Biosynthesis of coumarins in plants: a major pathway still to

be unravelled for cytochrome P450 enzymes

autori: F. Bourgaud A. Hehn R. Larbat S. Doerper
E. Gontier S. Kellner U. Matern

Abstract Coumarins (1,2-benzopyrones) are

ubiquitously found in higher plants where they
originate from the phenylpropanoid pathway.
They contribute essentially to the persistence of
plants being involved in processes such as defense
against phytopathogens, response to abiotic stresses,
regulation of oxidative stress, and probably
hormonal regulation. Despite their importance,
major details of their biosynthesis are still largely
unknown and many P450-dependent enzymatic
steps have remained unresolved. Ortho-hydroxylation
of hydroxycinnamic acids is a pivotal step
that has received insufficient attention in the
literature. This hypothetical P450 reaction is
critical for the course for the biosynthesis of
simple coumarin, umbelliferone and other
hydroxylated coumarins in plants. Multiple P450
enzymes are also involved in furanocoumarin
synthesis, a major class of phytoalexins derived
from umbelliferone. Several of them have been
characterized at the biochemical level but no
monooxygenase gene of the furanocoumarin
pathway has been identified yet. This review
highlights the major steps of the coumarin pathway
with emphasis on the cytochrome P450
enzymes involved. Recent progress and the outcomes
of novel strategies developed to uncover
coumarin-committed CYPs are discussed.

Traducere: Cumarinele Abstract (1,2-benzopyrones) sunt

ubicvitarian? (in general) gsite n plantele superioare n cazul n care acestea
provin din calea fenilpropanoidic.
Acestea contribuie n mod esenial la persistena
plante fiind implicate n procese, cum ar fi aprarea
mpotriva fitopatogeni, ca rspuns la solicitrile abiotice,
reglementarea stresului oxidativ, i, probabil,
reglarea hormonal. n ciuda importanei lor,
detalii majore ale biosintezei lor sunt nc n mare msur
necunoscute i multe P450 dependente enzimatic
pai au rmas nerezolvate. Orto-hidroxilarea
de acizi hydroxycinnamic este un pas esential
care a primit o atenie insuficient n
literatur. Aceast reacie de P450 este ipotetic
critic pentru cursul pentru biosinteza
cumarin simplu, umbeliferona i altele
cumarine hidroxilai n plante. P450 multiple
enzimele sunt, de asemenea, implicate n furanocumarina
sintez, o clas major de phytoalexins derivate

1
din umbeliferona. Mai multe dintre ele au fost
caracterizat la nivel biochimic, dar nu
gena monooxigenazei a furanocumarina
cale a fost nc identificat. aceast opinie
evideniaz etapele majore ale cii cumarin
cu accent pe citocromul P450
enzime implicate. Progrese recente i rezultatele
a unor strategii noi dezvoltate pentru a descoperi
Comis-cumarin CYP sunt discutate.

Introduction
Coumarins are derived from 1,2-benzopyrones.
These molecules are found in higher plants where
they originate from the general phenylpropanoid
pathway (Harborne 1999) and are subject to
numerous modifications. Coumarins continue to
receive attention for their diverse bioactivities.
Some natural coumarins have been used as
human therapeutics, while 4-hydroxycoumarins
are prominent examples of microbial modification
which gave rise to the first generation molecules
developed along with aspirin and heparin as anticoagulants
(Mueller 2004). Other applications
appear possible in the course of new developments
in various therapeutic fields, like
Phytochem Rev (2006) 5:293308
DOI 10.1007/s11101-006-9040-2
symptomatic treatment of multiple sclerosis
(Wulff et al. 1998), photochemotherapy of T cell
lymphoma (Plumas et al. 2003), chemotherapy of
multidrug resistant tumors (Kawase et al. 2005),
organ transplants (Damjanovich et al. 2004), or
treatment of smokers for nicotine addiction
(Malaiyandi et al. 2005).
Despite the importance of coumarins for plant
life and human uses, major details of their
biosynthesis have remained unresolved. This
review will give an update of coumarin biogenesis
in plants with emphasis on the cytochrome P450
enzymes involved.

Introducere:
Cumarine sunt derivate din 1,2-benzopyrones.
Aceste molecule se gsesc n plantele superioare n cazul n care
acestea provin de la fenilpropanoidic general
cale (Harborne 1999) i sunt supuse
numeroase modificri. Cumarine continu s
primesc o atenie pentru diverse bioactiviti lor.
Unele cumarine naturale au fost folosite ca
terapeutica umana, in timp ce 4-hydroxycoumarins
sunt exemple elocvente de modificare microbian
care a dat natere la primele molecule de generare
dezvoltat impreuna cu aspirina si heparina ca anticoagulani
(Mueller 2004). alte aplicaii
pare posibil n cursul noilor evoluii

2
n diferite domenii terapeutice, cum ar fi
Phytochem Rev (2006) 5: 293-308
DOI 10.1007 / s11101-006-9040-2
tratamentul simptomatic al sclerozei multiple
(Wulff i colab. 1998), photochemotherapy de celule T
limfom (Plumas et al. 2003), chimioterapia
tumori rezistente la multiple (Kawase et al. 2005),
transplanturi de organe (Damjanovich et al. 2004), sau
tratamentul dependentei de nicotina pentru fumatori
(Malaiyandi et al. 2005).
n ciuda importanei cumarine pentru plant
de via i umane utilizri, detalii majore ale acestora
biosintezei au rmas nerezolvate. Acest
revizuire va da o actualizare a biogenezei cumarin
n plante, cu accent pe citocromul P450
enzime implicate.

Occurrence and functions of coumarins in plants

Coumarins may be subclassified as simple coumarins
(benzo-a-pyrones syn 1,2-benzopyrone),
7-oxygenated coumarins (furanocoumarins syn.
furobenzo-a-pyrones or furocoumarins), pyranocoumarins
(benzodipyran-2-ones), and phenylcoumarins
(benzo-benzopyrones) (Estevez-
Braun and Gonzalez 1997; Murray 1991; Murray
et al. 1982). Simple coumarins, furanocoumarins
and pyranocoumarins derive from the same
pathway, whereas the most common phenylcoumarins
(i.e., coumestans) originate from
isoflavone metabolism and will not be considered
in this review.

Apariie i funciile cumarine n plante

Cumarine pot fi subclasificate in cumarine simplu
(Benzo-a-pirone syn 1,2-Benzopiron),
7-oxigenai cumarinelor (furanocumarinele Syn.
furobenzo-a-pirone sau furocoumarins), pyranocoumarins
(benzodipyran-2-one), i phenylcoumarins
(benzo-benzopyrones) (Este'vez-
Braun i Gonza'lez 1997; Murray 1991; Murray
i colab. 1982). cumarine simple, furanocumarinele
i pyranocoumarins deriv din aceeai
cale, n timp ce cele mai frecvente phenylcoumarins
(Adic, coumestans) provin de la
metabolismul isoflavone i nu vor fi luate n considerare
n aceast reexaminare.
Simple coumarins
These compounds are widespread in plants and

3
more than 700 structures have already been
described (Harborne 1999).

Cumarine simplu
Aceti compui sunt larg rspndite n plante i
mai mult de 700 de structuri au fost deja
descris (Harborne 1999).

Coumarin
Coumarin (Fig. 1) is a natural product wellknown
for its pleasant vanilla-like odor. It was
reported from many plants of a variety of families,
including Fabaceae i.e., Tonka bean (Coumarouna
odorata) (Ehlers et al. 1995) or
sweetclover (Melilotus alba) (Akeson et al.
1963), Lamiaceae i.e., lavender (Lavandula officinalis)
(Brown 1962), and Lauraceae i.e., cinnamon
(Cinnamonum verum) (Miller et al. 1996).
More recent studies have revealed the presence
of o-coumaric acid in Arabidopsis thaliana root
exudates (Walker et al. 2003). As cis-o-coumaric
acid is unstable under acidic or neutral conditions
and lactonizes spontaneously to coumarin it is
conceivable that coumarin is formed in Arabidopsis
thaliana.
There have been many reports on the effect of
coumarin in plants, at the organ, tissue and
cellular levels (Brown 1981). These observations
tend to demonstrate that coumarin acts as a plant
hormone. However, until now neither solid evidence
for a physiological function nor the molecular
mode of action of coumarin has been
provided in plant tissues.

