From the Institut de Biologie Moléculaire des Plantes, CNRS, Université Louis Pasteur, 12, rue du Général Zimmer, 67000 Strasbourg, France
Received for publication, September 12, 2002, and in revised form, October 11, 2002
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ABSTRACT |
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A protein hydrolyzing hydroxycinnamoyl-CoA esters
has been purified from tobacco stem extracts by a series of high
pressure liquid chromatography steps. The determination of its
N-terminal amino acid sequence allowed design of primers permitting the
corresponding cDNA to be cloned by PCR. Sequence analysis revealed
that the tobacco gene belongs to a plant acyltransferase gene family,
the members of which have various functions. The tobacco cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was
affinity-purified and cleaved to yield the recombinant enzyme for use
in the study of catalytic properties. The enzyme catalyzed the
synthesis of shikimate and quinate esters shown recently to be
substrates of the cytochrome P450 3-hydroxylase involved in
phenylpropanoid biosynthesis. The enzyme has been named
hydroxycinnamoyl-CoA: shikimate/quinate
hydroxycinnamoyltransferase. We show that
p-coumaroyl-CoA and caffeoyl-CoA are the best acyl group
donors and that the acyl group is transferred more efficiently to
shikimate than to quinate. The enzyme also catalyzed the reverse
reaction, i.e. the formation of caffeoyl-CoA from
chlorogenate (5-O-caffeoyl quinate ester). Thus,
hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase appears to control the biosynthesis and turnover of major plant phenolic compounds such as lignin and chlorogenic acid.
Phenylpropanoid compounds are derived from phenylalanine by the
action of phenylalanine ammonia-lyase, the branch point enzyme between
primary and secondary metabolism (see Fig. 1) (1, 2). The biosynthesis
of phenylpropanoids is developmentally activated in specific tissues
and cell types and also in response to biotic and abiotic stimuli such
as wounding, pathogen infection, and ultraviolet irradiation (1, 3-5).
Cinnamic acid, the reaction product of phenylalanine ammonia-lyase
catalysis, is further modified by the action of hydroxylases and
O-methyltransferases, leading to the synthesis of a wide
range of hydroxycinnamic acids (see Fig. 1). The enzyme
4-hydroxycinnamoyl-CoA ligase catalyzes the formation of CoA
esters of hydroxycinnamic acids, which are activated intermediates in
the biosynthesis of diverse compounds via specific branches of the
pathway leading to lignins, lignans, flavonoids and isoflavonoids,
stilbenes, coumarins, and numerous esters and amides (see Fig. 1) (6,
7). 4-Hydroxycinnamoyl-CoA ligase from various plant species accepts
p-coumarate, ferulate, and caffeate as substrates in this
decreasing preferential order, but does not accept sinapate (8, 9).
Thus, it is likely that sinapoyl-CoA is formed via the alternative
pathway involving caffeoyl-CoA O-methyltransferase, which
produces feruloyl-CoA (10, 11), which is the substrate of cinnamoyl-CoA
reductase, the first committed enzyme of monolignol synthesis (see Fig.
1). Recently, it has been shown that coniferaldehyde, the product resulting from cinnamoyl-CoA reductase action, is a preferential substrate for the cytochrome P450 hydroxylase that introduces the
hydroxyl group in the 5-position of the aromatic ring (see Fig. 1) (12,
13). Finally, the second methylation reaction leading to syringyl
lignin subunits may occur at the level of CoA ester, aldehyde, or
alcohol derivatives (see Fig. 1) (10, 13, 14).
Very recently, it has been demonstrated that C-3 hydroxylation
does not take place at the free acid level, as is the case for
C-4 hydroxylation. By a functional genomics approach,
p-coumarate 3-hydroxylase from Arabidopsis
thaliana has been shown to be a P450 enzyme that accepts the
shikimate and quinate esters of p-coumarate as substrates,
but not the free acid form or the p-coumaroyl-CoA ester
(15). Mutants tagged in the p-coumarate 3-hydroxylase gene
are characterized by a reduced epidermal fluorescence phenotype (and
called ref8) and have been shown to accumulate
p-coumarate esters and to be affected in lignin
biosynthesis, thus providing direct evidence that
p-coumaroyl shikimate and/or p-coumaroyl quinate
is probably an important intermediate in the phenylpropanoid pathway
(16, 17).
Here, we report the characterization of an acyltransferase from tobacco
that uses p-coumaroyl-CoA as acyl donor and shikimic acid or
quinic acid as acceptor, yielding the shikimate or quinate ester,
respectively. The enzyme has been purified from tobacco stems, and
determination of its N-terminal sequence allowed us to clone the
cognate cDNA by PCR. The tobacco enzyme shares 46% identity with
the carnation anthranilate benzoyl-CoA benzoyltransferase, which
catalyzes anthranamide phytoalexin biosynthesis (18) and belongs to a
large plant acyltransferase gene family (19). The recombinant enzyme
expressed in Escherichia coli efficiently synthesizes p-coumaroyl esters from p-coumaroyl-CoA, in
agreement with a putative role upstream of p-coumarate
3-hydroxylase in the phenylpropanoid pathway. It also catalyzes the
biosynthesis of chlorogenic acid (5-O-caffeoylquinic ester), one of the most
widespread soluble phenolic compound in the plant kingdom. The tobacco
acyltransferase can also catalyze the reverse reaction, i.e.
transfer of the caffeoyl moiety of chlorogenic acid to CoA to form
caffeoyl-CoA, the precursor of guaiacyl and syringyl units of lignin.
