FUNCTIONAL
CHARACTERIZATION
FIRST
PRENYLTRANSFERASE
THE
BIOSYNTHESIS
OF
INVOLVED
OF
IN
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
THE
What are coumarin?
Introduction
Secondary
metabolites
Phenylpropanoids
Coumarins
R5
R6
R7
Implication of coumarins
O
O
R8
Self defense system of plants (Insecticides, Bactericide and Allelochemicals)
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
Introduction
NH2
HOOC
Phenylalanine
/ HCT
/ C3’H…
C4H/4CL
PAL
HOOC
OH 4CL
Flavonoid
Chlorogenic
Acid…….
HOOC
Cinnamic acid
COCoA
Lignin
p-coumaroyl coA
C2’H
HO
O
Umbelliferone
Prenyltransferase
O
Prenyltransferase
O
HO
HO
Furanocoumarins
O
O
DMS
Linear form
Osthenol
Angular form
O
O
O
O
Séminaire RP2E - 19/01/2012 – F. Karamat
O
O
O
What is prenylation?
The addition of an isoprene unit
Prenyltransferase (PT) : enzyme that catalyze the prenylation step
Introduction
PPi
O
R
O
R
Isoprene group
polyphenol
O
O
Prenylated polyphenol
Prenylated phenols in plants
Anti-tyrosinase
Anti-glycosidase
Phytoalexin
Euphorbiaceae
Rutaceae
Leguminosae
Umbelliferae
Anti-oxidant
Drug enhancement
effects
Bactericide
Anti-poliferative activity
Moraceae
Anti-cancer
(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
SfN8DT
Umbelliferae
Rutaceae
Celery, parsley
Angelica
Lime, lemon,
Ruta
EST Sequencing
Séminaire RP2E - 19/01/2012 – F. Karamat
1) Identification of new candidate genes
Flavonoids
Experimental strategy
Vitamin E
Flavonoids or
others?
Séminaire RP2E - 19/01/2012 – F. Karamat
Experimental strategy
Experimental strategy
Identification of new
candidate genes
Done by Prof Yazaki
(collaboration)
Cloning of the genes in
plasmidic expression vectors
Heterologous expression of the
corresponding proteins
Functional characterization of
proteins
Séminaire RP2E - 19/01/2012 – F. Karamat
Plant Heterologous
expression
system
Nicotiana benthamiana.
2) Heterologous expression system
The principle of the approach
ORF
Promotor
Experimental strategy
Agrobacterium
N-Term
Plasmid with concerned gene
Protein extraction(4
days post inoculation)
(Voinnet et al., 2003; Kapila et al., 1997)
Séminaire RP2E - 19/01/2012 – F. Karamat
2) Heterologous expression system
Validation of the system in two steps
1) Test with the Jellyfish Green Fluorescent Protein (GFP)
Experimental strategy
Promotor
N-Term
GFP
Visible spectra
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)
Experimental strategy
Promotor
Infiltration
leaves
of
SfN8DT
N-Term
tobacco
4 days post
inoculation
Ultra-turrax was used to prepare a
homogeniezed mixture of fresh
plant material.
Centrifugation
and
ultracentrifugation was done
to get membrane pellets
Crude membrane extracts
Séminaire RP2E - 19/01/2012 – F. Karamat
Results
Validation of the system in two steps
2) Test with an enzyme : the Sophora flavescens prenyltransferase
Primary Results
Promotor
Reaction
SfN8DT
naringenin
N-Term
With enzyme
(presence of 8dN)
Without
enzyme
The heterologous expression system is functional
Séminaire RP2E - 19/01/2012 – F. Karamat
Expression of 6 candidate PTs genes
Promotor
N-Term
Putative PT ORF
Isoprenic moeity
Results
DMAPP
GPP
Xanthotoxol
-
-
-
Bergaptol
-
-
-
Herniarine
-
-
-
-
-
-
-
Umbelliferone
6- methoxy
coumarin
-
Test of the 6 candidate genes
FPP
PT-Ruta
PT-Lime
PT-Lemon
PT-Angelic
PT-Parsley
PT-Celery
uV
7- methoxy
coumarin
6,7- methoxy
coumarin
Coumarin
-
-
-
-
-
-
-
-
-
7-ethoxy coumarin
-
-
-
550000
500000
450000
400000
O
HO
O
O
HO
350000
O
300000
250000
200000
5-methoxypsoralen
-
-
-
8-methoxypsoralen
-
-
-
psoralen
-
-
-
150000
100000
50000
0
0.0
5.0
10.0
15.0
Without enzyme
Séminaire RP2E - 19/01/2012 – F. Karamat
20.0
25.0
30.0
35.0
With enzyme
m in
Results
Biochemical parameters?
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
O
Linear
furanocoumarins
12000
Osthenol
Angelicin
Angular
furanocoumarins
Quantity Of DMS in nM
Results
Prenyltransferase
Psoralen
O
Inhibition of the reaction
with psoralen
Umbelliferone
Demetylsuberosin
O
10000
8000
6000
Series1
4000
2000
0
0
20
40
60
80
100
Conc. Of Psoralen in µM
Séminaire RP2E - 19/01/2012 – F. Karamat
120
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
Prenyltransferase
 We showed a potential way
to regulate the production of
furanocoumarins in plants
thanks to the negative
feedback of psoralen
-
Demetylsuberosin
Psoralen
Linear
furanocoumarins
Séminaire RP2E - 19/01/2012 – F. Karamat
Prenyltransferase
Osthenol
Angelicin
Angular
furanocoumarins
Project perspective
Conclusion and perspective
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
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
PT- parsley is bifunctional?
Umbelliferone
Prenyltransferase
HO
Demetylsuberosin
O
O
HO
Osthenol
Psoralen
Linear
furanocoumarins
Without enzyme
With enzyme
O
O