Cumarinele
Cumarina (fig. 1), este un produs natural binecunoscut
pentru mirosul plcut de vanilie cum ar fi. A fost
raportat la multe plante din diferite familii,
inclusiv Fabaceae adic, fasole Tonka (Coumarouna
odorata) (Ehlers i colab., 1995) sau
sweetclover (Melilotus alba) (Akeson i colab.
1963), Lamiaceae adic, lavanda (Lavandula officinalis)
(Brown 1962) i Lauraceae adic, scorioar
(Cinnamonum verum) (Miller et al. 1996).
Mai multe studii recente au relevat prezena

4
de acid o-cumaric n Arabidopsis thaliana rdcin
exudate (Walker i colab., 2003). Ca cis-o-cumaric
Acidul este instabil n condiii acide sau neutre
i lactonizes spontan la cumarina este
de conceput c cumarina este format n Arabidopsis
Arabidopsis.
Au existat multe rapoarte privind efectul
cumarina n plante, la organe, esuturi i
nivel celular (Brown 1981). aceste observaii
tind s demonstreze c actele de cumarin ca plant
hormon. Cu toate acestea, pn n prezent nici o dovad solid
pentru o funcie fiziologic, nici molecular
Modul de aciune al cumarin a fost
furnizate n esuturile vegetale.

Hydroxylated and methoxylated coumarins

Prevalent hydroxylated coumarins are umbelliferone,
herniarin and scoparone (2 methoxylated
derivatives of umbelliferone), esculetin, fraxetin,
isofraxidin, isoscopoletin, daphnetin and their
corresponding glucosides (Fig. 1). As for scopoletin,
these molecules are involved in plant
responses to stressors like salicylic acid (Pastirova
et al. 2004; Repcak et al. 2001). Herniarin was
demonstrated to be demethylated to umbelliferone
by C4H from Helianthus tuberosus
(CYP73A1) heterologously expressed in yeast
(Pierrel et al. 1994); However, the Km was so
high compared to cinnamate substrate that the
implication of C4H for herniarin demethylation
remains questionable.
Scopoletin and scopolin (7-b-D-glucoside of
scopoletin, Fig.1) were reported from many
plants, e.g., rubber tree (Giesemann et al. 1986;
Silva et al. 2002) and cassava (Gomez-Vasquez
et al. 2004) or carrot (Coxon et al. 1973) and
cotton (Zeringue 1984), but have been mainly
studied in tobacco (Fraissinet-Tachet et al. 1998;
Maier et al. 2000) and sunflower (Cabello-Hurtado
et al. 1998; Gutierrez et al. 1995). Scopoletin is
a typical phytoalexin (Ku` c 1982). Its synthesis is
post-infectionally activated in plants (Sharan
et al. 1998), but can also be triggered by various
abiotic stresses (Gutierrez et al. 1995). Scopoletin
also displays radical scavenging properties toward
reactive oxygen species and may be involved in the reduction of oxidative stress in plant
cells
.(Chong et al. 2002). Until recently, there was no
report of hydroxylated coumarins in Arabidopsis,
however, recent metabolic studies have revealed
that this plant can accumulate scopolin in stems
(Rohde et al. 2004) and roots (Bednarek et al.
2005). These findings demonstrate that stressinduced
hydroxylated coumarins are more common
in higher plant species than previously

5
assumed. As frequently described for other secondary
metabolites (Harborne 1999), scopoletin
is glucosylated to scopolin (Fig. 1) in the cytosol
and then transferred to the vacuole (Taguchi
et al. 2000).
Derivatives of daphnetin have attracted most
attention recently. Cold acclimated rye expresses
an O-methyltransferase with attenuated specificity
for position 8. The product, 7-hydroxy-8-
methoxycoumarin (hydrangetin) (Fig. 1), had
been reported as a protein kinase inhibitor (Yang
et al. 1999), and the modulating effect on protein
kinases was proposed to function during exposure
of rye to high photosystem II excitation pressure
and cold acclimation (Ndong et al. 2003). This
might be the first example of a coumarin involved
in hormone-like signaling.
Polyhydroxylated coumarins, like 6,7,8-trihydroxycoumarin,
have been described from Pelargonium
sinoides (Kayser and Kolodziej 1995; Latte
et al. 2000), which demonstrates that plants are
capable of multiple-step hydroxylations leading to
more complex coumarin patterns.

Traducere: cumarinele hidroxilai i metoxilate

Cumarine hidroxilai sunt prevalente umbeliferon, herniarin i scoparone (2
metoxilate derivai de umbeliferon), esculetinei, fraxetin,
isofraxidin, isoscopoletin, daphnetin i lor glucozide corespunztoare (fig. 1). n
ceea ce privete scopoletin, aceste molecule sunt implicate n instalaii
raspunsuri la factorii de stres, cum ar fi acidul salicilic (Pastirova
i colab. 2004; Repcak i colab. 2001). Herniarin a fost
dovedit a fi demetilat la umbeliferona
prin C4H de la Helianthus tuberosus
(CYP73A1) heterolog exprimate n drojdie (Pierrel et al 1994.); Cu toate
acestea, Km a fost att mare n comparaie cu substratul cinamat c implicarea
C4H pentru demetilare herniarin rmne sub semnul ntrebrii. Scopoletin i
scopolin (7-b-D-glucozid de scopoletin, Fig.1) au fost raportate la mai multe
plante, spre exemplu, arbore de cauciuc (Giesemann et al 1986.;
Silva i colab. 2002) i cassava (Gomez-Vasquez i colab. 2004) sau morcov
(Coxon i colab., 1973) i
bumbac (Zeringue 1984), dar au fost, n principal a studiat la tutun (Fraissinet-
Tachet i colab 1998.; Maier i colab. 2000) i floarea-soarelui (Cabello-
Hurtado i colab. 1998; Gutierrez i colab. 1995). Scopoletin un phytoalexin
tipic (Ku` c 1982). Sinteza sa este post-infectionally activat n plante (Sharan i
colab. 1998), dar poate fi declanat de diferite subliniaz abiotice (Gutierrez i
colab., 1995). scopoletin
prezint, de asemenea, proprieti de captare a radicalilor fa specii reactive de
oxigen i pot fi implicate n reducerea stresului oxidativ n celulele vegetale.

6
(Chong et al. 2002). Pn de curnd, nu a existat Raportul de cumarine
hidroxilai n Arabidopsis, Cu toate acestea, studii recente au relevat metabolice
c aceast plant se poate acumula scopolin n tulpini (Rohde i colab., 2004) i
rdcinile (Bednarek et al. 2005). Aceste descoperiri demonstreaz c
stressinduced cumarine hidroxilai sunt mai frecvente n specii de plante mai
mari dect cele anterior asumat. Aa cum s-a descris n mod frecvent pentru alte
secundar metaboliti (Harborne 1999), scopoletin este glucosylated la scopolin
(fig. 1), n citosol i apoi transferat la vacuole (Taguchi i colab. 2000). Derivai
de daphnetin au atras cel mai mult atenia recent. Rece secar i exprim
aclimatizai o O-metiltransferaz cu specificitate atenuat pentru poziia 8.
Produsul, 7-hidroxi-8- metoxicumarin (hydrangetin) (Fig. 1), a avut au raportat
ca un inhibitor al protein kinazei (Yang i colab. 1999), iar efectul de modulare
asupra proteinei kinaze a fost propus pentru a funciona n timpul expunerii de
secar la presiune ridicat de excitaie fotosistemul II i aclimatizare la rece
(Ndong et al. 2003). Acest ar putea fi primul exemplu de cumarin implicat
hormon-ca de semnalizare. cumarine polihidroxilai, cum ar fi 6,7,8-
trihidroxicumarin, au fost descrise de Pelargonium sinoides (Kayser i
Kolodziej 1995; Latte i colab. 2000), ceea ce demonstreaz c plantele sunt
capabil s hydroxylations n mai multe etape care conduc la mai multe modele
de cumarin complexe.

Minor coumarins
There have been reports on many other minor
coumarins in the phytochemical literature, which
are beyond the scope of this review. Amongst this
vast chemical diversity, methylenedioxy-substituted
coumarins, i.e., ayapin (Fig. 1), and prenylated
coumarins, like osthole and puberulin
(Fig. 1), deserve mentioning. Ayapin has been
described from Asteraceae only (Cabello-Hurtado
et al. 1998; Scio et al. 2003) and was characterized
as a phytoalexin (Gutierrez et al. 1995).
Methylenedioxy bridge-formation commonly
occurs through cyclization of an ortho-methoxyphenol
and is catalyzed by cytochrome P450-
dependent activities (Clemens and Barz 1996;
Ikezawa et al. 2003). Such compounds are difficult
to detoxify by phytopathogenic fungi
(George and VanEtten 2001), and it is noteworthy
that the methylenedioxy moiety is known as a
potent P450 inhibitor group requiring bioactivation
(Murray and Redy 1990). Osthole and
puberulin have been frequently reported from Rutaceae (Brophy et al. 2002; Brown et al.
1984) and Apiaceae (Tosun et al. 2005). O-Prenylated coumarins may be desaturated
further to the
corresponding butenylethers (Fig. 1) as shown in
Ammi majus (Hamerski et al. 1990a), and these
reactions are likely also catalyzed through P450
enzymes. The butenylethers are labile and release
a potentially toxic aldehyde moiety, which contributes

7
to their role as phytoalexins. Thus, the
aliphatic substitution of umbelliferone may provide
new substrates for further cytochrome P450
modifications, but neither of these enzymes has so
far been identified. As in case of ayapin in
Asteraceae, the P450 monooxygenases must be
considered as essential ecological factors (see
below).