Thus, in plants, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase
(HCT)1 appears to play a
critical role in the phenylpropanoid pathway, both upstream and
downstream of the 3-hydroxylation step.
Chemicals and Enzymes
Commonly used chemicals and reagents were of the highest purity
readily available. Bradford protein dye reagent was purchased from
Bio-Rad (Marnes-la-Coquette, France). Restriction enzymes and buffers
were purchased from New England Biolabs, Inc. (Beverly, MA) or
Invitrogen (Cergy Pontoise, France). T4 DNA ligase, T4 polynucleotide
kinase, ATP, and purified oligonucleotides used for cloning and
DNA sequencing were purchased from Invitrogen. Glutathione-agarose,
isopropyl- Bacterial Strains and Plasmids
Cloning into the pGEX-KG vector (Amersham Biosciences) and PCR
screening for positive clones were carried out as described by Martz
et al. (20). Protein expression was performed using E. coli BL21-G612, a kanamycin-resistant strain. E. coli
BL21-G612 cells carry the plasmid pLysS and express rare prokaryotic tRNAs.
DNA Sequencing
DNA sequencing was performed using the rhodamine dye terminator
cycle ready kit with AmpliTaq DNA polymerase FS (PerkinElmer Life
Sciences) and an Applied Biosystems DNA sequencer (Model 373A).
Partial Purification of HCT from Tobacco Stems and
Amino Acid Sequence Analysis
Preparation of Crude Extracts--
Tobacco stems (200 g) were
ground at 4 °C in a Waring Blendor in 200 ml of 20 mM
Tris-HCl (pH 7.0) containing 15 mM Anion Exchange Chromatography--
The crude extract was loaded
onto a Mono Q column (0.5 × 5 cm; Amersham Biosciences)
equilibrated with buffer A. Proteins were eluted with 200 ml of a
0-0.3 M NaCl gradient in buffer A. Active fractions
exhibiting HCT activity were pooled.
Molecular Exclusion Chromatography under Fast Protein Liquid
Chromatography Conditions--
Pooled fractions were concentrated to
200 µl on Centricon 10 concentrators (Amicon, Inc., Beverly,
MA), filtered, injected onto a Superdex 75 HR10/30 column (Amersham
Biosciences), and eluted with buffer A at a flow rate of 0.2 ml/min.
Electrophoresis, Protein Staining, and Microsequencing--
The
basic procedures used for electrophoresis under denaturing conditions
and silver nitrate staining have been described (21). After
electrophoretic separation under denaturing conditions, proteins were
immobilized onto Problott membrane (Bio-Rad), stained with Coomassie
Blue (Bio-Rad) according to the manufacturer's instructions, and
microsequenced with an Applied Biosystems gas-phase sequencer (Model 470A).
Cloning of HCT cDNA
Reverse Transcription--
Reverse transcription with total RNA
from 2-month-old Nicotiana tabacum stems was carried out
using poly(dT) primer and SuperscriptTM (Invitrogen)
according to the manufacturer's instructions.
Generation of Partial cDNAs--
Partial cDNAs were
produced by PCR using cDNA generated by reverse transcription as
template. Based on the amino acid sequence of the purified protein, a
sense degenerate oligonucleotide primer (SP1) was synthesized (see also
Fig. 5): 5'-ATGGTIAARCCIGCIACIGARACICC-3', where I and R
indicate inosine and A/G, respectively. The antisense primer (ASP1) was
based upon a conserved region (DFGWG) near the C terminus of
acyltransferase proteins (see Fig. 6) and had the following sequence:
5'-CC CCA ICC RAA RTC-3'. DNA amplification was performed under the
following conditions: 94 °C for 3 min and then 35 cycles at 94 °C
for 1 min, 42 °C for 1 min, and 72 °C for 1 min. At the end of
the 35 cycles, the reaction mixture was incubated for an additional 10 min at 72 °C. The amplified DNA was resolved by agarose gel
electrophoresis, and the band of the expected size (1130 bp) was
isolated and subcloned into pCRII-TOPO (Invitrogen) prior to sequencing.
5'- and 3'-End Amplification--
Based on the nucleotide
sequence of the partial cDNA, new oligonucleotide primers were
synthesized for amplification of the 5' and 3'-ends of HCT
transcripts (see also Fig. 5): ASP2,
5'-GCGCCACACATTCCTGCCAACATCTCGTAGGAGC-3'; ASP3,
5'-GAAACTGAGGCTGAGGTGGATCACGAGCACGGA-3'; ASP4,
5'-GTCAATGAAAGGTGGGATGGTGAGGTCCAGACC-3'; and SP2,
5'-AGTAAATTACATGATGCATTGGC-3'. To identify the 5'-end of the HCT
transcript, the 5'-RACE System for Rapid Amplification of cDNA Ends
Version 2.0 (Invitrogen) was used. First, ASP2 and total RNA from
N. tabacum stems were used for cDNA synthesis. Then, a
first round of PCR was performed using the nested antisense primer ASP3
along with the anchored RACE-specific primer furnished by the
manufacturer. A second round of PCR was performed using 1/100th
of the first-round PCR mixture as template and the nested antisense
primer ASP4 along with the universal RACE-specific primer (Invitrogen).
