From the Risoe National Laboratory, Plant Research Department, PRD-301, Plant Products, DK-4000 Roskilde, Denmark
Received for publication, December 20, 2002, and in revised form, February 6, 2003
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ABSTRACT |
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Agmatine coumaroyltransferase (ACT), which
catalyzes the first step in the biosynthesis of antifungal
hydroxycinnamoylagmatine derivatives, was purified to apparent
homogeneity from 3-day-old etiolated barley (Hordeum
vulgare L.) seedlings. The enzyme was highly specific for
agmatine as acyl acceptor and had the highest specificity for
p-coumaroyl-CoA among various acyl donors with a specific
activity of 29.7 nanokatal × mg Agmatine coumaroyltransferase
(ACT,1 EC 2.3.1.64) was the
first amine N-hydroxycinnamoyltransferase characterized from
plants (1, 2). The enzyme catalyzes the synthesis of
hydroxycinnamoylagmatines from agmatine and hydroxycinnamoyl-CoA
thiolesters in barley (Hordeum vulgare).
Hydroxycinnamoylagmatines are direct precursor of hordatines (Fig.
1), which are antifungal compounds found
to be highly abundant in the young barley seedling (3). The hordatines
seem to be confined to the genus Hordeum as preformed
infection inhibitors (4, 5), and recent studies indicate that the
synthesis of hydroxycinnamoylagmatine derivatives are induced in
response to fungal infection of the leaves (6, 7). Additionally,
hydroxycinnamoylagmatine derivatives have been found in wheat (8), and
histochemical staining of epidermal leaf tissue confirms that these
compounds might accumulate in cereals in general as a response to
fungal infection (9). The function(s) of hydroxycinnamoylagmatine derivatives in plants is not known but may include cell wall
fortification, restriction of pathogen ingress, and cytotoxicity to the
invading pathogen (7, 9, 10). Related hydroxycinnamic acid amides are
found throughout the plant kingdom, and three plant
N-hydroxycinnamoyltransferases have been purified and
characterized: tyramine N-hydroxycinnamoyltransferase (THT, EC 2.3.1.110) (11-13), putrescine
N-hydroxycinnamoyltransferase (EC 2.3.1.138) (14), and
anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT, EC
2.3.1.144) (15). In general, the role of hydroxycinnamic acid amides
remains speculative, but conjugation compounds of tyramine have been
repeatedly reported to be present in cell wall fractions from several
plant species, and they are believed to produce a phenolic barrier
against pathogens by reducing cell wall digestibility (16-19) and
forming the phenolic domain of suberin (18, 20, 21). However,
anthranilic acid amides have been predominantly detected as soluble
compounds, and their synthesis can be induced in response to pathogen
attack (22). The purified transferases are all cytosolic proteins.
Putrescine N-hydroxycinnamoyltransferase and HCBT appeared
as monomers with native molecular masses of about 44 to 50 kDa, whereas
the THTs all appeared as dimers (11-15). The carnation HCBT cDNA
(15) and THT cDNAs from tobacco, potato, and red pepper (23-25)
have been cloned. The amino acid sequence identity is high between the
characterized THTs, but the HCBT shares an absolute minimum of sequence
similarity to the THTs (24). Thus, although these acyltransferases have
a number of related enzymatic properties, the primary structure of the
enzymes differs considerably.
1 protein. Barley
ACT was found to be a single polypeptide chain of 48 kDa with a pI of
5.20 as determined by isoelectric focusing. The 15 N-terminal amino
acid residues were identified by micro-sequencing of the native protein
and were used to clone a full-length barley ACT cDNA that predicted
a protein of 439 amino acid residues. The sequence was devoid of
N-terminal signal peptide, suggesting a cytosolic localization of
barley ACT. Recombinant ACT produced and affinity-purified from
Escherichia coli had a specific activity of 189 nanokatal × mg
1 protein, thus confirming the
identity of the purified native protein. A partial cDNA sequence
for ACT was obtained from wheat that predicted a protein of 353 amino
acid residues and had 95% sequence identity to barley ACT. Two motifs
in the amino acid sequence reveal that barley ACT represents a new
class of N-hydroxycinnamoyltransferases belonging to the
transferase superfamily. The barley ACT is unique in producing the
precursor of hordatine, a proven antifungal factor that may be directed
toward Blumeria graminis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Biosynthesis of hordatine. The two steps
depend on a reaction catalyzed by ACT followed by an oxidative
dimerization. The p-hydroxycinnamoyl-CoA is either
coumaroyl-CoA (R1 = H) or feruloyl-CoA (R1 = OMe). Hordatine A (R1 = R2 = H) is formed if
both cinnamic acid derivatives are p-coumaroyl-CoA and
hordatine B (R1 = OMe and R2 = H) is formed if
one is feruloyl-CoA. Hordatine M (R2 = D-glycopyranosyl) is a mixture of the glucosides of
hordatines A and B.