Traducere:
Cumarilene Minore
Au existat rapoarte cu privire la multe alte minore
cumarine n literatura fitochimic, care
sunt dincolo de sfera de aplicare a prezentei reexaminri. printre aceast
diversitate chimic vast, metilendioxi-substituit
cumarine, adic ayapin (fig. 1) i prenylated
cumarine, cum ar fi osthole i puberulin
(Fig. 1), menionnd merit. Ayapin a fost
s-a descris la Asteraceae numai (Cabello-Hurtado
i colab. 1998; SCIO i colab. 2003) i a fost caracterizat
ca phytoalexin (Gutierrez i colab., 1995).
Metilendioxi pod formarea frecvent
are loc prin ciclizarea unui orto-metoxifenol
i este catalizat de citocromul P450-
activiti dependente (Clemens i Barz 1996;
Ikezawa i colab. 2003). Astfel de compui sunt dificil
la detoxifia de ciuperci fitopatogene
(George i VanEtten 2001), i este de remarcat
c radicalul metilendioxi este cunoscut ca
potent inhibitor al grupului P450 care necesit bioactivare
(Murray i Redy 1990). Osthole i
puberulin au fost frecvent raportate la Rutaceae (Brophy et al 2002. Brown i colab 1984.) i
Apiaceae (Tosun et al 2005.). cumarine O-Prenylated poate fi desaturated mai departe asupra
butenylethers corespunztoare (fig. 1), aa cum se arat n
Ammi majus (Hamerski et al. 1990a), iar acestea
Reaciile sunt probabil, de asemenea, prin catalizat de P450
enzime. Cei butenylethers sunt labile i eliberare
un fragment aldehid cu potenial toxic, care contribuie
rolul lor ca phytoalexins. Astfel,
substituia alifatic umbeliferona poate furniza
noi substraturi pentru continuare a citocromului P450
modificri, dar nici una dintre aceste enzime are asa
departe a fost identificat. Ca i n cazul ayapin n
Asteraceae, a monooxygenases P450 trebuie s fie
considerai factori ecologici eseniali ca (a se vedea textul de
de mai jos).

Furanocoumarins
Furanocoumarins can be grouped into the linear
type, where the (dihydro)furan ring is attached at
C(6) and C(7), and the angular type, carrying the

8
substitution at C(7) and C(8). Linear furocoumarins
(syn. psoralens) are principally distributed in
four angiosperm families: Apiaceae, Moraceae,
Rutaceae and Leguminosae (restricted to Psoralea
and Coronilla generae). The angular (dihydro)
furanocoumarins are less widely distributed
and primarily confined to the Apiaceae and
Leguminosae (Bourgaud et al. 1989). The most
abundant linear furanocoumarins are psoralen,
xanthotoxin, bergapten and isopimpinellin,
whereas the angular type is mostly represented
by angelicin, sphondin, and pimpinellin (Fig 1).
As was mentioned for the simple coumarins,
numerous minor furocoumarins have been described
in the literature, like bergamottin (5-
geranoxy-psoralen) (Stanley and Vannier 1967)
which has received attention recently as a major
grapefruit component interfering with drug
metabolism by intestinal CYP3A4 (Paine et al.
2005; Wen et al. 2002).
Furanocoumarins are recognized as potent
phytoalexins (Beier and Oertli 1983) and allelochemical
compounds (Baskin et al. 1967; Beier
1990). An outstanding feature of linear furanocoumarins
is their ability to intercalate into
dsDNA and create covalent cross-links primarily
with thymidine residues (DallAcqua et al. 1978).
Crosslinking proceeds readily under photoactivation
and potentially blocks DNA replication and
transcription. Accordingly, psoralens exhibit
strong genotoxicity toward all living organisms,
whereas the angular furanocoumarins are just
capable of forming mono-adducts with DNA
creating much less damage (Wamer et al. 1995).
Another remarkable property of furanocoumarins
is their reactivity to inactivate P450 enzymes
as mentioned above for bergamottin. This kind of
enzyme inhibition has been demonstrated for
P450s from vertebrate (Koenigs and Trager
1998), insect (Zumwalt and Neal 1993) and plant
sources (Gravot et al. 2004). Psoralens inactivate
by a mechanism-based inhibition (also referred as
suicide inhibition) which requires their conversion
to reactive intermediates by the enzyme
itself. These intermediates form covalent links to
the apoprotein and permanently inactivate the
enzyme (Fouin-Fortunet et al. 1986; Mays et al.
1989). The reactivity of furanocoumarins bears considerable ecotoxicological
consequences, i.e.,
attributing these compounds an important role as allelochemicals during plant-insect
interactions (Schuler and Berenbaum 2003). Only herbivores able to tolerate
furanocoumarins can feed on
psoralen-rich plants, and xanthotoxin-insensitive P450 forms have been described from
Papilio
polyxenes, a papilionid butterfly adapted to furanocoumarin-
accumulating host plants. This
insensitivity was supposed to be the result of
coevolution of insect detoxifying enzymes and the
particular phytochemical defense since Papilio
glaucuswhose host-plants do not contain furanocoumarins

9
exhibits sensitive P450s (Zumwalt
and Neal 1993). The race of coevolution of
butterflies on Apiaceae host plants has been
studied in detail. The capacity of Papilio polyxenes
to detoxify furanocoumarins through
CYP6B1 follows the order xanthotoxin > psoralen
> angelicin (Wen et al. 2003), but a synergistic
effect has been described between angular
furanocoumarins and psoralen or xanthotoxin in
response to insect attack (Berenbaum and Zangerl
1993). Considering the minor direct toxicity
of angular furanocoumarins, the synergism is
conceivably based on the inhibition of psoralendetoxifying
CYP by angelicin. Furthermore, the
accumulation of angular furanocoumarins is confined
to a few taxons only. It was hypothesized,
therefore, that the capacity for angular furanocoumarin
biosynthesis has evolved later and
presumably as a consequence to compensate for
the success of herbivores in the detoxification of
psoralens. (Berenbaum and Zangerl 1998). It
remains to be established, whether the enzymes
for angular furanocoumarin biosynthesis have
evolved from the biosynthesis of linear
furanocoumarins.
Most plants accumulating furanocoumarins
possess a highly inducible biosynthetic pathway.
which can be triggered by various biotic
(Hagemeier et al. 1999; Hamerski and Matern
1988b) and abiotic stresses (Eckey-Kaltenbach
et al. 1994; Katz et al. 1998). Ruta graveolens, and
possibly other Rutaceae, are exceptional because
they do not respond to stressors and synthesize
constitutively furanocoumarins in all tissues (Eilert
1989). However, the elicitation is still possible
in Ruta graveolens dedifferentiated cells (Bohlmann
et al. 1995).
The tissue-specific distribution of furanocoumarins
has been studied in Apiaceae
(Nitao and Zangerl 1987) and Rutaceae (Milesi
et al. 2001). Obviously, these compounds accumulate
in cells as well on the surface of plants.
The pronounced accumulation on seeds and
reproductive organs matches the optimal defense
theory which predicts that defense compounds
are principally allocated to the organs that play a
key-role in plant fitness (McKey 1979). The subcellular
localization of furanocoumarins is still
unknown, but glucosylated forms have been
frequently reported (Nguyen et al. 1997; Zobel
and Brown 1988), suggesting a probable vacuolar
compartmentation.
Pyranocoumarins
Pyranocoumarins, like xanthyletin (Fig. 1), have
been mainly described from Rutaceae (Anaya
et al. 2005; Sarker et al. 2002) and Apiaceae
(Zgo rka et al. 1998). As for furanocoumarins,
linear and angular forms can be distinguished. To
our knowledge, there is no proposal on their
functions in plants, however, due to the structural

10
relationship with furanocoumarins, a role as phytoalexins
may be assumed. The biosynthesis of
pyranocoumarins has not yet been investigated.