The amplified DNA was resolved by agarose gel electrophoresis, and the
band of the correct size (600 bp) was isolated and subcloned into
pCRII-TOPO prior to sequencing. To identify the 3'-end of the HCT
transcript, PCR was performed using poly(dT) reverse transcription mixture as template and SP2 and poly(dT) oligonucleotides as primers.
Generation of Full-length HCT cDNA--
The sequence
information required for generation of a full-length cDNA was
derived from the nucleotide sequence of the 5'- and 3'-ends of HCT
transcripts (see also Fig. 5). The full-length clone was amplified
using the end-specific primers SP3
(5'-GCTCTAGACATGAAGATCGAGGTGAAAGAATCG-3') and ASP5
(5'-CCGCTCGAGTCGACCCATGGTCAAAAGTCATACAAGAACTTCTC-3'). SP3 and
ASP5 contain recognition sites for the restriction endonucleases XbaI and XhoI, which permit subcloning into the
pGEX-KG plasmid for heterologous expression in E. coli. DNA
amplification was performed under the following conditions: 94 °C
for 3 min and four cycles at 94 °C for 30 s, 46 °C for 1 min, and 72 °C for 1 min, followed by 30 cycles as above except that
the third step was at 55 °C for 1 min. After the 30 cycles, the
reaction mixture was incubated for an additional 10 min at 72 °C.
The amplified DNA was resolved by agarose gel electrophoresis, and the
band of the correct size (1347 bp) was isolated and subcloned into pCRII-TOPO prior to sequencing.
Heterologous Expression and Purification of HCT
The full-length cDNA generated by reverse transcription-PCR
was ligated into the pGEX-KG plasmid, which had been digested with the
restriction endonucleases XbaI and XhoI. The
pGEX-KG plasmids containing the HCT gene coding region were
electroporated into E. coli strain BL21-G612. A 10-ml
preculture was grown overnight at 37 °C in LB medium containing 50 mg/liter kanamycin and 100 mg/liter ampicillin. It was used to
inoculate 100 ml of fresh medium. Bacteria were grown for 3 h at
37 °C and then transferred to 18 °C overnight after addition of 1 mM isopropyl- Synthesis and Purification of Substrates
CoA esters were prepared according to the method of
Stöckigt and Zenk (27) with some modifications (23) and
identified and quantified by spectroscopy as described (24).
Enzyme Activity Measurements
Spectrophotometric Assay--
During enzyme purification from
plant extracts, 10 nmol of p-coumaroyl-CoA was added to 100 µl of protein mixture and incubated at 30 °C for 1 h. The
activity was determined by the decrease in the absorbance at 346 nm
measured against a blank reaction mixture containing a boiled protein extract.
Standard Assay Conditions for the Recombinant Enzyme--
The
reaction mixture contained (in a total volume of 20 µl) 100 mM phosphate buffer (pH 6.6), 1 mM
dithiothreitol, 20 ng to 1 µg of purified enzyme, and the different
substrates at 10 µM to 10 mM. The reaction
was initiated by enzyme addition, incubated at 30 °C for 20 min, and
terminated by addition of 20 µl of HPLC solvent. Reaction products
were analyzed by HPLC.
Determination of Kinetic Parameters--
For
Km determination, varying substrate and enzyme
concentrations were used depending on the substrate tested. For Km with quinate, 50 ng/µl purified enzyme, 1 mM p-coumaroyl-CoA, and 1-10 mM
quinate were used. For Km with shikimate, 1 ng/µl
purified enzyme, 1 mM p-coumaroyl-CoA, and
250-4000 µM shikimate were used. For
p-coumaroyl-CoA Km measurement, we used 5 ng/µl purified enzyme with quinate as acceptor and 1.8 ng/µl with
shikimate as acceptor, 4 mM quinate or shikimate, and 10-100 µM p-coumaroyl-CoA. For caffeoyl-CoA
affinity determination, 20 or 1 ng/µl purified enzyme with quinate or
shikimate as acceptor, respectively; 4 mM quinate or
shikimate; and 20-500 µM caffeoyl-CoA were tested.
Finally, for feruloyl-CoA Km measurement, 8 ng/µl
purified enzyme, 4 mM shikimate, and 20-200
µM feruloyl-CoA were used. Km and
Vmax values were calculated from the Lineweaver-Burk plots.
Assay Conditions with Other Putative Acyl Donors or
Acceptors--
Cinnamoyl-CoA, sinapoyl-CoA, and benzoyl-CoA were
tested as acyl donors at a concentration of 100 µM each
in the presence of 50 ng/µl purified enzyme and 4 mM
quinate or shikimate. Anthranilate, glucose, malate, tyramine,
spermidine, spermine, putrescine, and agmatine were tested as possible
acyl acceptors at a concentration of 4 mM each in the
presence of 50 ng/µl purified enzyme and 4 mM
p-coumaroyl-CoA.
