Download Chemical Inactivation of the Cinnamate 4

Chemical Inactivation of the Cinnamate 4-Hydroxylase
Allows for the Accumulation of Salicylic Acid in
Elicited Cells1
Guillaume A. Schoch, Georgi N. Nikov, William L. Alworth, and Danièle Werck-Reichhart*
Department of Plant Stress Response, Institute of Plant Molecular Biology, Centre National de la Recherche
Scientifique-Unité Propre de Recherche 2357, Université Louis Pasteur, 28 Rue Goethe, F–67000 Strasbourg,
France (G.A.S., D.W.-R.); and Department of Chemistry, Tulane University, New Orleans, Louisiana 70118
(G.N.N., W.L.A.)
The cinnamate (CA) 4-hydroxylase (C4H) is a cytochrome P450 that catalyzes the second step of the main phenylpropanoid
pathway, leading to the synthesis of lignin, pigments, and many defense molecules. Salicylic acid (SA) is an essential trigger
of plant disease resistance. Some plant species can synthesize SA from CA by a mechanism not yet understood. A set of
specific inhibitors of the C4H, including competitive, tight-binding, mechanism-based irreversible, and quasi-irreversible
inhibitors have been developed with the main objective to redirect cinnamic acid to the synthesis of SA. Competitive
inhibitors such as 2-hydroxy-1-naphthoic acid and the heme-coordinating compound 3-(4-pyridyl)-acrylic acid allowed
strong inhibition of C4H activity in a tobacco (Nicotiana tabacum cv Bright Yellow [BY]) cell suspension culture. This
inhibition was however rapidly relieved either because of substrate accumulation or because of inhibitor metabolism.
Substrate analogs bearing a methylenedioxo function such as piperonylic acid (PIP) or a terminal acetylene such as
4-propynyloxybenzoic acid (4PB), 3-propynyloxybenzoic acid, and 4-propynyloxymethylbenzoic acid are potent
mechanism-based inactivators of the C4H. PIP and 4PB, the best inactivators in vitro, were also efficient inhibitors of the
enzyme in BY cells. Inhibition was not reversed 46 h after cell treatment. Cotreatment of BY cells with the fungal elicitor
␤-megaspermin and PIP or 4PB led to a dramatic increase in SA accumulation. PIP and 4PB do not trigger SA accumulation
in nonelicited cells in which the SA biosynthetic pathway is not activated. Mechanism-based C4H inactivators, thus, are
promising tools for the elucidation of the CA-derived SA biosynthetic pathway and for the potentiation of plant defense.
Phenylpropanoids form a large family of plantspecific compounds implicated in a broad range of
functions. Among the numerous chemical structures
stemming from the pathway, lignin is a quantitatively major biopolymer that plays a key role in plant
mechanical support and water transport and as physical barrier against pathogen infection. Other chemical classes of phenylpropanoids such as flavonoids,
isoflavonoids, stilbenes, or coumarins have essential
functions as antimicrobials, UV protectants, signaling
molecules mediating interaction with insect or symbiotic bacteria, or pathogen response (Dixon and
Paiva, 1995). An essential mediator of pathogen response and systemic acquired resistance is salicylic
acid (SA; Dempsey et al., 1999).
Although much is known about the diversity and
accumulation of the phenylpropanoid products, less
is understood about networking and control of their
biosynthesis. In particular, the diversity of enzymes
catalyzing the same reactions, evolution of some biosynthetic branches believed to be resulting from spe1
This work was supported by Ministère de la Recherche et de
l’Enseignement Supérieur (grant to G.A.S.).
* Corresponding author: e-mail [email protected]; fax 33–3–90 –24 –18 – 84.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.004309.
1022
ciation, and equilibration/compensation mechanisms between the different branches of the pathway
remain elusive. In addition, some biosynthetic
branches such as those of SA or coumarins are not yet
elucidated either at the biochemical or at the molecular level.
The objective of this work was to develop new
chemical effectors of the phenylpropanoid pathway.
Chemicals allowing inactivation or enhancement of
selected steps of the pathway are useful tools for both
biochemical and molecular investigations, constituting alternatives or complements to mutation or transgenic techniques for gene up- or down-regulation.
The main advantages of such chemical approaches
are the simultaneous inhibition of all isoenzymes
catalyzing the same reaction (provided that a reaction does not involve different families of proteins)
and easy transposition to orthologous gene products.
The upstream part of the phenylpropanoid metabolism consists of three enzymatic steps leading to
4-coumaroyl CoA (Fig. 1). The cinnamate (CA)
4-hydroxylase (C4H) catalyzes the second step, i.e.
the conversion of CA into p-coumarate. This enzyme
is a member of the structural family of cytochrome
P450 heme thiolate proteins, which catalyze monooxygenation of a broad range of substrates within all
organisms (Werck-Reichhart and Feyereisen, 2000).
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Cinnamate 4-Hydroxylase Inhibition in Vitro and in Vivo
Figure 1. C4H and branching in the upper phenylpropanoid pathway. PAL, Phe ammonialyase; 4CL, 4-hydroxycinnamate CoA ligase;
AOPP, amino-␤-phenyl-propionic acid is an inhibitor of PAL (Amrhein et al., 1983); MDCA,
methylene dioxocinnamic acid is an inhibitor of
4CL (Funk and Brodelius, 1990).
In plant biosynthetic pathways, P450s catalyze critical hydroxylation steps and a variety of more complex reactions (Kahn and Durst, 2000). The C4H gene
CYP73A1 was isolated from Jerusalem artichoke (Helianthus tuberosus; Teutsch et al., 1993). CYP73A1 orthologs have then been isolated from more than 20
plant species including tobacco (Nicotiana tabacum cv
Bright Yellow [BY]; http://drnelson.utmem.edu/
P450dbplant.html; Ralston et al., 2001). All of them
belong to the CYP73A subfamily of P450 genes, and
when the proteins were expressed in heterologous
systems, they all displayed C4H activity.
The substrate specificity and several inhibitors of
the recombinant CYP73A1 expressed in yeast (Saccharomyces cerevisiae) have been described in detail
(Pierrel et al., 1994; Batard et al., 1997). CYP73A1
substrates are planar, aromatic, molecules with an
anionic site opposite (i.e. at about 8.5 Å) to the position of oxidative attack (Schalk et al., 1997, 1998).