Furanocumarinele
Furanocumarinele pot fi grupate n liniar
tip, n cazul n care (dihidro) inelul furan este ataat
C (6) i C (7), precum i tipul unghiular, care transport
substituie la C (7) i C (8). furocoumarins liniare
(Sin. Psoralens) sunt distribuite n principal n
patru familii angiosperme: Apiaceae, Moraceae,
Rutaceae i Leguminosae (limitat la Psoralea
i Coronilla generae). Unghiular (dihidro)
furanocumarinele sunt distribuite pe scar larg mai puin
i n primul rnd limitat la Apiaceae i
Leguminosae (Bourgaud i colab., 1989). Cel mai
furanocumarinele liniare abundente sunt psoralen,
xanthotoxin, bergapten i isopimpinellin,
n timp ce tipul unghiular este reprezentat n principal
prin angelicin, sphondin i pimpinellin (figura 1).
Dup cum a fost menionat pentru cumarine simplu,
numeroase furocoumarins minore au fost descrise
n literatura de specialitate, cum ar fi bergamottin (5-
geranoxy-psoralen) (Stanley i Vannier 1967)
care a primit o atenie recent ca un important
componenta de grapefruit interfereaz cu medicamentul
metabolism, de ctre CYP3A4 intestinale (Paine i colab.
2005; Wen i colab. 2002).
Furanocumarinele sunt recunoscute ca fiind puternic
phytoalexins (Beier i OERTLI 1983) i allelochemical
Compuii (Baskin i colab 1967;. Beier
1990). O caracteristic remarcabil a furanocumarinele liniare
este capacitatea lor de a intercala n
dsADN i de a crea covalente legturi ncruciate n primul rnd
cu reziduuri timidina (Dall'Acqua i colab. 1978).
Reticularea cu uurin veniturile sub fotoactivrii
si, posibil, blocheaz replicarea ADN-ului i
transcriere. n consecin, psoralens prezint
genotoxicitate puternic fa de toate organismele vii,
ntruct furanocumarina unghiulare sunt doar
capabil s formeze mono-aduci cu ADN
crend mai puine pagube (Wamer i colab., 1995).
O alt proprietate remarcabil a furanocumarinele
este reactivitatea lor de a inactiva enzimele P450
aa cum sa menionat mai sus pentru bergamottin. Acest tip de
inhibarea enzimelor a fost demonstrat pentru
P450s de la animale vertebrate (i Koenigs Trager1998), insecte (Zumwalt i Neal 1993) i a
plantelor
Surse (Gravot et al. 2004). psoralens inactiva

11
printr-o inhibare bazat pe mecanism (denumit, de asemenea, ca
inhibare de suicid), care necesit o conversie a acestora
la intermediari reactivi de ctre enzima
n sine. Aceti intermediari formeaz legturi covalente cu
Apolipoproteina i inactiva permanent
enzim (Fouin-Fortunet i colab 1986;. Mays i colab.
1989). Reactivitatea furanocumarinele are consecine ecotoxicologie considerabile, adic,
atribuirea acestor compui un rol important ca n timpul interaciunilor allelochemicals-plante
insecte (Schuler i Berenbaum 2003). Numai erbivore capabil s tolereze furanocumarinele
poate alimenta pe
Plante psoralen bogate i formele P450-xanthotoxin insensibil au fost descrise de la Papilio
polyxenes, un fluture papilionid adaptat la furanocoumarin-
acumularea de plante gazd. Acest
insensibilitate trebuia s fie rezultatul
coevoluiei enzimelor detoxifiante de insecte, iar
special n aprare fitochimica, deoarece Papilio
Glaucus-gazd ale cror-plante nu conin furanocoumarins-
prezint P450s sensibile (Zumwalt
i Neal 1993). Cursa coevoluiei de
fluturi pe plante gazd Apiaceae a fost
a studiat n detaliu. Capacitatea polyxenes Papilio
pentru a detoxifia furanocumarinele prin
CYP6B1 urmeaz comanda xanthotoxin> psoralen
> Angelicin (Wen i colab. 2003), ci o sinergie
efect a fost descris ntre unghiulara furanocumarinele i psoralen sau xanthotoxin n
ca rspuns la atacul insectelor (Berenbaum i Zangerl
1993). Avnd n vedere toxicitatea direct minor
de furanocumarinele unghiulare, sinergismul este
teoretic bazat pe inhibarea psoralendetoxifying
CYP de angelicin. Mai mult,
acumularea de furanocumarinele unghiulare este limitat
la doar cteva taxoni. Sa emis ipoteza,
prin urmare, c capacitatea de furanocumarina unghiulare
biosintez a evoluat mai trziu i
probabil ca o consecin a compensa
succesul erbivore n detoxifierea
psoralens. (Berenbaum i Zangerl 1998). Aceasta
Rmne de stabilit dac enzimele
pentru biosinteza furanocumarina unghiulare au
evoluat de biosinteza liniare
furanocumarinele.
Cele mai multe plante care acumuleaz furanocumarinele
posed o cale de biosintez extrem de inductibile.
care poate fi declanat de diferite biotice
(HAGEMEIER i colab 1999. Hamerski i Matern
1988b) i subliniaz abiotici (Eckey-Kaltenbach
i colab. 1994; Katz i colab. 1998). Ruta graveolens, i
eventual, alte Rutaceae, sunt excepionale, deoarece
ei nu rspund la factorii de stres i de a sintetiza
furanocumarinele n toate esuturile n mod constitutiv (Eilert

12
1989). Cu toate acestea, elicitare este nc posibil
n Ruta graveolens celulele dedifereniate (Sholmann
i colab. 1995).
Distribuia esutului specific furanocumarinele
a fost studiat n Apiaceae
(Nitao i Zangerl 1987) i Rutaceae (Milesi
i colab. 2001). Evident, aceti compui se acumuleaz
n celule precum pe suprafaa plantelor.
Acumularea pronunat asupra seminelor i
organele de reproducere se potrivete aprare optim
Teoria care prezice c compuii de aprare
sunt alocate n principal la organele care joac un
rol-cheie n sala de fitness a plantelor (McKey 1979). subcelular
localizarea furanocumarinele este nc
, dar formele glucosylated necunoscute au fost
n mod frecvent raportate (Nguyen i colab 1997;. Zobel
Brown i 1988), sugernd o vacuolar probabil compartimentare.
Pyranocoumarins
Pyranocoumarins, cum ar fi xanthyletin (fig. 1), au
a fost descris n principal, de la Rutaceae (Anaya
i colab. 2005; Sarker i colab. 2002) i Apiaceae
(Zgo' RKA i colab., 1998). n ceea ce privete furanocumarinele,
Formele liniare i unghiulare pot fi distinse. La
cunotinele noastre, nu exist nicio propunere privind acestora
funcii n plante, cu toate acestea, din cauza structurale
relaie cu furanocumarinele, un rol phytoalexins
poate fi asumat. Biosinteza
pyranocoumarins nu a fost nc investigat.

Biosynthesis of coumarins in plants

Main enzymes and genes implicated in coumarins
biosynthesis and that have been sufficiently documented
are presented in Table 1.
Cinnamic acid to coumarin
The pathway of coumarin biosynthesis has been
largely outlined during the 60s and 70s, with the
help of tracer feeding experiments (Brown 1981).
Radiolabeled cinnamic acid was incorporated
into coumarin and 7-hydroxycoumarins (Brown
et al. 1960). Other tracer experiments conducted
with Lavandula officinalis, a plant that produces
coumarin as well as 7-hydroxylated coumarins,
revealed that in the latter instance para-hydroxylation
preceded the ortho-hydroxylation required
for lactonization (Brown 1962). This
indicated that umbelliferone (Fig. 1) is derived
from cis-p-coumaric acid, whereas coumarin
originates from cis-cinnamic acid (Fig. 2), and
may imply different enzymes for the orthohydroxylation/
lactonization of coumarin versus
umbelliferone.
The ortho-hydroxylation is a key step of

13
coumarin biosynthesis, that has received insufficient
attention. In initial experiments, doublelabeled
(ortho-3H, ring-1-14C) cinnamic acid was
fed to Melilotus alba shoots or Gaultheria procumbens
leaves, and the retention of label was
monitored upon conversion to o-coumaric acid
(Ellis and Amrhein 1971). An NIH shift was
proposed because of insignificant decrease of the
3H:14C ratio, which is an indication of a cytochrome
P450 monooxygenase reaction mechanism.
A following report addressed the formation
of coumarin with extracts from Melilotus alba, a
plant that produces high levels of coumarin. This
study allocated the ortho-hydroxylation of cinnamic
acid to the chloroplast and again suggested
a P450-dependent hydroxylation mechanism
(Gestetner and Conn 1974). Unfortunately, the
in vitro results could not be reproduced, and the
class of the enzyme involved as well as its
subcellular site remain to be established. As
revealed later, the early experiments may have
suffered from fundamental analytical problems,
since the chromatography and recrystallization
techniques employed were likely insufficient to
separate the various cinnamic acids. Nevertheless,
the proposed conversion of cinnamic to o-coumaric
acid received some support by precursor
feeding studies done with Petunia chloroplasts,
which ascribed cinnamate 2-hydroxylase, including
the formation of coumarin, and lack of
cinnamate 4-hydroxylase to these organelles
(Conn 1984; Ranjeva et al. 1977). In light of the
studies done since with Ammi majus microsomes
(Hamerski and Matern 1988a, b; Stanjek et al.
1999a) on the biosynthesis of furanocoumarins it
appears possible that the ortho-hydroxylase is an
exceptionally labile CYP enzyme, in contrast to
the CYPs hydroxylating cinnamic acids in paraor
meta-position (Fig. 2). Overall, the orthohydroxylation
of cinnamic (or 4-coumaric) acid,
being of pivotal importance for all coumarins,
remains a missing link in the network of phenylpropanoid
biosynthesis.