Assay for Caffeoyl-CoA Synthesis from Chlorogenate--
10
ng/µl purified enzyme was incubated in the presence of 100 µM chlorogenate and 100 µM CoA.
Identification of Reaction Products by HPLC
Incubation mixtures were diluted with 1 volume of 0.1% formic
acid and 5% acetonitrile in water and resolved on a Waters
reverse-phase C18 column (Novapak, 4 µm, 4.6 × 250 mm) using an increasing gradient of acetonitrile (5-50%) in
water containing 0.1% formic acid. For the characterization of
caffeoyl-CoA formed after incubation of HCT in the presence of
chlorogenate and CoA, 20 mM phosphate (pH 5.3) was
dissolved in water, and a 5-25% acetonitrile gradient was applied for
column elution. Reaction products were characterized by their elution
time, and their UV absorption spectra were recorded with a Waters
photodiode array detector.
3-O- and 4-O-caffeoylquinic acids were produced
from chlorogenic acid (5-O-caffeoylquinic acid; Fluka) by
heating for 30 min at 90 °C in 0.2 M phosphate buffer
(pH 7.0) (25). The isomers were separated by HPLC and collected.
Sequence Alignment
Sequence alignment and analysis were performed with ClustalW
software. The phylogenetic tree was built using the TreeView program.
Occurrence of an Activity Degrading Caffeoyl-CoA in Tobacco
Stems--
As shown in Fig. 1,
hydroxycinnamoyl-CoA esters are important metabolic intermediates in
the phenylpropanoid pathway. We have previously shown that caffeoyl-CoA
is methylated by tobacco caffeoyl-CoA O-methyltransferase to
yield feruloyl-CoA (10). Surprisingly, when a crude extract from
tobacco stems was used as the enzyme preparation, TLC analysis of
reaction products revealed the presence of feruloyl-CoA, ferulic acid,
and another unknown compound (data not shown). These results suggest
that ferulic acid arises from hydrolysis of either feruloyl-CoA or
caffeoyl-CoA, followed by the methylation of caffeic acid, which is
known to be catalyzed by caffeic/5-hydroxyferulic acid
O-methyltransferase I in vitro (10, 26). The
reactions involved are indicated by the dotted box in Fig.
1.
To discriminate between these two possibilities, we first investigated
the stability of hydroxycinnamoyl-CoA esters in the presence of protein
extracts from tobacco stems. As shown in Fig. 2A, the UV absorption spectrum
of caffeoyl-CoA presents three maxima of absorption. The peak at 346 nm
is characteristic of the presence of the thioester bond (27). After a
1-h incubation of caffeoyl-CoA at 30 °C in the presence of tobacco
stem extract (Fig. 2B), the absorbance of the two first
peaks remained roughly unchanged, whereas the third absorption peak had
markedly decreased. These observations indicate that hydrolysis of
caffeoyl-CoA is catalyzed by the crude enzyme extract. No change in the
absorption spectrum was recorded in the absence of protein extract
(data not shown) or in the presence of a boiled protein extract (Fig. 2A).
Purification of Tobacco Thioesterase--
The total protein
extract from tobacco stems was clarified by centrifugation and
filtration. Proteins were first fractionated by anion exchange
chromatography under fast protein liquid chromatography conditions
(Fig. 3A). Fractions
containing thioesterase activity (as measured by the decrease in
absorbance at 346 nm) were then pooled, concentrated, and
submitted to two successive molecular sieving chromatography steps
(Fig. 3, B and C). Table
I summarizes the purification factor and
yield values measured at each purification step. Likely due to some
enzyme instability, a final enzyme activity recovery of 0.6% was
observed, resulting in a low apparent purification factor. All our
attempts to further purify the enzyme were unsuccessful and led to a
complete loss of thioesterase activity. However, when the content of
active protein fractions from the second molecular sieving step was
analyzed by electrophoresis on SDS gels, only a limited number of
protein species were detected (Fig. 3D). Among the protein
bands, those in the 45-51-kDa range displayed intensity variations, which correlated with enzyme activity levels. Therefore, fractions 34-40 were pooled, concentrated, and submitted to
preparative SDS gel electrophoresis, followed by blotting on membrane.
Every protein band in the 45-51-kDa range was microsequenced, and one amino acid sequence revealed important homology to protein sequences available in the data banks: as shown in Fig.
4, the N-terminal amino acid sequence of
a 51-kDa tobacco protein (Fig. 3D, arrow) displayed 12 of 18 residues identical to the N terminus of a carnation protein that has been characterized as a
hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase involved in the
biosynthesis of dianthramides (18).