To enable further analysis of the hydroxycinnamoylagmatine derivatives
and their synthesis and potential significance in plant defense, a
biochemical approach was taken to enable molecular characterization of
ACT. In this study, we develop an enzyme purification strategy and
report the purification of a barley ACT isoform to apparent
homogeneity. Furthermore, we describe some new characteristics of the
protein and the sequencing of the 15 N-terminal amino acids. Full-length ACT cDNAs were cloned, and the identity of the purified protein was confirmed by heterologous expression of an ACT cDNA in
Escherichia coli showing in vitro ACT activity.
Finally, based on protein sequence alignment of the ACT, HCBT, and
THTs, we suggest that ACT is a new class of the amine
N-hydroxycinnamoyltransferases belonging to a diverse
transferase family.
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EXPERIMENTAL PROCEDURES |
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Plant Material--
Barley (H. vulgare L., cv.
Triumph) was sown on top of an ~3-cm layer of soil and covered with a
thin layer of fine gravel. The moistened trays were wrapped in black
plastic bags and placed in a growth chamber (22 °C, 24 h of
darkness). Seedlings were harvested 3 days after sowing, frozen in
liquid N2, ground to a fine powder with a mortar and a
pestle, and stored at 80 °C for subsequent protein purification.
Chemicals and Substrates--
The hydroxycinnamoyl-CoA
thiolesters were enzymatically synthesized using recombinant tobacco
4-coumarate:coenzyme A ligase (4CL). The E. coli strain
carrying the Nt4CL-19 plasmid was used to prepare crude E. coli protein extracts essentially as described (26). The extracts
containing 4-coumarate:coenzyme A ligase were adjusted to 30% glycerol
and stored at 20 °C. The ligase reaction was as described (27),
and after 15 min of incubation at 37 °C, synthesized cinnamoyl-CoA
derivatives were purified on LC-18 solid phase extraction
columns (Supelco) as described (27) except that MOPS was replaced by
0.2 M Tris, pH 7.5, in all procedures. The
hydroxycinnamoyl-CoA derivatives were concentrated in a vacuum
centrifuge to ~0.5 mM and stored at
20 °C.
Mass Spectrometry-- The molecular weights of compounds were determined by nanoelectrospray ionization tandem mass spectrometry. ACT assay solution containing synthesized p-coumaroylagmatine was loaded on micro-scale reverse phase columns (~0.2 × 8 mm) packed with Poros Oligo R3 (PerSeptive Biosystems, Framingham, MA) in 10-µl Gel-loader pipette tips (Eppendorf). Columns were washed with 40 µl of 5% (v/v) formic acid and eluted directly into precoated borosilicate nanoelectrospray needles (Protana, Denmark) with 2.5 µl of 5% (v/v) formic acid, 50% (v/v) methanol. The nanoelectrospray ionization tandem mass spectrometry was performed on a Finnigan LCQ quadropole ion storage mass spectrometer (San Jose, CA). A potential of 0.71 kV and manual syringe pressure was applied to the needles. Conditions for the nanoelectrospray ionization mass spectrometry were as follows: capillary temperature 240 °C, capillary voltage 34 V, maximum ion injection time 50 ms, and 3 microscans. Collision-induced dissociation was performed on the [M + H]+ ions formed by nanoelectrospray using argon as the collision gas. Mass spectrometry spectra of p-coumaroyl-CoA and p-coumaroylagmatine were acquired in the m/z range from 800 to 1100 and 75 to 300, respectively.
Buffers-- The following buffers were used in the enzyme purification: Buffer A, 100 mM Tris, pH 8.5, containing 1 mM EDTA, 10 mM 2-mercaptoethanol, 50 mM KCl, and 250 mM sucrose; Buffer B, 50 mM Tris, pH 7.5, containing 1 mM EDTA, 10 mM 2-mercaptoethanol, and 50 mM KCl; Buffer C, same as B but 2.5 M KCl instead of 50 mM KCl; Buffer D, same as B but 2.0 M CH3COOK replacing KCl; Buffer E same as B but 0.5 M KCl instead of 50 mM KCl; Buffer F, 100 mM Tris, pH 7.5, containing 1 mM EDTA, 10 mM 2-mercaptoethanol, and 50 mM KCl. Buffers (100 mM) used to determine the pH optimum of the enzyme were MES (pH 5.5-6.7), Bis-Tris (pH 5.8-7.2), MOPS (pH 6.5-7.9), TES (6.8-8.2), HEPES (pH 7.0-8.4), Tris (pH 7.5-8.9), glycine (pH 8.8-10.2), and CAPS (pH 9.7-11.1).