Traducere: Biosintezei de cumarine n plante

enzime principale si gene implicate in cumarine
biosinteza i care au fost suficient de documentate
sunt prezentate n tabelul 1.
acidul cinamic la cumarin
Calea de biosintez cumarin a fost
n mare parte conturat n anii '60 i '70, cu
ajutorul experimentelor de marcare de alimentare (Brown, 1981).
Acidul cinamic radiomarcat a fost ncorporat
n cumarin i 7-hydroxycoumarins (Brown
i colab. 1960). Alte experimente efectuate trasor
cu officinalis Lavandula, o planta care produce
cumarin precum i cumarine 7-hidroxilai,

14
a relevat faptul c, n acest din urm caz para-hidroxilare
precedat orto-hidroxilare necesare
pentru lactonizare (Brown, 1962). Acest
indicat c umbeliferona (fig. 1) este derivat
de la cis-p-cumaric acid, n timp ce cumarina
provine din acid cis-cinamic (fig. 2), i
pot implica enzime diferite pentru orthohydroxylation /
lactonizarea cumarin versus
umbeliferona.
Orto-hidroxilare este un pas cheie
biosinteza cumarin, care a primit insuficient
Atenie. n experimentele iniiale, doublelabeled
(Orto-3H, inel-1-14C) cinamic a fost
hrnite la Melilotus muguri alba sau procumbens Gaultheria
frunze, iar pstrarea etichetei a fost
monitorizate la conversie o-cumaric acidului
(Ellis i G.T.Amrheim 1971). O schimbare de NIH a fost
a propus, datorit diminurii nesemnificativ a
3H: raportul 14C, care este un indiciu al unei citocromul
P450 monooxigenaza mecanism de reacie.
Un raport urmtor adresat formarea
de cumarin cu extracte din sulfin alba, o
plant care produce un nivel ridicat de cumarin. Acest
Studiul a alocat orto-hidroxilarea cinamic
acid la cloroplast i sugera din nou
un mecanism dependent de hidroxilare P450
(Gestetner i Conn 1974). Din pcate,
in vitro, rezultatele nu au putut fi reproduse, iar
clasa a enzimei implicate precum i sa site-ul subcelular rmn a fi stabilite. La fel de
dezvluit mai trziu, primele experimente pot avea
a suferit de probleme analitice fundamentale,
deoarece cromatografia i recristalizarea
Tehnicile utilizate au fost probabil insuficiente pentru a
separarea diverilor acizi cinamici. Cu toate acestea,
conversia propus a cinamic la o-cumaric
Acidul a primit un sprijin de precursor
Studiile de hrnire efectuate cu cloroplaste petunia,
care atribuie cinamat 2-hidroxilaza, inclusiv
formarea de cumarin i lipsa
cinamat 4-hidroxilaz acestor organite
(Conn 1984; Ranjeva et al 1977.). Avnd n vedere cele
Studiile efectuate ncepnd cu microzomii Ammi majus
(Hamerski i Matern 1988a, b; Stanjek i colab.
1999a) privind biosinteza furanocumarinele-l
pare posibil ca "orto-hidroxilaza" este o
enzima CYP excepional de labil, n contrast cu
a hidroxilarea acizilor cinamic CYP n paraor
meta-poziie (fig. 2). n ansamblu, orthohydroxylation
de cinamic (sau 4-cumaric) acid
fiind de o importan crucial pentru toate cumarine,

15
rmne o veriga lips n reeaua de fenilpropanoidic
biosintez.

Cinnamic acid to umbelliferone and other

hydroxylated coumarins
The formation of umbelliferone proceeds from
4-coumaric acid or its ester derivatives (Fig. 2).
The conversion of cinnamic acid to 4-coumaric
acid is catalyzed by cinnamate 4-hydroxylase, a
cytochrome P450 monooxygenase from the
CYP73A family (Teutsch et al. 1993). This
enzyme constitutes the P450 enzyme most studied
to date and sets the stage for several branch
pathways, such as the lignification (Anterola and
Lewis 2002) or flavonoid biosynthesis (Harborne
1999) (see Ehlting et al. 2006 in this issue for a
review).
Following the pertaining literature, 4-coumaric
acid is ortho-hydroxylated to 2,4-dihydroxycinnamic
acid. The respective enzyme activity was
reported exclusively from Hydrangea macrophylla
and assigned to the chloroplasts (Kindl 1971).
This enzyme fraction was demonstrated to slowly
convert cinnamic acid to o-coumaric acid but was
more active to transform p-coumaric acid and
ferulic acid respectively to umbelliferone and

Traducere: Acidul cinamic la umbeliferona i altele

cumarine hidroxilate
Formarea veniturilor din umbeliferona
Acid 4-cumaric sau derivaii si ester (Fig. 2).
Transformarea acidului cinamic 4-cumaric
Acidul este catalizat de cinamat 4-hidroxilaz, un
citocromului P450 monooxigenazei From
Familia CYP73A (Teutsch i colab., 1993). Acest
enzim constituie enzima P450 cea mai studiat
pn n prezent i stabilete scena pentru mai multe sucursale
ci, cum ar fi lignification (Anterola i
Lewis 2002) sau biosinteza (Harborne
1999) (a se vedea Ehlting et al. 2006, n aceast problem pentru o
revizuire).
Ca urmare a literaturii referitoare, 4-cumaric
Acidul este orto-hidroxilat la 2,4-dihydroxycinnamic
acid. Activitatea enzimei respective a fost
raportate exclusiv din Hydrangea macrophylla
i atribuite cloroplaste (Kindl 1971).
Aceast fracie enzim a fost demonstrat lent
conversia acidului cinamic la acid o-cumaric, dar era
mai activ pentru a transforma acidul p-cumaric i
Acidul ferulic respectiv umbeliferona i....

16
scopoletin. Although this report is unique in
describing the ortho-hydroxylation of a hydroxylated
coumarin in vitro and suggesting one plastidic
fraction for the o-hydroxylation of both
p-coumaric and ferulic acid as well as benzoic
acid, the conversion of ferulic acid to scopoletin
had been postulated before from precursor feeding
studies in tobacco tissue cultures (Fritig et al.
1970). This biosynthetic course of scopoletin/
scopolin has been recently established in Arabidopsis
thaliana (Kai et al. 2006). T-DNA insertion
mutants within the gene encoding CYP98A3,
which catalyzes 3-hydroxylation of p-coumarate,
revealed a dramatic decrease in both scopoletin
and scopolin contents, confirming the origin from
ferulic acid in Arabidopsis. This is in contrast to
the results obtained for puberulin (Fig. 1) biosynthesis
in Agathosma puberula (Brown et al.
1988). Here, as well as in Daphne mezereum,
ferulic acid was not readily incorporated as
opposed to umbelliferone, therefore making
esculetin (Fig. 1) a likely precursor for the
synthesis of scopoletin (Brown 1986). The details
were discussed before (Murray et al. 1982), and
different pathways may operate in different
plants.
The formation of esculetin (Fig. 1; 6,7-dihydroxy
coumarin) was examined in Cichorium intybus
(Brown 1985). These studies revealed that
umbelliferone was an efficient precursor but not