The most efficient inhibitors of the C4H share the
same structural characteristics as the substrates. Depending on the functions present in the molecule,
they behave as simple competitors or mechanismbased irreversible or quasi-irreversible inactivators
(Pierrel et al., 1994; Schalk et al., 1997, 1998). The
most potent inhibitors reported so far are 2-hydroxy1-naphthoate (2HN) and piperonylic acid (PIP). 2HN
is a very potent competitive inhibitor that binds the
active site with a 10-fold higher affinity than the
substrate (Ks ⫽ 0.28 ␮m) but is not metabolized
(Schalk et al., 1997). PIP is a natural compound isolated from the bark of Paracoto tree that behaves as a
selective and potent mechanism-based quasiirreversible inhibitor of the C4H (Schalk et al., 1998).
Oxidative attack of the PIP methylenedioxo function
leads to a carbene that forms a coordination with the
P450 heme iron. The enzyme-inhibitor complex is
Plant Physiol. Vol. 130, 2002
fairly stable, but the carbene can be displaced at
high-substrate concentration. In elicitor-treated BY2
cells, PIP also behaves as an effective C4H inactivator
and inhibits the formation of p-coumarate and accumulation of the downstream metabolite scopoletin
(Schalk et al., 1998).
A single branch point at CA is reported in the
upstream phenylpropanoid pathway, which leads to
benzoic acids such as the defense signal SA by a
mechanism that is not yet elucidated (Chong et al.,
2001). Preliminary experiments have suggested that
PIP inhibition of C4H activity promotes the accumulation of SA in tobacco BY2 cells (Chong et al., 2001).
PIP and 2HN are both at least partially reversible
inhibitors of C4H. Irreversibly binding inactivators of
the enzyme should thus be more efficient for obtaining strong inhibition and redirection of metabolic
fluxes in vivo. Compounds containing a terminal
alkyne are known as potent irreversible inactivators
of P450s (Ortiz de Montellano and Komives, 1985).
2,4-Dichlorophenoxypropyne was described as such
an inactivator of C4H (Pierrel et al., 1994). This compound was however not designed to target C4H, and
its inhibition and binding constants are far from optimal. In this paper, we report new competitive and
mechanism-based
irreversible
inactivators
of
CYP73A1, and compare their relative efficiencies at
inhibiting C4H in tobacco cell cultures and promoting accumulation of SA with those of PIP and 2HN.
RESULTS
Binding of Inhibitors to CYP73A1
The set of CA analogs tested as inhibitors of
CYP73A1 is shown in Figure 2. New types of P450
inhibitors were added to PIP and 2HN in these ex-
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1023
Schoch et al.
mechanism-dependent irreversible inhibitors of the
enzyme.
The binding of ligands near the heme of the oxidized P450 influences the spin state and the redox
potential of the heme iron, which triggers the reaction or makes it more difficult, and results in a
change in visible absorbance of the heme protein
(Jefcoate, 1978). The binding properties of compounds in Figure 2 were investigated with yeast
microsomes expressing CYP73A1 (Table I). All three
propynyl derivatives led to a shift of the maximum of
absorbance of the oxidized P450 from 420 to 390 nm,
similar to that observed with cinnamic acid or PIP
(Schalk et al., 1998). The resulting “type I” difference
spectrum reflects the displacement of a water sixth
ligand to the heme iron, correlated to an increase in
spin and redox potential that favors electron supply
and activates the catalytic cycle (Raag and Poulos,
1989). The amplitude of the binding spectra obtained
with 4PB, 3PB, and 4PO is larger than that previously
reported for 2,4-dichlorophenoxypropyne (Pierrel et
al., 1994). These new compounds are thus better positioned for oxidative attack in the active site and are
therefore expected to behave as more efficient
mechanism-based inactivators of the C4H. Displacement of water from the active site, however, is incomplete, and dissociation constants remain high
compared with the Ks for CA (8 ␮m; Pierrel et al.,
1994), indicating that these inhibitors are not yet fully
optimized.
As expected for a compound forming a coordination to ferric heme iron, the binding of 3PA induces a
shift of the maximum of absorbance of CYP73A1
from 414 to 428 nm, giving rise to a “type II” differ-
Figure 2. Molecules tested as inhibitors of CYP73A1.
periments, including trans-3-(pyrid-4-yl)-acrylic acid
(3PA), the sp2 hybridized ring nitrogen of which is
positioned to coordinate as the sixth ligand to the
heme iron. Such a compound, anchored both on the
protein and the heme, is expected to behave as a
tight-binding but reversible inhibitor of the C4H
(Ortiz de Montellano and Correia, 1995). Analogs
bearing a terminal alkyne function and more closely
mimicking the structure of cinnamic acid than
2,4-dichlorophenoxypropyne, 4-propynyloxybenzoic
acid (4PB), 4-propynyloxymethylbenzoic acid (4PO),
and 3-propynyloxybenzoic acid (3PB) were also synthesized. Oxidative attack of the triple bond of such
compounds is likely to lead either to heme alkylation
or to the formation of a ketene that can react with the
protein or be hydrolyzed to a carboxylic acid metabolite (Ortiz de Montellano and Komives, 1985; Ortiz
de Montellano and Correia, 1995; Regal et al., 2000).
These terminal acetylenes are expected to behave as
Table I. Inhibitor binding and inhibition constants determined with recombinant C4H (CYP73A1)
Binding and inhibition constants were determined using H. tuberosus C4H (CYP73A1) in microsomes
from yeast W(R). Binding constants were determined from the shift of heme maximum of absorbance
detected upon binding of increasing concentrations of inhibitor. Inhibition constants were calculated
from residual C4H activity.
Binding
Inhibitor
Mechanism-based irreversible inactivator
3PB
4PO
4PB
3-(2,4-Dichlorophenoxy)-1-propyne
Mechanism-based quasi-irreversible inactivator
PIP
Reversible inhibition
3PA
2HN
Suicide
Inhibition
Competitive
Inhibition
Ks
High spina
Ki
Kinact
Ki
␮M
%
␮M
min⫺1
␮M
10.5
580
53
94
0.346
0.22
0.41
0.122
17
0.064
204
40
86
11
390
78
Type I (low shiftb)
45
34
1.7
0.28
Type IIc
58
2
0.048
a
A 100% conversion of CYP73A1 to high spin is obtained for saturating cinnamic acid concentrab
tion, with an ␧type I of 125 mM⫺1 cm⫺1 (Urban et al., 1994).