Determination of ACT Activity--
The reaction mixture
contained 100 mM Tris, pH 7.5, 1 mM EDTA, 15 mM -monothioglycerol, 10% (v/v) ACT extract, 10 µM hydroxycinnamoyl-CoA, and 0.2 mM agmatine.
Assays were started by the addition of agmatine. ACT activity was
determined spectrophotometrically by monitoring the decrease in
A333 (2). Hydroxycinnamoyl-CoA was slowly
degraded in the absence of agmatine: thus, controls were run, and the
rates were subtracted from those for the samples. For the blank,
agmatine were replaced by double distilled H2O. Assays were
performed at 25 °C. The documented extinction coefficients for
hydroxycinnamoyl-CoA derivatives (28) were used for ACT activity
calculations. All assays were performed with five replicates.
Purification of ACT-- All procedures were performed at 4 °C, and chromatography was done on a fast protein liquid chromatography system from Amersham Biosciences. All columns and chromatographic materials used for ACT purification were purchased from Amersham Biosciences. The Coomassie Plus protein assay reagent (Pierce) was used for quantification of protein concentration throughout the purification steps using bovine serum albumin as the standard. Frozen ground barley seedlings (200 g) were mixed with Buffer A (1:5 w/v) in a mortar and gently stirred with a pestle for 15 min. The slurry was filtered through two layers of nylon mesh and centrifuged at 20,000 × g for 90 min. The supernatant was then filtered through a 0.20-µm filter and collected as crude extract (Step 1). The crude protein extract was chromatographed in 50-ml aliquots on a blue Sepharose column (HiTrap Blue HP, 5 ml) equilibrated in Buffer B. After loading, the column was washed in 20 column volumes (CV) of Buffer B before a step gradient (5 CV of 50% and 20 CV of 100%) of Buffer C was applied at a flow rate of 5 ml/min. Fractions of 1 ml were collected and subsequently assayed for ACT activity (Step 2). Fractions containing ACT activity from the individual runs were pooled and adjusted to 2 M CH3COOK. The slurry was gently stirred for 2 h and then centrifuged at 10,000 × g for 10 min. The supernatant was chromatographed in aliquots of 50 ml on a t-butyl hydrophobic interaction chromatography column (25-ml butyl-Sepharose 4 Fast Flow in a XK 26 column) previously equilibrated in Buffer D. The column was washed in 5 CV of Buffer D before bound protein was eluted with a linear gradient (100-0% in 16 CV) of Buffer B at a flow rate of 5 ml/min. Fractions of 8 ml were collected and assayed for ACT activity (Step 3).
Active protein fractions collected from the individual runs of Step 3 were pooled and dialyzed twice for 24 h against Buffer B. The dialyzed protein was chromatographed in aliquots of 50 ml on a RESOURCE Q (1 ml) column equilibrated in Buffer B. The column was washed in 10 CV of Buffer B before a linear gradient (0-100% in 50 CV) of Buffer E was applied at a flow rate of 2.5 ml/min. Fractions of 1 ml were collected and assayed for ACT activity (Step 4).
Active protein fractions from the individual runs of Step 4 were pooled and concentrated to ~100 µl in two steps using Centriplus YM-10 and Microcon YM-10 centrifugal filter devices (Amicon, Millipore), respectively. The ACT concentrate was loaded onto a Superose 12 HR 10/30 equilibrated in Buffer F. Protein was eluted with buffer F at a flow rate of 0.4 ml/min, and fractions of 0.3 ml were collected and assayed for ACT activity (Step 5).
Gel Electrophoresis-- SDS-PAGE was carried out in a Hoefer Mighty Small II SE 260 vertical minigel system using NOVEX pre-cast 4-12% NuPAGE Bis-Tris 1.0-mm gels. Running buffer was 50 mM MOPS, pH 7.7, containing 50 mM Tris base, 0.1% SDS, and 1 mM EDTA. The isoelectric point of ACT was determined with a 2117 Multiphor II flatbed electrophoresis system using pre-cast polyacrylamide gels containing ampholines in the pH range 3.5-9.5 (Ampholine PAGplate, Amersham Bioscience). Isoelectric focusing gels were fixed in 3.45% sulfosalicylic acid, 11.5% trichloroacetic acid, and 30% ethanol. To visualize proteins, gels were subjected to silver staining using the method reported in Morrissey (29) except that ethanol replaced methanol, and glutaraldehyde fixation was omitted.
Molecular Exclusion-- The molecular mass of native ACT was estimated by chromatography on the Superose 12 HR 10/30 using a molecular weight marker kit (Sigma, MW-GF-200). The molecular mass of denatured ACT was estimated by SDS-PAGE by use of molecular weight standards (NOVEX, Mark12; Mo Bi Tec, BOA001).