Traducere:
scopoletin. Cu toate c acest raport este unic n
descrie orto-hidroxilarea unui hidroxilat
cumarin in vitro i care sugereaz o plastidic
fraciune de o-hidroxilarea ambele
p-cumaric i acidul ferulic precum benzoic
Acid, conversia acidului ferulic la scopoletin
a fost postulat nainte de hrnire de la precursorul
Studiile in culturi de esuturi de tutun (Fritig i colab.
1970). Acest curs de biosintez de scopoletin /
scopolin a fost recent nfiinat n Arabidopsis
Arabidopsis (Kai et al. 2006). inseria ADN-T
mutante n CYP98A3 gena care codific,
care catalizeaz 3'-hidroxilarea p-coumarate,
a artat o scdere dramatic att scopoletin
i scopolin coninutul, confirmnd originea din
Acidul ferulic n Arabidopsis. Acest lucru este n contrast cu
rezultatele obinute pentru puberulin (fig. 1) biosinteza
n Agathosma puberula (Brown i colab.
1988). Aici, precum i n Daphne mezereum,
Acidul ferulic nu a fost uor de reinut,
spre deosebire de umbeliferon, prin urmare, face

17
esculetinei (fig. 1), un precursor al mai probabil pentru
Sinteza scopoletin (Brown 1986). Detaliile
Au fost discutate mai nainte (Murray i colab. 1982) i
ci diferite pot funciona n diferite
plante.
Formarea esculetinei (fig. 1, 6,7-dihidroxi
cumarin) a fost examinat n Cichorium intybus
(Brown 1985). Aceste studii au relevat faptul c
umbeliferona a fost un precursor eficient, dar nu.....

caffeic acid, suggesting 6-hydroxylation of umbelliferone,

probably by the action of a P450 monooxygenase.
This deserves mentioning, because the
conversion of caffeic acid to esculetin is readily
accomplished in vitro with various plant extracts
containing phenoloxidase activity (Kneusel 1987;
Sato 1967), but has not been confirmed in planta.
Similar to esculetin, daphnetin (Fig. 1, 7,8-dihydroxycoumarin)
in Daphne mezereum, was shown
to be derived from umbelliferone rather than
caffeic acid.

acidul cafeic, sugernd 6-hidroxilarea umbeliferon,

probabil prin aciunea unui monooxigenazei P450.
Acest lucru merit menionat, pentru c
conversia acidului cafeic la esculetinei este uor
realizat in vitro, cu diferite extracte din plante
coninnd activitatea phenoloxidase (Kneusel 1987;
Sato 1967), dar nu a fost confirmat n pianta.
Similar cu esculetinei, daphnetin (fig. 1, 7,8-dihidroxicumarin)
n Daphne mezereum, s-a artat
s fie derivate din umbeliferona, mai degrab dect
Acidul cafeic.
The ortho-hydroxylation: a common route
with salicylic acid
Analogous to C2H, another major ortho-hydroxylation
step in phenolic metabolism is still controversial.
Salicylic acid is a pivotal signal
molecule in plant defense mechanisms (Shah
2003) but the biosynthesis pathway is still matter
of debate. Two routes have been proposed. A
pathway already shown to occur in bacteria has
been proposed in tobacco through chorismate and
isochorismate, via the general shikimic acid
metabolism (Wildermuth et al. 2001). Another
route has been documented in tobacco (Coquoz
et al. 1998; Yalpani et al. 1993) and rice (Silverman
et al. 1995), via decarboxylation of transcinnamic
acid to benzoic acid and subsequent
2-hydroxylation. This benzoic acid 2-hydroxylase
was characterized as a P450 enzyme but important
biochemical characteristics are atypical for
an eucaryotic P450 as it appears to be soluble and
it exhibits an unusually high molecular weight
(Leon et al. 1995). The corresponding P450 gene

18
has not been reported so far. This benzoic acid
2-hydroxylase is unable to transform cinnamic
acid into o-coumaric acid (Yalpani et al. 1993)
and consequently is unlikely to interfere with the
coumarin pathway.

Orto-hidroxilare: o cale comun

cu acidul salicilic
Analoaga C2h, un alt mare orto-hidroxilarea
pas n metabolismul fenolic este nc controversat.
Acidul salicilic este un semnal de pivot
molecula in mecanismele de aparare a plantelor (Shah
2003), dar calea de biosintez este nc materie
dezbaterii. Au fost propuse dou rute. A
cale demonstrat deja sa apara in bacterii are
a fost propus n tutun prin chorismate i
isochorismate, prin intermediul acidului shikimic general
Metabolismul (Wildermuth i colab., 2001). O alta
traseu a fost documentat n tutun (Coquoz
i colab. 1998; Yalpani i colab. 1993) i orez (Silverman
i colab. 1995), prin decarboxilarea transcinnamic
acid la acidul benzoic i ulterior
2-hidroxilare. Acest acid benzoic 2-hidroxilaza
a fost caracterizat prin aceea ca o enzim P450 dar important
Caracteristicile biochimice sunt atipice pentru
un P450 eucariot aa cum pare s fie solubil i
acesta prezint o greutate molecular neobinuit de mare
(Leon i colab., 1995). Gena P450 corespunztoare
Nu au fost raportate pn n prezent. Acest acid benzoic
2-hidroxilaza este n imposibilitatea de a transforma cinamic
acid in acid o-cumaric (Yalpani i colab. 1993)
i, prin urmare, este puin probabil s interfereze cu
cale cumarin.

Biosynthesis of furanocoumarins in plants

While coumarin biosynthesis remains a black box,
several enzymes of the furanocoumarin pathway
have been isolated and characterized (Fig. 3).
Entry of umbelliferone into the
furanocoumarin pathway
Umbelliferone rather than coumarin is the parent
compound of furanocoumarins, as was reported a
long time ago (Floss and Mothes 1964). It is first
prenylated in 6- (for linear furanocoumarins) or
8-position (for angular furanocoumarins) to yield
demethylsuberosin and osthenol, respectively
(Fig. 3). Dimethylallyl diphosphate required for
the 6-prenylation at least is provided in celery
(Apium graveolens) by the deoxy-D-xylulose
pathway and not through the mevalonate-dependent
pathway (Stanjek et al. 1999b). This is
conceivably also the case in other plants, because
the prenyltransferase has been identified in Ruta
graveolens as a plastidic enzyme (Dhillon and

19
Brown 1976; Ellis and Brown 1974), and the
activity was also documented in Ammi majus
(Hamerski et al. 1990b) The homologous enzyme
for the angular furanocoumarins has not been
isolated so far.

Biosintezei de furanocumarinele n plante

n timp ce cumarina biosintezei rmne o cutie neagr,
mai multe enzime ale cii furanocumarina
au fost izolate i caracterizate (fig. 3).
Intrare umbeliferona n
cale furanocumarina
Umbeliferona mai degrab dect cumarina este printele
compus din furanocumarinele, aa cum a fost raportat un
mult timp n urm (Floss i Mothes 1964). Este mai nti
prenylated n 6- (pentru furanocumarinele liniare) sau
8 poziii (pentru furanocumarinele angulare) pentru a produce
demethylsuberosin i osthenol, respectiv
(Fig. 3). Dimetilalil difosfat necesar pentru
6-prenilarea cel puin este prevzut n elin (Apium graveolens) de deoxi-D-xylulose cale
i nu prin mevalonat dependente cale (Stanjek i colab., 1999b). Asta e teoretic, de asemenea,
cazul n alte plante, deoarece preniltransferaza a fost identificat n Ruta graveolens ca o
enzim plastidic (Dhillon i Brun 1976; Ellis i Brown 1974), iar De asemenea, activitatea a
fost documentat n Ammi majus (Hamerski et al. 1990b) omolog Enzima pentru
furanocumarina unghiulare nu a fost izolate pn acum.