A shift of the maximum of absorbance
c
was observed, but ⌬Amax was too small for determination of the binding parameters.
A type II
difference spectrum was recorded with an ␧type II of 95 mM ⫺1 cm⫺1.
1024
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Plant Physiol. Vol. 130, 2002
Cinnamate 4-Hydroxylase Inhibition in Vitro and in Vivo
ence spectrum. The resulting high- to low-spin transition is accompanied by a change in redox potential
that makes P450 reduction more difficult. The dissociation constant for 3PA is slightly lower than that
measured for CA, and the maximum amplitude of
the difference spectrum (⑀type II of 95 mm⫺1 cm⫺1) is
high, which indicates that 3PA is a nearly optimized
ligand of CYP73A1. 3PA should thus behave as a
strong competitive inhibitor of C4H. In agreement
with these data, no metabolism of 3PA is detected
upon incubation with recombinant CYP73A1.
In Vitro Mechanism-Based Inactivation of CYP73A1
4PB, 3PB, and 4PO were tested as mechanismbased inactivators of CYP73A1. Recombinant yeast
microsomes were incubated with NADPH and various concentrations of each of the molecules before
determination of residual C4H activity. C4H inactivation was concentration- and time-dependent for all
three compounds and did not occur in the absence of
NADPH. A high concentration of CA in the assay for
residual activity did not reverse the inhibition.
Pseudo-first-order kinetics were obtained in the initial phase of the inactivation (Fig. 3). The time required for half-maximal inactivation (t1/2) at each
inactivator concentration was calculated and plotted
versus the reciprocal of the inhibitor concentration.
The plots were typical of an inactivation that proceeds with saturation (Silverman, 1996). Kinetic constants calculated from the plots are summarized in
Table I and compared with those previously determined for 2,4-dichlorophenoxypropyne and PIP
(Pierrel et al., 1994; Schalk et al., 1998). Higher Kinact
observed for 4PB and 3PB were correlated to lower KI
and to large changes in heme iron spin state upon
Figure 3. Time- and concentration-dependent inactivation of CYP73A1 by 4PB, 3PB, and 4PO. Recombinant CYP73A1 in
W(R) yeast microsomes was preincubated in the presence of NADPH and different concentrations of the propynyl derivatives
before assaying residual C4H activity. A, Time course of the C4H inactivation measured upon incubation with 4PB. Similar
pseudo-first-order kinetics of inactivation were obtained with 3PB and 4PO. B, Plot of the half-inactivation times, estimated
from linear regression analysis of curves shown in A, versus reciprocal of inhibitor concentration. C and D, Half-inactivation
time plots, as in B, for 3PB and 4PO.
Plant Physiol. Vol. 130, 2002
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1025
Schoch et al.
binding of the inhibitors. On the basis of these data,
inactivation of CYP73A1 with 100 ␮m inhibitor occurs in 2.5 min with 4PB or 3PB, compared with 10
min with 2,4-dichlorophenoxypropyne or PIP and 21
min with 4PO. 4PB and 3PB thus appear to be the
most potent irreversible inactivators of C4H
enzymes.
Attempts to detect carboxylic acid metabolites of
4PB upon incubation with CYP73A1 were unsuccessful, indicating either a further modification of the
product of the reaction or a very low partition ratio.
In favor of the second hypothesis, only a very small
proportion of the inhibitor was processed after complete inactivation.
Competitive Inhibition of CYP73A1
3PA does not bear any function likely to lead to
P450 inactivation but very closely mimics cinnamic
acid. Combined with this strong structural homology
to CA, coordination of its 4-nitrogen to the heme iron
should lead to a very tight binding to the active site.
No time-dependent inhibition was observed with
3PA that, as expected, behaved as a typical and
strong competitive inhibitor (Fig. 4). The KI determined from the kinetic analysis is 2 ␮m, which is in
good agreement with the KS measured in binding
experiments.
C4H Activity and Inhibition in Vivo
The best inhibitor of each type, i.e. 2HN (simple
competitive), 3PA (iron-coordinating, competitive),
PIP (quasi-irreversible, mechanism-dependent), and
4PB (irreversible, mechanism-dependent) was selected for comparison of C4H inhibition efficiency in
planta.
To set up a test for the evaluation of in vivo C4H
activity, a tobacco BY cell suspension culture was fed
with 20 ␮m [14C]CA. Free phenolic acid content was
Figure 4. Inhibition of C4H activity by 3PA. Lineweaver-Burk representation of the inhibition of C4H activity of recombinant
CYP73A1 (3 nM in the assay) by 3PA (0, 1, 3, or 10 ␮M). Data are
means of duplicate experiments.
1026
Figure 5. Time course of the conversion of CA into p-coumarate in
a tobacco BY cell suspension culture. One-milliliter aliquots of 5-dold cells were incubated with 20 ␮M [14C]CA in Eppendorf tubes at
30°C. CA to p-coumarate conversion was evaluated after TLC analysis of extracted free phenolics, and identity of radiolabeled spots
was confirmed by HPLC-diode array analysis. Data are expressed as
a proportion of initial CA recovered as p-coumarate at the end of the
incubation.
extracted, and radiolabeled CA and p-coumarate
were quantified by thin-layer chromatography (TLC)
and HPLC. p-Coumarate was the only radiolabeled
metabolite recovered in these assays. Its identity was
confirmed by HPLC-diode array analysis. The timedependent formation of p-coumarate relative to initial CA is represented in Figure 5. Approximately
25% of the initial [14C]CA is converted into
p-coumarate in 30 min. p-Coumarate then declines,
probably because of further conversion into nonether-soluble esters or other metabolites. C4H inhibition in BY cells was therefore evaluated by measuring the formation of p-coumarate after 30 min of
incubation with radiolabeled substrate.
The inhibitors were added to the BY cell suspension culture, and aliquots were collected for monitoring of residual activity (Fig. 6). Strong C4H inhibition
was observed with all four molecules in aliquots
collected immediately after addition of the inhibitors.