Cloning and Purification of the Recombinant Protein--
The
N-terminal sequence of the HCT protein purified from tobacco and the
consensus sequences found in the acyltransferase family (19) were used
to design primers for the first step of PCR cloning (see
"Experimental Procedures"). At the end of the cloning procedure, a
nearly full-length tobacco cDNA clone was obtained, the sequence of
which is shown in Fig. 5. The closest match found in the data banks (considering only characterized gene
products) was still with the anthranilate
N-hydroxycinnamoyl/benzoyl transferase from carnation, which
shares 46% identity and 62% similarity with the tobacco protein. Both
proteins belong to a multifunctional superfamily of plant
acyltransferases (19). Fig. 6 presents a
few sequences of typical members of the family, the catalytic
properties of which have been well characterized. They catalyze the
transfer of acetyl, benzoyl, or hydroxycinnamoyl groups onto a variety
of acceptor molecules, including alkaloids (salutaridinol
7-O-acetyltransferase and deacetylvindoline
4-O-acetyltransferase), anthranilate (anthranilate
N-hydroxycinnamoyl/benzoyltransferase), benzyl alcohol
(benzyl-alcohol acetyltransferase), anthocyanins (anthocyanin
5-aromatic acyltransferase), and diterpenoids
(3'-N-debenzoyl-2'-deoxytaxol N-benzoyltransferase). As shown in Fig. 6, all these
proteins have structural features in common, in particular an
HXXXD motif (His153-Asp157) that
may function in acyl transfer catalysis and the DFGWG block (Asp382-Gly386) that was used to design
cloning primers (see "Experimental Procedures"). To investigate the
potential catalytic activity of the tobacco protein, the cloned
cDNA was transformed into E. coli bacterial cells, and
expression of a GST fusion protein was induced as described under
"Experimental Procedures." The fusion protein, present in the
soluble fraction of transformed bacteria (Fig.
7, lane 6), was purified by
affinity chromatography on glutathione-agarose beads and then cleaved
by thrombin, yielding the recombinant protein as illustrated in
lane 8. The purified recombinant enzyme was used for
substrate specificity studies.
Substrate Specificity of Tobacco HCT--
As mentioned above, the
importance of an acyltransferase step in the phenylpropanoid
biosynthetic pathway has been strongly suggested by the recent
characterization of a cytochrome P450 3'-hydroxylase that displays a
high level of specificity for depsides versus free
coumaric acid (15). Because p-coumaroyl quinate and
p-coumaroyl shikimate were shown to be substrates for the P450 hydroxylase, we investigated the possibility that the tobacco enzyme catalyzes the synthesis of these compounds. Thus, we incubated the purified recombinant enzyme in the presence of
p-coumaroyl-CoA and quinic acid or shikimic acid and
analyzed the reaction products by HPLC (Fig.
8, A and B). HPLC
profiles recorded after incubation of substrates with (right
panels) or without (left panels) active enzyme
preparations are shown. In the presence of active enzyme, a new product
was detected in both cases, the absorbance spectrum of which was
similar to that of p-coumaric acid due to the presence of
the p-coumaroyl moiety (data not shown). The reaction
products were further identified by comparing their retention times
with those of authentic quinate and shikimate esters of
p-coumaric acid (15). In planta,
these compounds are converted by the P450 hydroxylase into the
corresponding caffeoyl esters, which, in turn, must be transformed into
caffeoyl-CoA ester, which is the substrate of caffeoyl-CoA
O-methyltransferase, the next enzyme in the pathway (Fig.
1). Because plant acyltransferases have been described as having
reversible activity (28-31), we investigated whether the tobacco
enzyme can also catalyze the reverse reaction, i.e. the
formation of caffeoyl-CoA from quinate ester. When chlorogenic acid
(5-O-caffeoylquinic ester) was incubated in the presence of HCT
and CoA (Fig. 8C), it decreased in quantity (reflected by
diminished peak height), whereas a small peak of caffeoyl-CoA appeared.
The caffeoyl-CoA peak was identified by comparing the elution time and
spectroscopic properties with those of an authentic standard. To
quantify the reverse HCT reaction, we also used another solvent system
allowing the precise estimation of newly formed caffeoyl-CoA ester (see
"Experimental Procedures"). These results indicate that transferase
activity may be implicated at different levels in the phenylpropanoid
metabolic grid.
To gain insight into HCT function, we evaluated the kinetic parameters
of the purified enzyme for a range of substrates. As shown in Table
II, the affinity of the enzyme for
shikimate as acceptor was ~100-fold higher than for quinate. From the
data presented in Table II, it also appears that various CoA esters can
be used by HCT to transfer the acyl group to shikimate, but with
different efficiencies. Caffeoyl-CoA was the most efficient donor, with
a Vmax/Km of 0.3, followed by
p-coumaroyl-CoA, which displayed a value of 0.11, whereas
feruloyl-CoA was a poor donor, with a 10-fold lower efficiency (Table
II). The activity with sinapoyl-CoA was even lower, and the kinetic
parameters were not measured with this substrate. When quinate was
tested as the acceptor molecule, p-coumaroyl-CoA was a
better donor compared with caffeoyl-CoA (Table II), whereas no activity
at all was recorded with feruloyl-CoA or sinapoyl-CoA (data not
shown).