Linear furanocoumarins
Demethylsuberosin is transformed to marmesin
and further to psoralen by two separate cytochrome
P450 enzymes (Hamerski and Matern
1988b; Wendorff and Matern 1986). The enzymes
were biochemically characterized, and evidence
for their P450 nature was obtained from characteristic
blue-light-reversible inhibition of the
activities by carbon monoxide, and the use of
specific inhibitors. The two enzymes formally
catalyze very different reactions, the first forming
the dihydrofuran-ring from the ortho-prenylated
phenol (marmesin synthase) and the second
catalyzing the oxidative carbon-carbon chain
cleavage reaction (psoralen synthase). The mechanism
of marmesin synthase has not been solved
yet, but it might be speculated that some analogy
exists to menthofuran synthase from Mentha
piperita which belongs to the CYP71 family
(Bertea et al. 2001; Croteau et al. 2005). Psoralen
synthase was found to operates by syn-elimination
of acetone and one hydrogen from position 3
(Fig. 3) (Stanjek et al. 1999a). This release of
acetone is unique in plants. Psoralen synthase is.

furanocumarinele liniare
Demethylsuberosin este transformat n marmesin

20
i mai departe la psoralen de dou citocromul separate,
enzimele P450 (Hamerski i Matern
1988b; Wendorff i Matern 1986). enzimele
au fost caracterizate biochimic, i probe
pentru natura lor P450 a fost obinut din caracteristica
albastru-deschis-reversibil inhibiie a
activiti ale monoxidului de carbon, precum i utilizarea
inhibitori specifici. Cele dou enzime formal
catalizeaz reacii foarte diferite, formarea primul
dihidrofuran-ring din orto-prenylated
fenol (sintaza marmesin), iar al doilea
catalizarea catena carbon-carbon oxidativ
reacia de scindare (psoralen sintetaza). mecanismul
de marmesin sintetazei nu a fost rezolvat
cu toate acestea, dar s-ar putea specula c unele analogie
exist pentru a mentofuran sintaz din Mentha
piperita care aparine familiei CYP71
(Bertea i colab 2001;. Croteau et al 2005.). psoralen
sintaza a fost gsit la acioneaz prin sin-eliminare
de aceton i un hidrogen din poziia 3 '
(Fig. 3) (Stanjek i colab., 1999a). Aceast versiune
aceton, este unic n plante. Psoralen sintaza este

very specific for (+)-marmesin and does not

accept the ()-stereoisomer (nodakenetin) as a
substrate. Neither of the two P450s has been
characterized at the gene level.
Psoralen 5-monooxygenase catalyzes the subsequent
hydroxylation of psoralen to bergaptol
and was also characterized as a cytochrome P450
enzyme (Hamerski and Matern 1988a) from
Ammi majus cell suspensions. Nevertheless, there
is still the possibility that bergaptol could be
formed from 5-hydroxymarmesin. This possibility
was strongly favored by the work of Caporale and
coll. (Caporale et al. 1981). Different plants might
thus have developed a slightly different sequences.
Bergaptol is then O-methylated to bergapten. The
cDNA encoding the O-methyltransferase catalyzing
this reaction was recently cloned and functionally
characterized from Ammi majus (Hehmann
et al. 2004). The enzyme was shown to be highly
specific for bergaptol and does not accept xanthotoxol,
the C(8) corresponding phenol. This
corroborates previous reports on the separation of
bergaptol and xanthotoxol methyltransferases

21
from Ruta graveolens (Sharma et al. 1979) or
Petroselinum crispum (Hauffe et al. 1986).
The path for isopimpinellin formation
(5, 8-dimethoxypsoralen) is uncertain. It was studied
in Heracleum lanatum. In this plant xanthotoxin
was the most efficient precursor (Brown and
Sampathkumar 1977). However, bergapten was

foarte specifice pentru (+) - marmesin i nu

accepta (-) - stereoizomer (nodakenetin) drept
substrat. Nici unul dintre cele dou P450s a fost
caracterizat la nivelul genei.
Psoralen 5-monooxigenaza catalizeaz ulterioare
hidroxilarea psoralen la bergaptol
i a fost caracterizat ca citocromul P450
enzim (Hamerski i Matern 1988a) din
Suspensiile de celule Ammi majus. Cu toate acestea, exist
este n continuare posibilitatea ca bergaptol ar putea fi
format din 5-hydroxymarmesin. aceast posibilitate
a fost puternic favorizat de activitatea Caporale i
coll. (Caporale i colab. 1981). plante diferite s-ar putea
Astfel, s-au dezvoltat un uor diferite secvene.
Bergaptol este apoi O-metilat la bergapten.
ADNc care codific cataliza O-metiltransferaz
aceast reacie a fost recent clonat i funcional
caracterizat prin aceea de la Ammi majus (Hehmann
i colab. 2004). Enzima a fost dovedit a fi extrem
specifice pentru bergaptol i nu accept xanthotoxol,
C (8) fenol corespunztor. Acest
coroboreaz rapoartele anterioare privind separarea
bergaptol i xanthotoxol methyltransferases
din Ruta graveolens (Sharma i colab., 1979) sau
Petroselinum crispum (Hauffe i colab., 1986).
Calea pentru formarea isopimpinellin
(5, 8-dimethoxypsoralen) este incert. Acesta a fost studiat
n Heracleum lanatum. n aceast xanthotoxin plant
a fost cel mai eficient precursor (Brown i
Sampathkumar 1977). Cu toate acestea, bergapten era...

found to be converted into isopimpinellin,

although at a lower rate. Both 5- and 8-hydroxylation
pathways can thus lead to final product, but
5,8-dihydroxypsoralen was also demonstrated to
be a possible precursor in Ruta graveolens (Innocenti
et al. 1983). Enzymatic turnover of the
pathways could simply explain the prevalence of
one of the three routes in a given plant.

22
dovedit a fi convertite n isopimpinellin,
dei la o rat mai sczut. Att 5 i 8-hidroxilarea
ci poate duce astfel la produsul final, dar
5,8-dihydroxypsoralen a fost demonstrat
s fie un posibil precursor n Ruta graveolens (Innocenti
i colab. 1983). Cifra de afaceri enzimatica a
cai ar putea explica pur si simplu prevalenta
una dintre cele trei rute ntr-o anumit plant.

Angular furanocoumarins
The transformation of columbianetin to angelicin
is very similar from a mechanistic and stereochemical
point of view to the conversion of
marmesin to psoralen (Stanjek and Boland
1998). As demonstrated by feeding studies using
fluor- or deuterium-labeled columbianetin with
plants or leaf tissues. It is, thus, conceivable that
the enzymes for angular furanocoumarin biosynthesis
may have emerged by evolutionary adaptation
from the linear pathway. This would be
consistent with the fact that angular furanocoumarins
are less abundant in plants than
the linear type and that angular furanocoumarins
are always found concomitantly with linear furanocoumarins.
This hypothesis will be investigated
once the genes for marmesin synthase and
psoralen synthase, as well as those for umbelliferone
6- and 8-prenyltransferases, will be identified
(Fig. 3). Unfortunately, no information is
available yet at the genetic level.

furanocumarinele unghiulare
Transformarea columbianetin la angelicin
este foarte similar dintr-un mecanicista si stereochimica
punct de vedere transformrii
marmesin la psoralen (Stanjek i Boland
1998). Aa cum este demonstrat prin studii de hrnire folosind
fluor- sau columbianetin cu marcate cu deuteriu
plante sau esuturi de frunze. Este, deci, posibil ca
enzimele pentru biosinteza furanocumarina unghiulare
este posibil s fi aprut prin adaptarea evolutiv
din calea liniar. Acest lucru ar fi
n concordan cu faptul c furanocumarinele unghiulare
sunt mai puin abundente n plante dect
tipul liniar i c furanocumarinele unghiulare
sunt ntotdeauna gsite concomitent cu furanocumarinele liniare.
Aceast ipotez va fi investigat
odat ce genele pentru sintaza marmesin i
sintaza psoralen, precum i cele pentru umbeliferona
6- i 8-prenyltransferases, vor fi identificate
(Fig. 3). Din pcate, nici o informaie este
nc disponibile la nivel genetic.

23
Implication of P450s in furanocoumarin
synthesis
Cytochrome P450 enzymes are pivotal enzymes
of furanocoumarin biosynthesis, i.e., the formation
of xanthotoxin relies, at least, on four
sequential P450 reactions catalyzed by C4H,
marmesin synthase, psoralen synthase and psoralen
8-monooxygenase. This was at a first glance
puzzling because of the intrinsic capacity of
furanocoumarins to inhibit very different cytochrome
P450 enzymes, irrespective of the species,
through a mechanism-based inactivation process
(Fouin-Fortunet et al. 1986).
To understand how plants cope with this
problem Gravot and co-workers compared
inactivation by furanocoumarins (Gravot et al.
2004) of three different C4H: one from a plant that
does not contain furanocoumarins (Helianthus
tuberosus, CYP73 A1) and two from plants that
synthesize furanocoumarins (Ruta graveolens,
CYP73A32; Petroselinum crispum CYP73A10).
They showed that Kinact and 1/Ki in presence of
psoralen were lower for CYP73A32 and
CYP73A10 compared to CYP73A1, and, accordingly,
the cinnamate hydroxylation activity of
CYP73A32 and CYP73A10 appears more resistant
to mechanism-based inactivation than that of
CYP73A1 (respectively kinact/Ki 7, 4.4, and
45 min1mM1). This would suggest that plants
producing furanocoumarins have adapted their
P450 enzyme repertoire to the need for reduced
inactivation while retaining the high catalytic
efficiency. It is reasonable to expect a similar
adaptation of all the P450 enzymes in the same
pathway.
The evolution toward furanocoumarin accumulation
must have occurred under strong selection
pressure, since the biocidal and enzyme
inactivation properties of furanocoumarins appear
to be lethal to plants unless quick adaptation
can be accomplished. This pressure might have
built up by the exposure to herbivores and the
need for efficient antifeedant metabolites. This
would be fully compatible with the scheme of
furanocoumarins as allelochemicals in the warfare
with insects only adapted to hatch on furanocoumarin
producing plants (Schuler and Berenbaum
2003). It will be interesting to compare the
cytochrome P450 families recruited for the synthesis
of furanocoumarins in the plant and their
detoxification in insects.