After 4 h, 2HN, 3PA and PIP inhibition was more
than 95%. After 22 h, competitive inhibition by 2HN
and 3PA was largely reversed, either because of an
accumulation of substrate and displacement of the
inhibitor or because of an inhibitor conjugation
and/or compartmentation. Both mechanism-based
inactivators, 4PB and PIP, remained fully effective for
at least 46 h. Slow displacement by CA of the inhibitory carbene formed upon PIP metabolism was previously reported (Schalk et al., 1998). The present
data thus suggests that if CA concentrations reached
in this experiment are high enough for reversion to
occur, the enzyme inactivation by PIP in vivo was
probably faster than the displacement of the derived
carbene by CA. This would be in agreement with
previous observations in a recombinant system in
vitro (Schalk et al., 1998).
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Plant Physiol. Vol. 130, 2002
Cinnamate 4-Hydroxylase Inhibition in Vitro and in Vivo
PAL activity, and no PAL inhibition from 3PA, PIP,
and 4PB is expected.
SA Accumulation in Inhibitor-Treated BY Cells
Figure 6. Inhibition of C4H activity in tobacco BY cells. Inhibitors in
DMSO were added to a 5-d-old suspension culture. DMSO was
added to the control. Final concentrations in the culture were 0.06%
(v/v) DMSO, 40 ␮M 2HN, 40 ␮M 3PA, 100 ␮M 4PB, and 10 ␮M PIP.
Aliquots collected after 0.5, 4, 22, and 46 h of incubation were
assayed for conversion of [14C]CA (20 ␮M). Data are means ⫾ SD of
four measurements.
The same cell treatment was performed after 30
min of elicitation with ␤-megaspermin, a proteinaceous fungal elicitor triggering hypersensitive response (Baillieul et al., 1995). C4H inhibition with all
four compounds was similar to that observed with
nonelicited cells at 0.5 h. Only 4PB provided effective
inhibition (70%) after 22 h. After 46 h, none of the
inhibitors influenced CA conversion anymore. Increased flux of metabolites and induced enzyme synthesis thus relieve the inhibition, probably because of
displacement of reversible inhibitors and complete
metabolism of mechanism-dependent inactivators. In
agreement with this hypothesis, the irreversible
mechanism-dependent and poorly metabolized inhibitor 4PB is the compound that provides the longer
lasting inhibition.
The expected impacts of an in vivo C4H inactivation are a decreased formation of downstream metabolites, such as scopoletin (Schalk et al., 1998), and
the accumulation of CA and derived compounds
such as benzoic and SAs (Chong et al., 2001). To
compare the relative efficiencies of 4PB and PIP for
triggering accumulation of SA, BY cell suspension
cultures were treated with inhibitor concentrations of
10 ␮m. Low inhibitor concentrations were used in
this experiment to ensure selectivity of the inhibition
and to avoid interfering with the benzoate hydroxylase activity that was previously suggested to be a
P450 enzyme (Leon et al., 1995). Total (free plus
conjugated) SA accumulation was monitored with
and without a prior treatment of the cells with
␤-megaspermin (Fig. 8). The basal content in control,
dimethylsulfoxide (DMSO)-treated cultures was 300
ng total SA g⫺1 wet cells. No significant variation of
this basal SA level was detected over 30 h in the
absence of ␤-megaspermin treatment. Upon treatment with elicitor, SA accumulation was induced to
reach a maximum of 1,800 ng g⫺1 at 24 h.
When the BY cells were treated with inhibitors,
only very slight increases in SA, never exceeding 45%
of the control, were detected in the absence of
␤-megaspermin. In the presence of the elicitor, SA
accumulated with a time course similar to that observed in the absence of inhibitor, but levels of total
SA at 24 h were about 8-fold that of the control for
cells treated with PIP and 3.6-fold with 4PB. The
highest levels of SA reached after treatment with PIP
and ␤-megaspermin is close to 13 ␮g g⫺1 wet cells. In
Inhibition of the PAL
Cinnamic acid and structurally related molecules
were reported to behave as PAL inhibitors (Sato et
al., 1982). Because our aim was to develop C4Hspecific inhibitors allowing redirection of the flux of
metabolites from cinnamic acid toward that of SA,
the effects of 2HN, 3PA, PIP, and 4PB on PAL activity
were also investigated (Fig. 7). PAL activity was
tested on a whole extract of tobacco BY cells pretreated with ␤-megaspermin to induce the enzyme
expression. With PIP, 4PB, and 3PA, no PAL inhibition was observed at concentrations up to 500 ␮m. A
concentration-dependent inhibition was however observed with 2HN, with PAL inhibition reaching 20%
with 100 ␮m of the compound. The concentration of
2HN used for subsequent C4H inhibition in vivo (40
␮m) thus should not very significantly interfere with
Plant Physiol. Vol. 130, 2002
Figure 7. Inhibition of PAL activity. Whole extracts of
␤-megaspermin pretreated BY cells were diluted 5-fold in 100 mM
borate buffer, pH 8.8, and incubated with 85 ␮M [14C]Phe and
various concentrations of inhibitors for 1 h at 37°C. Radiolabeled
cinnamic acid was extracted and counted by liquid scintillation.
Data are means ⫾ SD of two experiments. 100% activity ⫽ 19 pmol
h⫺1␮L⫺1.
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1027
Schoch et al.
Figure 8. Accumulation of total SA in inhibitor-treated cells. A 5-dold tobacco BY cell culture was treated with 10 ␮M PIP, 10 ␮M 4PB,
or DMSO. Identical treatment was performed 30 min after elicitation
with ␤-megaspermin (␤-meg). Free and conjugated SA were extracted from aliquots and hydrolyzed, and total SA was purified and
quantified by fluorometry. The experiment was performed on two
subcultures maintained independently to evaluate variations of cell
response.
addition to higher SA accumulation, PIP also triggers
an earlier response compared with 4PB, already close
to maximal at 12 h. Upon 4PB treatment, however,
SA accumulation persist longer and is still 3-fold that
of the control after 30 h.
DISCUSSION
New Reversible and Irreversible C4H Inhibitors
The main objective of this work was to develop
new C4H inhibitors to control the flow of the metabolites in the main phenylpropanoid pathway and to
investigate the impact of a redirection of this flow on
the synthesis of SA and plant defense. Efficient C4H
inhibitors may in particular constitute very useful
tools to help characterization of the SA biosynthetic
routes that remain elusive.