A thioesterase activity hydrolyzing caffeoyl-CoA was detected in
tobacco extracts and purified. From the N-terminal amino acid sequence
of the purified protein, we cloned the corresponding cDNA, which
proved homologous to acyltransferase genes from various origins and
with diverse functions (Fig. 6). Plant acyltransferases are encoded by
a large gene family, the members of which are involved in the
biosynthesis of a wide variety of secondary metabolites (18, 19, 32)
and display common structural features that enabled us to clone a
tobacco homolog. Heterologous expression of the cDNA in E. coli yielded a recombinant protein that was purified and
characterized. The enzyme was shown to be the most active with
p-coumaroyl-CoA and caffeoyl-CoA esters as acyl donors, to
also use feruloyl-CoA (but less efficiently), and to have very low
activity with sinapoyl-CoA. Surprisingly, cinnamoyl-CoA was a good
donor (data not shown), but the functionality of the resulting cinnamic
esters in the lignin biosynthetic route is not demonstrated because it
is not known whether these esters are substrates for cinnamate
4-hydroxylase or p-coumarate 3-hydroxylase. It is noteworthy that the tobacco acyltransferase has a pronounced preference for shikimic acid versus quinic acid as acceptor (Table II), but
can, however, efficiently catalyze the synthesis of quinate esters (Fig. 8). This contrasts with the strict specificity of
acyltransferases partially purified from various plants, which have
been reported to transfer the acyl group of CoA esters either to
quinate or to shikimate, but not to both (29-31). We have also tested
other potential acceptors shown to be active with other plant enzymes, viz. anthranilate, glucose, malate, tyramine, spermidine,
spermine, putrescine, agmatine, and benzyl alcohol; but no activity
could be detected with these compounds (data not shown). Thus, the
tobacco enzyme appears to be specialized in the synthesis of quinate
and shikimate esters.
HCT belongs to a versatile plant acyltransferase family that shares
structural motifs (Fig. 6) and that comprises several members (the
catalytic properties of which have been determined) that are involved
in diverse secondary metabolisms of plants. We aligned the sequences of
all these biochemically characterized acyltransferases and constructed
the phylogenetic tree presented in Fig.
9. It appears that the gene sequences
from different plant species cluster within four distinct groups.
Groups A and B (Fig. 9) include acyltransferases involved in Taxol and
anthocyanidin biosynthesis, respectively. Gene products of group C
catalyze the esterification of the hydroxyl moiety of metabolically
unrelated molecules, whereas subgroup D comprises HCT and the related
enzyme anthranilate N-hydroxycinnamoyl/benzoyltransferase,
both of which transfer hydroxycinnamoyl groups to acceptors issued from
the shikimate pathway (viz. shikimate, quinate, and
anthranilate). A survey of the Arabidopsis genome has
uncovered ~60 genes putatively encoding acyltransferases of the same
family.2 Among them, three
are located in group D, and the closest gene (AT5G48930) (Fig. 9) codes
for an acyltransferase with biochemical activity similar to that of
HCT.3 Thus, phylogenetic
analysis of the acyltransferase family defines four evolutionary groups
and should provide a useful framework for studying the functions of the
numerous members of this plant gene family.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside, and LB broth were
purchased from Sigma. DNA amplification using Taq polymerase
(Invitrogen) was performed in the iCyclerTM thermocycler
(Bio-Rad). Plasmid and PCR products were extracted and purified from
agarose gels using kits purchased from QIAGEN Inc. (Hilden, Germany).
-mercaptoethanol and
0.2 g of charcoal (Merck). After filtration through four layers of
cheesecloth, the homogenate was centrifuged at 10,000 × g for 30 min. The supernatant was desalted on a Sephadex
G-25 column (4 × 70 cm; Amersham Biosciences) equilibrated with
20 mM Tris-HCl (pH 7.0) containing 15 mM
-mercaptoethanol (buffer A). The clear supernatant obtained after
centrifugation constituted the crude extract.
-D-thiogalactopyranoside to
induce protein expression. After centrifugation for 10 min at 4000 rpm,
the bacteria were resuspended in 5 ml of phosphate-buffered saline
containing 1% Triton X-100, 2 mM EDTA, 0.1%
-mercaptoethanol, and protease inhibitor mixture tablets (Roche
Molecular Biochemicals, Mannheim, Germany). Cells were lysed by two
passages through a French press (Aminco). The bacterial lysate was
centrifuged at 11,000 rpm for 30 min; the pellet was discarded; and
glutathione-agarose beads (Sigma) were added to the supernatant
containing soluble proteins. After 2 h at room temperature, the
beads were washed three times with cold phosphate-buffered saline, and
the fusion protein was directly cleaved by incubation of the beads with
thrombin for 1 h at room temperature. The supernatant contained
the recombinant HCT protein. The different steps of purification were
assessed by electrophoresis on 12% SDS-polyacrylamide gel. The amount
of recombinant HCT protein was quantified by densitometry of the bands
on polyacrylamide gels stained with Coomassie Brilliant Blue R-250
(Fluka) and by the method of Bradford (22) using the Bio-Rad reagent.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Outline of the phenylpropanoid biosynthetic
pathway. Routes to major products are indicated. The dotted
box includes the reactions that may explain the appearance of
ferulic acid after caffeoyl-CoA incubation with tobacco extract (see
"Results"). 4CL, 4-hydroxycinnamoyl-CoA ligase;
C3H, p-coumarate 3-hydroxylase; C4H,
cinnamate 4-hydroxylase; CAD, cinnamyl-alcohol
dehydrogenase; CCoAOMT, caffeoyl-CoA
O-methyltransferase; CCR, cinnamoyl-CoA
reductase; COMTI, caffeic/5-hydroxyferulic acid
O-methyltransferase I; F5H, ferulate
5-hydroxylase; PAL, phenylalanine ammonia-lyase;
SAD, sinapyl-alcohol dehydrogenase.