Traducere: Implicaie de P450s n furanocumarina

sintez
enzimele citocromului P450 sunt enzime pivot
biosintezei furanocumarina, adic formarea
din xanthotoxin se bazeaz, cel puin, pe patru
Reaciile P450 secveniale catalizate de C4H,

24
marmesin sintaza, sintaza psoralen i psoralen
8-monooxigenaza. Aceasta a fost la prima vedere
ncurcat din cauza capacitii intrinseci a
furanocumarinele de a inhiba foarte diferite citocromului
enzimele P450, indiferent de specie,
printr-un proces de inactivare mecanism bazat pe
(Fouin-Fortunet i colab., 1986).
Pentru a intelege modul in care plantele face cu acest
problema Gravot i co-lucrtorilor n comparaie
inactivarea de ctre furanocumarinele (Gravot i colab.
2004) de trei C4H diferite: unul dintr-o plant care
nu conine furanocumarinele (Helianthus
tuberosus, CYP73 A1) i dou din plante care
sintetiza furanocumarinele (Ruta graveolens,
CYP73A32; Petroselinum crispum CYP73A10).
Ei au artat c Kinact i 1 / Ki n prezena
psoralen au fost mai mici pentru CYP73A32 i
CYP73A10 comparativ cu CYP73A1, i, n consecin,
activitatea cinamat hidroxilarea
CYP73A32 i CYP73A10 apare mai rezistent
la inactivare mecanism bazat pe dect cea a
CYP73A1 (respectiv Kinact / Kj 7, 4.4 i
45 min-1 mM-1). Acest lucru ar sugera c plantele
furanocumarinele productoare s-au adaptat lor
P450 enzima repertoriu la necesitatea redus
inactivarea de reinere, n timp ce ridicat catalitic
eficien. Este rezonabil s se atepte o similar
adaptarea toate enzimele P450 n aceeai
cale.
Evoluia spre acumulare furanocumarina
trebuie s fi avut loc n conformitate cu selecie puternic
presiune, deoarece biocid i enzim
Proprietile de inactivare ale furanocumarinele apar
s fie letal pentru plante cu excepia cazului n adaptarea rapid
poate fi realizat. Aceast presiune poate avea o
construit prin expunerea la erbivore, iar
au nevoie de metabolii antifeedant eficieni. Acest
ar fi pe deplin compatibil cu sistemul de
furanocumarinele ca allelochemicals n rzboiul
cu insecte adaptate numai pentru trapa furanocumarina
plante care produc (Schuler i Berenbaum
2003). Acesta va fi interesant de comparat
familiile citocromului P450 recrutai pentru sinteza
din furanocumarinele n plant i lor
detoxifiere in insecte.

Perspectives
Although no monooxygenase of the furanocoumarin
pathway stricto sensu has been
characterized at the gene level, techniques such
as differential display and RT-PCR strategies

25
have been developed for P450s (Schoendorf
et al. 2001; Schopfer and Ebel 1998) which
should be readily applicable to furanocoumarin
pathway. Such techniques already led to the
characterization of the C4H and C3H in the
relevant plants. Inducible systems are needed to

Perspective
Chiar daca nu monooxigenazei a furanocumarina
cale stricto sensu a fost
caracterizat la nivelul genei, tehnici
ca afiare diferenial i strategii RT-PCR
au fost dezvoltate pentru P450s (Schoendorf
i colab. 2001; Schopfer i Ebel 1998), care
ar trebui s fie uor aplicabil furanocumarina
cale. Astfel de tehnici condus deja la
caracterizarea C4H i C3'H n
plante relevante. Sistemele inductibile sunt necesare pentru....

differentiate and correlate the individual transcript

abundances with product accumulation
Elicitor-treated Ammi majus cultures appear to
qualify for this purpose.
Numerous recent studies focused on the role of
furanocoumarins as key allelochemicals, but the
physiological relevance of coumarins reaches far
beyond in the producing plants. This includes the
potential role of simple coumarins as hormones
and signaling molecules, which were shown in the
past decade to be much more widespread in plant
kingdom than previously assumed. More functional
insight should be obtained once the mechanism,
regulation of their biosynthesis and their
subcellular localization will be known. Biosynthesis
of L-phenylalanine proceeds in plastids
while phenylalanine ammonia-lyase and C4H
activities reside in the cytosol and endoplasmic
reticulum. Subcellular localization of the pivotal
ortho-hydroxylation of cinnamic or 4-coumaric
acid, so far, remains unresolved. Investigation in
Ruta graveolens assigned the subsequent 6-prenylation
of umbelliferone to plastidial membranes
(Dhillon and Brown 1976). Clarification of the
localization of the 2-hydroxylation will be the
further step to understand the physiological role
of coumarins.
It is probable that different routes to coumarins
will be discovered to operate in plants, some
of them might be confined to a taxonomic group.
The formation of scopoletin is an example and
derives either from esculetin or ferulic acid
according to the plant species considered. It is
currently unknown, whether the P450s involved
in the furocoumarin pathway belong to a single
family, as is the case with CYP71s in benzoxazine
synthesis, or to multiple P450 families as shown
for biosynthesis of cyanogenic glucosides

26
(CYP71E1 and CYP79A1). In either case, the
discovery of genes involved in coumarin synthesis
will add another stage of complexity to the
phenylpropanoid pathway.
The recent detection of coumarin and hydroxylated
coumarins in Arabidopsis thaliana have
opened the way for new approaches. Metabolomics
in conjunction with screening of mutant
libraries is likely to reveal new players in the
coumarin pathway.

...diferenierea i corelarea transcriptului individuale

abundances cu acumulare de produs
Culturile Ammi majus tratate cu Elicitor par
se calific pentru acest scop.
Numeroase studii recente s-au concentrat asupra rolului
furanocumarinele ca allelochemicals-cheie, dar
Importana fiziologic a cumarinicelor ajunge mult mai departe
dincolo n fabricile productoare. Aceasta include
Rolul potenial al cumarine simplu ca hormoni
i molecule de semnalizare, care au fost prezentate aici n
deceniu trecut pentru a fi mult mai rspndit n plant
regat dect sa presupus anterior. mai funcional
insight trebuie obinut o dat mecanismul,
reglementarea biosintezei a acestora i a acestora
localizare subcelular va fi cunoscut. biosintez
din veniturile L-fenilalanin n plastide
n timp ce fenilalanin amoniac-liaza i C4H
activiti de edere n citosol i endoplasmatic
reticulului. Localizarea subcelular a pivot
orto-hidroxilarea cinamic sau 4-cumaric
de acid, pn n prezent, rmne nerezolvat. investigaie
Ruta graveolens atribuit ulterior 6-prenilarea
de umbeliferona la plastidial membrane
(Dhillon i Brown, 1976). Clarificarea domeniului
localizarea 2-hidroxilare va fi
pas mai departe pentru a nelege rolul fiziologic
din cumarine.
Este probabil ca rute diferite pentru cumarine
vor fi descoperite pentru a funciona n plante, unele
dintre ele ar putea fi limitat la un grup taxonomic.
Formarea scopoletin este un exemplu i
deriv fie din esculetinei sau acid ferulic
n funcie de speciile de plante luate n considerare. Este
n prezent necunoscut, dac P450s implicate
n calea furocoumarin aparin unei singure
familie, aa cum se ntmpl n cazul CYP71s n benzoxazin
sintez, sau a mai multor familii P450 dup cum se arat
pentru biosinteza de glucozide cianogenice
(CYP71E1 i CYP79A1). n ambele cazuri,
Descoperirea de gene implicate n sinteza cumarin
se va aduga o alt etap de complexitate

27
calea fenilpropanoidic.
Recenta detectare a cumarin i hidroxilat
cumarine din Arabidopsis thaiiana au
a deschis calea pentru noi abordri. metabolomica
coroborat cu screening-ul mutant
biblioteci este de natur s dezvluie noi juctori n cadrul
caii cumarinelor.

28