Different types of inhibitors have been described,
based on specific properties of cytochromes P450. In
addition to classical competitive inhibitors, the synthesis of structural analogs of the substrate bearing
activatable functions allows the design of molecules
selectively inactivating specific P450 isoforms (Ortiz
de Montellano and Correia, 1995). Once metabolized,
such compounds can behave as tight-binding or irreversible ligands of the enzyme. Another type of
inhibitor, which simultaneously binds the protein
and the prosthetic heme iron, is often also more
1028
effective than molecules that rely on only one of these
interactions. The different types of P450 inhibitors
were tested and compared in this study.
3PA was assayed for its potential ability to coordinate heme iron by the intermediate of the electron
pair of its ring nitrogen and to simultaneously bind
through its carboxylate the substrate docking region
of the protein. It behaved as a good ligand and
formed the expected heme coordination resulting in
characteristic absorption spectra. As expected for
such a type of direct heme ligand inducing a high- to
low-spin shift of the iron, it was not metabolized but
behaved as a competitive inhibitor. 3PA dissociation
constant was however not significantly lower than
that measured for the substrate and much higher
than that of the classical competitive inhibitor 2HN.
A possible explanation of this relatively weak interaction of 3PA can be found in NMR measurements of
the distances of the substrate protons to the heme
iron, which indicate that the initial positioning of the
substrate and its analogs in the active site is approximately parallel with the heme plane (Schoch, 2001).
Such a positioning does not favor stable 3PA coordination to the heme iron. In good agreement with
measured binding constants, 2HN behaved as a better inhibitor of the C4H than 3PA. A stronger anchoring of 2HN to the C4H is probably provided by its
additional phenolic function that does not compromise other interactions with the protein.
We showed previously that PIP is a potent quasiirreversible mechanism-dependent inhibitor of the
C4H (Schalk et al., 1998). Tight heme-binding of the
inhibitory carbene formed during PIP metabolism
can however be slowly reversed by high concentrations of CA in vitro. An attempt was thus made at
designing completely irreversible mechanismdependent inhibitors. 4PB, 3PB, and 4PO were designed taking into account the C4H substrate pharmacophore deduced from substrate specificity
experiments (Pierrel et al., 1994; Schalk et al., 1998),
including size, aromatic ring, and carboxylate group
for anchoring in the active site. An activatable terminal acetylene was grafted in different positions onto
oxybenzoic or oxytoluic acids. The resulting molecules partially mimic cinnamic and naphthoic acids,
which are the best substrates of the C4H, and the
acetylenic function should be positioned to favor its
oxidative attack in the active site of the CAH. A
limitation for the optimization of such molecules and
to adapt them to the C4H substrate pocket was however the linear structure of the alkyne carbons.
As expected, 4PB, 3PB, and 4PO all behaved as
mechanism-dependent inactivators of recombinant
C4H in yeast microsomes. 4PB and 3PB, being better
positioned in the active site, were more effective inhibitors of C4H than 4PO and than previously described
compounds (Pierrel et al., 1994). 2-Ethynylnaphthalene, an effective P450 inactivator of several mammalian enzymes (Hopkins et al., 1992; Roberts et al.,
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Plant Physiol. Vol. 130, 2002
Cinnamate 4-Hydroxylase Inhibition in Vitro and in Vivo
1993; Strobel et al., 1999), which was included in our
assays, did not bind C4H nor inhibit its activity,
confirming the need for an appropriate design to
achieve efficient and selective enzyme inactivation.
Compared Inhibition Efficiency in Vivo
All types of inhibitors efficiently blocked the C4H
activity in cell culture experiments. Efficiency of 3PA
and 2HN was correlated to their respective enzyme
affinities. Inhibition by such competitive inhibitors
was however rapidly reversed. This was possibly
attributable to elevated CA concentrations in the
treated cells or resulted from their inactivation by
detoxification systems (conjugation and compartmentation). Fast reversion of C4H inhibition is a
strong limitation to the use of 3PA and 2HN for in
vivo experiments.
Very similar responses to the quasi-irreversible and
irreversible mechanism-dependent inactivators, PIP
and 4PB, were obtained when C4H inhibition was
assayed in cell cultures. PIP was still effective 46 h
after cell treatment, when the experiment was carried
on in the absence of elicitor. This suggests that the
CA concentrations in the PIP-treated cells are not
sufficient to dissociate the enzyme-inhibitor complex
or that the dissociation rate of the complex is slower
than the further inactivation of the enzyme. PIP thus
remains a useful C4H inactivator, effective at a low
concentration, and is therefore likely to be specific.
Its efficiency is further proved by the SA accumulation triggered by PIP concentrations as low as 10 ␮m.
C4H inhibition by 3PA and 2HN being very transient, only PIP and 4PB were tested for their ability to
redirect CA to the synthesis of SA. In cells cultured
under normal axenic conditions, low SA concentrations were detected and these concentrations were
only slightly increased upon treatment with C4H
inhibitors (Fig. 8). Under such conditions, the SA
biosynthetic pathway thus seemed almost inactive,
possibly because of low precursor supply (Chong et
al., 2001). Upon treatment with ␤-megaspermin, the
SA pathway was activated, and the activation was
transient, reaching a maximum around 24 h after
treatment. Addition of both PIP and 4PB to the elicited cells led to a dramatic increase in SA accumulation and to an effective reorientation of CA metabolism. At a 10 ␮m concentration, PIP allowed for an
almost complete C4H inhibition and a very strong
(8-fold) but also early (within less than 6 h) increase
in SA accumulation compared with untreated cells.
At 30 h, SA accumulation then dropped very sharply.
Because PIP-inhibition of C4H activity is already reversed after 22 h in elicited cells (data not shown),
this is more likely to reflect fast C4H recovery resulting from enzyme-inhibitor dissociation or PIP inactivation than a decrease in CA to SA conversion, in
PAL activity, or in inhibition of an upstream step in
the shikimate pathway. Used at the same low conPlant Physiol. Vol. 130, 2002
Figure 9. General synthetic route for the synthesis of propynyl inhibitors of CYP73A1.
centration, 4PB leads to a smaller increase in SA than
PIP, most likely as a result of its lower affinity for
C4H and only partial enzyme inhibition (Fig. 3). The
low metabolism of this inhibitor and its small partition ratio (i.e. high inactivation efficiency) however
ensure a longer lasting effect.