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Fig. 2.
UV absorbance spectra of caffeoyl-CoA.
Absorbance was recorded from 200 to 400 nm after incubation in the
presence of boiled (A) or native (B) tobacco stem
protein extract.
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Fig. 3.
Purification of tobacco thioesterase by
HPLC. Protein elution profiles (solid lines) and
thioesterase activity profiles (dashed lines) after ion
exchange chromatography (A) and two successive molecular
sieving steps (B and C) are shown. Active
fractions from the separation shown in C were analyzed by
SDS-PAGE, followed by silver staining (D). In
D, the arrow indicates the position of the 51-kDa
protein that proved to be HCT; protein markers of the given molecular
masses appear on the right.
Summary of the purification of HCT from tobacco stems
A346 nm), and protein amount was estimated by
the Bradford method (22) as described under
"Experimental Procedures."
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Fig. 4.
Alignment of N-terminal sequences of tobacco
and carnation proteins. Vertical lines indicate the
amino acid residues strictly conserved in the two proteins.
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Fig. 5.
Nucleotide and deduced amino acid sequences
of tobacco HCT cDNA. Initiation and termination codons are in
boldface. Arrows indicate the positions of sense
(SP1-3) and antisense (ASP1-5) primers used for cloning (see
"Experimental Procedures").
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Fig. 6.
Sequence alignment of representative members
of the plant acyltransferase family. HCT was from N. tabacum (this work). HCBT, anthranilate
N-hydroxycinnamoyl/benzoyltransferase from
Dianthus caryophyllus; BEAT,
benzyl-alcohol acetyltransferase from Clarkia breweri;
SALAT, salutaridinol 7-O-acetyltransferase from
Papaver somniferum; DAT, deacetylvindoline
4-O-acetyltransferase from Catharanthus roseus;
SAAT, strawberry alcohol acyltransferase from
Fragaria ananassa; DBTNBT,
3'-N-debenzoyl-2'-deoxytaxol N-benzoyltransferase
from Taxus cuspidata; Gt5AT,
anthocyanin-5-O-glucoside 6"-O-acyltransferase
from Gentiana triflora. Black and gray
boxes indicate conserved and similar residues, respectively, in at
least four of the eight sequences shown. In the consensus
row, asterisks indicate residues conserved in the
eight sequences. GenBankTM/EBI accession numbers are given
in the legend to Fig. 9.
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Fig. 7.
Purification of the recombinant
protein expressed in E. coli. Bacterial proteins
were analyzed by SDS-PAGE and Coomassie Blue staining. Protein contents
of uninduced (lane 1) and induced (lane
2) non-transformed cells were compared with those of uninduced
(lane 3) and induced (lane 4) transformed cells.
The GST-HCT fusion protein (80-kDa protein) strongly accumulated after
induction of transformed bacteria (lane 4). The fusion
protein was not detected in the insoluble fraction (lane 5),
but was present in a large amount in the soluble fraction (lane
6). After fixation on glutathione-agarose beads, the fusion
protein was either eluted with a glutathione solution (lane
7) or cleaved by thrombin to liberate the purified HCT protein
(lane 8). The arrows indicate the positions of
the GST-HCT fusion protein (80 kDa) and HCT (51 kDa). The positions of
markers of known molecular masses are indicated on the right.
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Fig. 8.
HPLC analysis of HCT reaction products.
An aliquot of the incubation medium without (left
panels) or with (right panels) HCT was analyzed. The
nature of the substrates tested is presented in the left
panels, and that of the reaction products detected at 320 nm in
the right panels. Products were characterized by their
retention times, and UV absorbance spectra were recorded with a
photodiode array detector.
Kinetic parameters of recombinant HCT
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Phylogenetic tree of acyltransferases
revealing four evolutionary sequence clusters. The tree was
constructed by neighbor-joining distance analysis. Line lengths
indicate the relative distances between nodes. Sequences of
biochemically characterized enzymes were used for alignment,
DBTNBT, 3'-N-debenzoyl-2'-deoxytaxol
N-benzoyltransferase from T. cuspidata
(GenBankTM/EBI accession number AF466397); DBAT,
deacetylbaccatin III 10-O-acetyltransferase from T. cuspidata (accession number AF193765); TAT, taxadienol
acetyltransferase from T. cuspidata (accession number
AF190130); TBT, taxane benzoyltransferase from T. cuspidata (accession number AF297618); Gt5AT,
anthocyanin-5-O-glucoside 6"-O-acyltransferase
from G. triflora (accession number BAA74428);
Pf3AT, anthocyanin-3-O-glucoside
6"-O-acyltransferase from P. frutescens
(accession number BAA93475); Ss5MaT1,
anthocyanin-5-O-glucoside
6 -O-malonyltransferase from Salvia
splendens (accession number AF405707); Pf5MAT,
anthocyanin
5-O-glucoside-6
-O-malonyltransferase from
Perilla frutescens (accession number AF405204);
DAT, deacetylvindoline 4-O-acetyltransferase from
C. roseus (accession number AF053307);
SAAT, strawberry alcohol acyltransferase from
F. ananassa (accession number AF193789); SALAT,
salutaridinol 7-O-acetyltransferase from P. somniferum (accession number AF339913); BEAT,
benzyl-alcohol acetyltransferase from C. breweri (accession
number AF043464); AT2G19070 and AT5G57840,
A. thaliana genes encoding putative acyltransferases;
HCBT, anthranilate
N-hydroxycinnamoyl/benzoyltransferase from D. caryophyllus (accession number Z84383); AT5G48930,
A. thaliana gene product with activity similar to that of
HCT (L. Hoffmann, S. Besseau, P. Geoffroy, and M. Legrand, unpublished
data). HCT is from N. tabacum (this work).
