lime, lemon, RutaAngelica
1) Identification of new candidate genes
Experi
menta
l st
rate
gy
Séminaire RP2E - 19/01/2012 – F. Karamat
Flavonoids
Vitamin E
Flavonoids or others?
Done by Prof Yazaki (collaboration)
Plant Heterologous expression system
Nicotiana benthamiana.
Experimental strategy
Experi
menta
l st
rate
gy
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
N-TermORFPromotor
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
Experi
menta
l st
rate
gy
Agrobacterium
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
Experi
menta
l st
rate
gy
GFPPromotor N-Term
Séminaire RP2E - 19/01/2012 – F. Karamat
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-TermSfN8DTPromotor
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
Experi
menta
l st
rate
gy
The heterologous expression system is functional
Reaction
Without enzyme
With enzyme (presence of 8dN)naringenin
Validation of the system in two steps2) Test with an enzyme : the Sophora flavescens prenyltransferase
Results
SfN8DTPromotor N-Term
Séminaire RP2E - 19/01/2012 – F. Karamat
Pri
mary
R
esu
lts
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 m in0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
550000
uV
Test of the 6 candidate genesDMAPP 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 - - -
Isoprenic moeity
PT-Lime
O OOHO OOH
Putative PT ORF Promotor N-Term
Without enzyme With enzyme
Séminaire RP2E - 19/01/2012 – F. Karamat
Resu
lts
Expression of 6 candidate PTs genes
PT-Ruta
PT-Lemon PT-Angelic
PT-Parsley PT-Celery
Optimal pH=8.2 Km=24 +/-3 µM
Biochemical parameters?
Resu
lts
Séminaire RP2E - 19/01/2012 – F. Karamat
In agreement with already characterized prenyltransferase (Sfn8DT, SfG6DT)
O OO
Inhibition of the reaction with psoralen
Biochemical parameters?
Resu
lts
Séminaire RP2E - 19/01/2012 – F. Karamat
Umbelliferone
Demetylsuberosin Osthenol
Linear furanocoumarins
Angular furanocoumarins
Psoralen Angelicin
Prenyltransferase
Phenylalanine
Qua
ntit
y O
f D
MS
in n
M
Conc. Of Psoralen in µM
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 Prenyltransferase
Concl
usi
on a
nd
pers
pect
ive
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
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
Concl
usi
on a
nd
pers
pect
ive
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
Umbelliferone
Demetylsuberosin Osthenol
Prenyltransferase
Without enzyme With enzymeLinear furanocoumarins
Psoralen
O OOHO OOH
PT- parsley is bifunctional?