CONCLUSIONS
A set of C4H inhibitors has been developed, allowing short to long term modulation of the enzyme
activity in plant tissues. Such inhibitors should be
useful tools to help elucidate the molecular mechanisms of the conversion of CA to SA and for understanding regulation of the SA and general phenylpropanoid pathways. Their reported effect on SA
accumulation in elicited tobacco cell cultures (this
work; Chong et al., 2001) strongly suggests that, in
this plant material, SA biosynthesis branches on CA
and not chorismate as recently reported for Arabidopsis (Wildermuth et al., 2002). If such inhibitors
cannot trigger SA accumulation per se, they might be
very useful to potentiate and/or accelerate the onset
of defense systems in elicited or pathogen-infected
plants.
MATERIALS AND METHODS
Chemicals
CA, PIP, 2-HN, NADPH, SA, and DMSO were from Sigma-Aldrich
(L’Isle d’Abeau Chesnes, France), 3PA was from Maybridge (Tintagel, UK),
trans-[3⬘-14C]CA was from Isotopchim (Ganagobie, France), [1⬘-14C]SA and
l-[14C(U)]-Phe were from NEN (Zaventem, Belgium). 4-Hydroxybenzoic
acid, 3-hydroxybenzoic acid, 4-(hydroxymethyl) benzoic acid, 4-hydroxycinnamic acid, 3-hydroxycinnamic acid, propargyl bromide, and acetyl chloride were from Aldrich (Milwaukee). ␤-Megaspermin purified from culture
filtrates of Phytophthora megasperma (Baillieul et al., 1995) was kindly provided by Dr. S. Kauffmann (Institut de Biologie Moléculaire des Plantes,
Strasbourg, France).
Synthesis of the Propynyl Derivatives (Fig. 9)
Flash column chromatography techniques were performed by using silica gel (mesh-230–400). The gas chromatography/mass spectroscopy (GC/
MS) analyses of the synthesized products were carried out on a gas chro-
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
1029
Schoch et al.
matograph/mass spectrometer (Chem Station 5997, Hewlett Packard, Palo
Alto, CA). NMR spectra were recorded on an Omega 400 MHz FT-NMR
spectrometer (General Electric, Fairfield, CT).
4-Hydroxybenzoate
Acetyl chloride (3.6 g, 46 mmol) and methanol (15 mL) were mixed
together at 0°C and then 4-hydroxybenzoic acid (5 g, 30.5 mmol) was added.
After the mixture was stirred at room temperature overnight, the methanol
was removed, and the residue was dissolved in CH2Cl2. The solution was
washed with a saturated aqueous solution of NaHCO3 and water and then
dried over magnesium sulfate. The solvent was removed under reduced
pressure to give 4-hydroxybenzoic acid methyl ester (4.8 g, 88% yield). 1H
NMR ␦ 7.99 (d, 2H, Ar-H), 7 (d, 2H, Ar-H), 4.6 (s, 1H, OH), and 4.3 (s, 3H,
CH3); GC/MS m/z 152.
4-Propynyloxybenzoate
Solution of powdered KOH (6 g, 108 mmol) in DMSO (12 mL) was
prepared. After stirring the mixture for 5 min, 4-hydroxybenzoic acid
methyl ester (4.8 g, 27 mmol) was added followed immediately by propargyl
bromide (7 g, 48 mmol). Stirring continued for 1 h at room temperature. The
reaction mixture was poured into water (40 mL) and extracted with CH2Cl2
(2 ⫻ 30 mL). The combined organic extracts were washed with water, dried
over magnesium sulfate and reduced to dryness under reduced pressure.
Chromatography on silica gel with eluent hexane:ethyl acetate (3:1, v/v) gave
4PB methyl ester (3 g, yield 59%). 1H NMR ␦ 7.99 (d, 2H, Ar-H), 7 (d, 2H,
Ar-H), 4.9 (s, 2H, CH2), 4.3 (s, 3H, CH3), and 3.1 (s, 1H, CH); GC/MS m/z 190.
Treatment of Tobacco (Nicotiana tabacum) Cell
Suspension Culture
The tobacco BY cell suspension was maintained as described for BY-2
lines (Nagata et al., 1992). For measurement of SA accumulation or Phe
ammonia-lyase (PAL) activity, 50 nm ␤-megaspermin was added to the
culture 30 min or 12 h before the experiment, respectively. Inhibitors were
added solubilized in DMSO. The final DMSO concentration in the culture
was 0.06%.
In Vivo Enzyme Assays
4PB
4PB methyl ester (3 g, 16 mmol) was added to a solution of NaOH (1.6 g,
40 mmol) in ethanol (25 mL) and water (8 mL). After stirring overnight at
25°C the alcohol was removed under reduced pressure, and the reaction
mixture was extracted with methylene chloride (2 ⫻ 30 mL). The combined
organic extracts were washed with water and acidified with 2 n HCl. The
product precipitated as white crystals, which were recrystallized from
ethanol-water to give 4PB (2.4 g, yield 84%). Melting point (mp) 195°C, 1H
NMR ␦ 7.99 (d, 2H, Ar-H), 7 (d, 2H, Ar-H), 4.9 (s, 2H, CH2), 3.1 (s, 1H, CH).
GC/MS m/z 176.
3PB
This compound was prepared from 3-hydroxybenzoic acid following the
chemical procedure above. mp 130°C, 1H NMR ␦ 7.65–7.40 (m, 4H, Ar-H),
4.8 (s, 2H, CH2), and 3.1 (s, 1H, CH); GC/MS m/z 176.
4PO
In vivo conversion of trans-CA into p-coumarate was evaluated using 1
mL of cell culture (about 0.3 g wet cells) incubated with 20 ␮m of [14C]CA
(140 Bq/nmol) at 30°C under agitation. The reaction was stopped by addition of 500 mm HCl from a 4 n solution. Free phenolic acids were extracted
three times with 2 volumes of ether:petroleum ether (50:50, v/v), concentrated under nitrogen and analyzed by TLC. Radiolabeled spots were
scraped, eluted with ether, and further analyzed by HPLC on a LiChrosorb
RP-18 column (Merck 4 ⫻ 125 mm, 5 ␮m) at a flow rate of 1 mL min⫺1, with
elution 5 min isocratic, followed by a 20-min linear gradient from 10% to
52% (v/v) acetonitrile, in water containing 0.2% (v/v) acetic acid. trans-CA
and 4-coumarate were identified from their retention time, UV absorbance,
and radioactivity, with reference to commercial standards.