The unsuspected importance of p-coumaroylquinic and
p-coumaroylshikimic esters in phenylpropanoid metabolism was
uncovered by characterization of the Arabidopsis
p-coumarate 3-hydroxylase, which very efficiently
hydroxylates the two esters, but not p-coumaric acid or
p-coumaroyl-CoA (15). In the present work, we cloned and
expressed the acyltransferase situated upstream of the 3-hydroxylation step, which furnishes the substrates to the P450 hydroxylase. We have
shown that chlorogenic acid (one hydroxylation reaction product), which
is the most abundant phenolic compound in tobacco, can yield
caffeoyl-CoA when incubated in the presence of CoA and HCT. Thus, HCT
plays a dual role in the phenylpropanoid pathway, upstream as well as
downstream of the 3-hydroxylation step, as schematically summarized in
Fig. 10. Then, caffeoyl-CoA is
methylated by caffeoyl-CoA O-methyltransferase to yield
feruloyl-CoA, the precursor of guaiacyl and syringyl lignins (see Fig.
1). In fact, although free hydroxycinnamic acids have long been thought
to be key intermediates in the pathway, it has now been clearly
demonstrated that many enzymatic conversions occur instead at the level
of hydroxycinnamic esters, aldehydes, and alcohols (33).
|
The shikimate pathway provides precursors not only of aromatic amino acids, but also of a vast array of secondary metabolites specific to plants (34). Chlorogenic acid (5-O-caffeoylquinic ester) is the most widespread depside in the plant kingdom (7) and is particularly abundant in Asteracaea, Solanaceae, and Rubiacaea (25). In Nicotianaea, its biosynthesis has been studied using radiolabeling methods (35) and cell-free preparations of cell suspensions (36). These studies have shown that 5-O-caffeoylquinic ester is the precursor of 3- and 4-isomers. Their biosynthesis has been shown to be affected by environmental cues (7, 37), thus pointing to a regulatory role for the acyltransferase step. Moreover, the recently elucidated major role of quinate and shikimate esters as committed intermediates in the biosynthesis of phenylpropanoids demonstrates that quinate and shikimate have a dual role in plant metabolism, both as precursors of aromatic amino acids as stressed above and as acceptors in acyltransferase reactions. Because acyltransferase efficiency is lower with quinate than with shikimate, regulation of transferase activity may depend on the relative importance of the pools of the two acceptors. In this respect, it is interesting to note that the two compounds are directly convertible in the shikimate pathway (34), thus allowing fine-tuned control of their relative amounts. Compared with the considerable amounts of quinate esters found in some plants, it is striking that shikimate ester accumulation has never been reported (31). This may indicate that small quantities of shikimate are sufficient to permit 3-hydroxylation of the aromatic ring at the level of shikimate ester because the subsequent hydrolysis of the ester into caffeoyl-CoA recycles shikimic acid (Fig. 10). No doubt, future in-depth studies of HCT regulation in various physiological situations will uncover a new checkpoint of the phenylpropanoid flux.
New insights into phenylpropanoid pathway have come recently through
antisense repression of several enzymes (4, 5, 33, 38, 39). Such data
are now needed to fully understand the function of tobacco HCT in
planta. Because HCT is likely implicated in the biosynthesis of
guaiacyl and syringyl units of lignin, but also in that of
caffeoylquinic esters, the predominant soluble phenolic compounds in
many plants, one can anticipate that HCT activity changes will have an
important impact on plant cell metabolism.
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ACKNOWLEDGEMENTS |
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We are grateful to Monique Leret for amino acid sequence determination and to Philippe Hamman and Malek Alioua for DNA sequencing. Drs. Kenneth Richards and T. Heitz (Institut de Biologie Moléculaire des Plantes) are gratefully acknowledged for careful reading of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ507825.
Present address: Lab. de Biologie des Ligneux et des Grandes
Cultures, UPRES EA 1207, Faculté des Sciences, Université
d'Orléans, 45067 Orléans Cedex 2, France.
§ Present address: Finnish Forest Research Inst., Rovaniemi Research Station, 96301 Rovaniemi, Finland.
¶ To whom correspondence should be addressed. Tel.: 33-3-8841-7280; Fax: 33-3-8861-4442; E-mail: michel.legrand@ibmp-ulp.u-strasbg.fr.
Published, JBC Papers in Press, October 14, 2002, DOI 10.1074/jbc.M209362200
2 L. Hoffmann, unpublished data.
3 L. Hoffmann, S. Besseau, P. Geoffroy, and M. Legrand, unpublished data.
![]() |
ABBREVIATIONS |
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The abbreviations used are: HCT, hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase; RACE, rapid amplification of cDNA ends; HPLC, high pressure liquid chromatography; GST, glutathione S-transferase.
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