For C4H inhibition experiments, 15 mL of 5-d-old-BY cell suspension
were subcultured 46 h in the presence of the inhibitor. At time intervals,
1-mL aliquots were assayed for residual C4H activity. For PAL inhibition
measurements, filtered BY cells were extracted and enzyme activity was
monitored, as described previously (Pellegrini et al., 1994).
SA Quantification
The compound was prepared from 4-(hydroxymethyl) benzoic acid following the chemical procedure above. mp 135°C, 1H NMR ␦ 8 (d, 2H, Ar-H),
7.5 (d, 2H, Ar-H), 4.65 (s, 2H, CH2), 4.2 (s, 2H, CH2), and 3 (s, 1H, CH);
GC/MS m/z 190.
Yeast (Saccharomyces cerevisiae) Expression and
Microsome Preparation
In vitro inhibition experiments were performed using microsomal preparations from yeast W(R) transformed with the expression plasmid pCA4H/
V60, allowing co-expression of the yeast P450 reductase and CYP73A1
(Urban et al., 1994). Microsomal fractions were prepared as described by
Pompon et al. (1996).
Spectrometric Measurements and Enzyme Assays
P450 content was calculated from reduced P450-CO versus reduced
difference UV-visible spectra (Omura and Sato, 1964). Low- to high-spin
conversion and dissociation constants of enzyme-ligand complexes were
1030
evaluated from type I ligand binding spectra as described by Schalk et al.
(1997). Efficiency of ligand-induced high- to low-spin conversion was deduced from type II difference spectra (Jefcoate, 1978).
trans-Cinnamic acid hydroxylation was assayed using radiolabeled
trans-[3⬘-14C]cinnamic acid as described by Pierrel et al. (1994), except that
the TLC analysis was performed using ether:petroleum ether:formic acid
(50:50:1, v/v) as a solvent. 4-Coumaric (RF 0.3) and cinnamic acids (RF 0.6)
were scraped, and radioactivity was counted by liquid scintillation. For
determination of the kinetic constants, data were fitted using the nonlinear
regression program DNRPEASY derived from DNRP53 (Duggleby, 1984).
The time-dependent inactivation of the C4H in yeast microsomes was
evaluated using a general dilution procedure (Silverman, 1996). CYP73A1
(400 nm) was preincubated at 28°C in 100 mm sodium phosphate pH 7.4, in
the presence of 500 ␮m NADPH, 1 mm Glc-6-phosphate, 0.2 unit of Glc-6phosphate dehydrogenase, and various concentrations of inactivator. For
each preincubation time, an aliquot of the inactivation medium was diluted
20-fold into a second incubation mixture containing 150 ␮m of [14C]CA to
assay residual activity.
For evaluation of SA accumulation, 40 mL of 5-d-old BY cell suspension
culture was treated with inhibitors simultaneously in the presence or absence of elicitor. At time intervals, 3-mL aliquots were filtered, weighted,
and frozen in liquid nitrogen. After addition of 2 nCi of [14C]SA as internal
standard, total phenolics were extracted successively with 2 mL g⫺1 cells of
90% and 100% (v/v) methanol and vacuum dried. Conjugated phenols were
hydrolyzed 1 h at 80°C after the addition of 1 mL of 4 n HCl. Free acids were
then extracted three times with 2.5 volumes of ether:petroleum ether (50:50,
v/v). Organic phases were pooled, dried under nitrogen, and resuspended
in solvent A (97% 20 mm sodium acetate, pH 5, and 3% [v/v] acetonitrile).
Samples were injected on a Nova Pak RP-C18 column (3.9 ⫻ 150 mm, 5
␮m; Waters, Milford, MA) equilibrated in solvent A, at a flow rate of 1 mL
min⫺1, with elution 10 min isocratic and then a 2-min linear gradient to 80%
(v/v) acetonitrile. SA was detected by fluorometry (␭ex315 nm, ␭em 405 nm).
To obtain a consistent signal and to avoid saturation of the detector, fractions from 0.1 to 0.005 of each sample were injected until a suitable response
was obtained. Quantification of the peak areas was performed relative to
commercial standards. An aliquot (0.25) of each fraction injected was
counted by liquid scintillation to determine recovery of the internal
standard.
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Plant Physiol. Vol. 130, 2002
Cinnamate 4-Hydroxylase Inhibition in Vitro and in Vivo
ACKNOWLEDGMENTS
We thank Monique Le Ret and Laurent Levallois for technical assistance.
The W(R) yeast strain and the pYeDP60 expression vector were kindly
provided by Drs. D. Pompon and P. Urban (Centre National de la Recherche
Scientifique [CNRS], Gif-sur-Yvette), and ␤-megaspermin by Dr. S. Kauffmann (CNRS, Institut de Biologie Moléculaire des Plantes, Strasbourg).
Helpful discussion and advice from Dr. P. Saindrenan are very gratefully
acknowledged.
Received February 14, 2002; returned for revision May 10, 2002; accepted
June 23, 2002.
LITERATURE CITED
Amrhein N, Frank G, Lemm G, Luhmann HB (1983) Inhibition of lignin
formation by l-alpha-aminooxy-beta-phenylpropionic acid, an inhibitor
of phenylalanine ammonia-lyase. Eur J Cell Biol 29: 139–144
Baillieul F, Genetet I, Kopp M, Saindrenan P, Fritig B, Kauffmann S (1995)
A new elicitor of the hypersensitive response in tobacco: a fungal glycoprotein elicits cell death, expression of defence genes, production of
salicylic acid, and induction of systemic acquired resistance. Plant J 8:
551–560
Batard Y, Schalk M, Pierrel MA, Zimmerlin A, Werck-Reichhart D (1997)
Regulation of the cinnamate 4-hydroxylase (CYP73A1) in Helianthus tuberosus tuber in response to wounding and chemical treatments. Plant
Physiol 113: 951–959
Chong J, Pierrel MA, Atanassova R, Werck-Reichhart D, Fritig B, Saindrenan P (2001) Free and conjugated benzoic acid in tobacco plants and
cell cultures: induced accumulation upon elicitation of defense responses
and role as salicylic acid precursors. Plant Physiol 125: 318–328
Dempsey DA, Shah J, Klessig DF (1999) Salicylic acid and disease resistance in plants. Crit Rev Plant Sci 18: 547–575
Dixon RA, Paiva NL (1995) Stress-induced phenylpropanoid metabolism.
Plant Cell 7: 1085–1097
Duggleby RG (1984) Regression analysis of nonlinear Arrhenius plots: an
empirical model and a computer program. Comput Biol Med 14: 447–455
Funk C, Brodelius PE (1990) Phenylpropanoid metabolism in suspension
cultures of Vanillia planifolia Andr.: II. Effects of precursor feeding and
metabolic inhibitors. Plant Physiol 94: 95–101
Hopkins NE, Foroozesh MK, Alworth WL (1992) Suicide inhibitors of
cytochrome P450 1A1 and P450 2B1. Biochem Pharmacol 44: 787–796
Jefcoate CR (1978) Measurements of substrate and inhibitor binding to
microsomal cytochrome P450 by optical-difference spectroscopy. Methods Enzymol 27: 258–279
Kahn R, Durst F (2000) Function and evolution of plant cytochrome P450. In
JT Romeo, R Ibrahim, L Varin, V De Luca, eds, Evolution of Metabolic
Pathways, Vol 34. Elsevier Science Publishing, New York, pp 151–189
Leon J, Shulaev V, Yalpani N, Lawton MA, Raskin I (1995) Benzoic acid
2-hydroxylase, a soluble oxygenase from tobacco, catalyzes salicylic acid
biosynthesis. Proc Natl Acad Sci USA 92: 10413–10417
Nagata T, Hasezawa S, Nemoto Y (1992) Tobacco BY-2 cell line as the
“Hela” cell in the biology of higher plants. Int Rev Cytol 132: 1–30
Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver
microsomes: I. Evidence for its hemoprotein nature. J Biol Chem 239:
2370–2378
Ortiz de Montellano PR, Correia MA (1995) Inhibition of cytochrome P450
enzymes. In P. Ortiz de Montellano, ed, Cytochrome P450: Structure,
Mechanism, and Biochemistry. Plenum Press, New York, pp 305–364
Ortiz de Montellano PR, Komives EA (1985) Branchpoint for heme alkylation and metabolite formation in the oxidation of arylacetylenes by
cytochrome P-450. J Biol Chem 260: 3330–3336
Plant Physiol. Vol. 130, 2002
Pellegrini L, Rohfritsch O, Fritig B, Legrand M (1994) Phenylalanine
ammonia-lyase in tobacco: molecular cloning and gene expression during
the hypersensitive reaction to tobacco mosaic virus and the response to a
fungal elicitor. Plant Physiol 106: 877–886
Pierrel MA, Batard Y, Kazmaier M, Mignotte-Vieux C, Durst F, WerckReichhart D (1994) Catalytic properties of the plant cytochrome P450
CYP73 expressed in yeast: substrate specificity of a cinnamate hydroxylase. Eur J Biochem 224: 835–844
Pompon D, Louerat B, Bronine A, Urban P (1996) Yeast expression of
animal and plant P450s in optimized redox environment. Methods Enzymol 272: 51–64
Raag R, Poulos TL (1989) The structural basis for substrate-induced changes
in redox potential and spin equilibrium in cytochrome P450CAM. Biochemistry 28: 917–922
Ralston L, Kwon ST, Schoenbeck M, Ralston J, Schenk DJ, Coates RM,
Chappell J (2001) Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum). Arch Biochem Biophys 393: 222–235
Regal KA, Schrag ML, Kent UM, Wienkers LC, Hollenberg PF (2000)
Mechanism-based inactivation of cytochrome P450 2B1 by 7-ethynylcoumarin: verification of apo-P450 adduction by electrospray ion trap mass
spectrometry. Chem Res Toxicol 13: 262–270
Roberts ES, Hopkins NE, Alworth WL, Hollenberg PF (1993) Mechanismbased inactivation of cytochrome P450 2B1 by 2-ethynylnaphthalene:
identification of an active-site peptide. Chem Res Toxicol 6: 470–479
Sato T, Kiuchi F, Sankawa U (1982) Inhibition of phenylalanine ammonialyase by cinnamic derivatives and related compounds. Phytochemistry
21: 845–850
Schalk M, Batard Y, Seyer A, Nedelkina S, Durst F, Werck-Reichhart D
(1997) Design of fluorescent substrates and potent inhibitors of CYP73As,
P450s that catalyze 4-hydroxylation of cinnamic acid in higher plants.
Biochemistry 36: 15253–15261
Schalk M, Cabello-Hurtado F, Pierrel MA, Atanossova R, Saindrenan P,
Werck-Reichhart D (1998) Piperonylic acid, a selective, mechanismbased inactivator of the trans-cinnamate 4-hydroxylase: a new tool to
control the flux of metabolites in the phenylpropanoid pathway. Plant
Physiol 118: 209–218
Schoch G (2001) Cinnamate hydroxylase et coumaroyl ester 3⬘-hydroxylase:
spécificité de substrat, études structurales, inhibition in vitro et in vivo.
PhD thesis, Université Louis Pasteur, Strasbourg, France
Silverman RB (1996) Mechanism-based enzyme inactivators. In DL Purich,
ed, Contemporary Enzyme Kinetics and Mechanism. Academic Press,
New York, pp 291–334
Strobel SM, Szklarz GD, He Y, Foroozesh M, Alworth WL, Roberts ES,
Hollenberg PF, Halpert JR (1999) Identification of selective mechanismbased inactivators of cytochromes P-450 2B4 and 2B5, and determination
of the molecular basis for differential susceptibility. J Pharmacol Exp
Ther 290: 445–451
Teutsch GH, Hasenfratz MP, Lesot A, Stoltz C, Garnier JM, Jeltsch JM,
Durst F, Werck-Reichhart D (1993) Isolation and sequence of a cDNA
encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant
cytochrome P450 involved in the general phenylpropanoid pathway.
Proc Natl Acad Sci USA 90: 4102–4106
Urban P, Werck-Reichhart D, Teutsch HG, Durst F, Regnier S, Kazmaier
M, Pompon D (1994) Characterization of recombinant plant cinnamate
4-hydroxylase produced in yeast: kinetic and spectral properties of the
major plant P450 of the phenylpropanoid pathway. Eur J Biochem 222:
843–850
Werck-Reichhart D, Feyereisen R (2000) Cytochromes P450: a success story.
Genome Biol 1: reviews3003.1–3003.9
Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2002) Isochorismate
synthase is required to synthesize salicylic acid for plant defence. Nature
414: 562–565
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
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