Protein Sequencing-- The ACT fraction purified to apparent homogeneity was concentrated on an Amicon centrifugal filter device with a 10-kDa nominal molecular weight limit (Microcon, Millipore). Concentrated protein (1-10 µg) was subjected to SDS-PAGE and transferred to Immobilon-P polyvinylidene fluoride membrane (Millipore) using the Hoefer Semiphor (Amersham Biosciences) semidry blotting system. Transfer buffer (25 mM Tris, 200 mM glycine, and 20% (v/v) methanol per liter) was used for electroblotting for 1 h at 0.8 mA/cm2 gel. The transferred protein was stained with Coomassie (0.25% Coomassie Blue R-250, 10% acetic acid, and 40% ethanol) for 3 min and then washed in destaining solution (10% acetic acid and 40% ethanol) for several changes. Finally the membrane was washed several times in double distilled H2O and then allowed to air dry. The stained protein band was cut out and frozen in Eppendorf tubes until sequencing was performed by Edman degradation on a Procise 494 protein sequencer (Applied Biosystems). Two runs of Edman sequencing (15 cycles of automated pulse liquid chemistry) were carried out.
Cloning of ACT cDNA from Barley and Wheat--
A TBlastN
data base search (30) using the 15 N-terminal amino acid residues
obtained from purified ACT identified five EST clones (accession
numbers BF259608, BI959297, BF628198, BI955449, BF619699, and
BM137380). Two sets of primers were designed for 5'- and 3'-RACE,
respectively. Oligonucleotides for 3'-RACE (CATGAAGATCACCGTGCACTC,
ATCCTGCTCAACGACGCC) and for 5'-RACE (GAGTGCACGGTGATCTTCATG,
GGCGTCGTTGAGCAGGATCG) were used in combination with vector primers for
nested amplification from cDNA libraries. Copy DNA libraries of
leaf mRNA from barley and wheat in phage (ZAP-XR and ZAP,
Stratagene, respectively) were used as template in the amplification
reactions. RACE was performed with 500 ng of cDNA as template using
the editing Expand Long Template PCR System (Roche Molecular
Biochemicals) as described by the manufacturer. Amplification was
initiated by 2 min of denaturing at 94 °C followed by 25 cycles of
denaturing at 94 °C for 30 s, annealing at 58 °C for 20 s, and elongation at 68 °C for 60 s plus 2 s/cycle.
Amplification was terminated by a 10-min incubation at 68 °C. PCR
products were cloned in pCR4-TOPO vector and sequenced on both strands
using vector and internal primers. Sequencing was carried out on an ABI
310 DNA sequencer (Applied Biosystems) using the DYEnamic ET terminator
cycle sequencing kit (Amersham Biosciences). Sequences were proofread
using Sequencher software Version 3.1.1 (Gene Codes Corp., MI).
Sequences and contigs were analyzed by MacVector 7.0 (Oxford Molecular software).
Expression of Recombinant ACT in E. coli--
Two
NdeI adapter primers (GGAATTCCATATGAAGATCACCGTGCACTCTTC,
GGAATTCCATATGCTAGGCAAGTGGCTAACGTTGATCC) were used to amplify the coding
region of pHV-ACT5-28-6 using Dynazyme EXT polymerase (Finnzymes,
Finland). Digested PCR fragments were cloned into the NdeI
site of pET15b (Novagen). Fidelity and orientation was confirmed by
sequencing. E. coli BL21 CodonPlus (DE3)-RIL (Stratagene) harboring the pET-ACT construct was grown in Luria Bertoni medium at
37 °C to a cell density of 1.3 (A600). The
culture was cooled to 28 °C before induction of expression, which
was carried out at 28 °C for 3 h after the addition of
isopropyl-1-thio--D-galactopyranoside to 0.5 mM. Cells were harvested and resuspended in 100 mM Tris, pH 8.5, 1 mM EDTA, 15 mM
-monothioglycerol, 50 mM NaCl, and lysed by incubation
with 100 µg/ml lysozyme (Sigma) at 30 °C for 15 min followed by
sonication and centrifugation (20,000 × g, 4 °C, 20 min). The supernatant buffer was changed to 20 mM Tris, pH 8.0, 5 mM imidazole, and 500 mM NaCl by gel
filtration (HiTrap desalting, 5 ml, Amersham Biosciences), and soluble
histidine-tagged recombinant ACT was purified by nickel
nitrilotriacetic acid-agarose (Qiagen) affinity chromatography as
described by the manufacturer. After loading, the column was washed
with 20 mM Tris, pH 8.0, containing 20 mM
imidazole and 500 mM NaCl, and the His-tagged protein was
eluted with 20 mM Tris, pH 8.0, containing 500 mM imidazole and 500 mM NaCl. Protein samples
were stored on ice. Fractions were assayed for ACT activity, and purity
was analyzed by SDS-PAGE.
Chemical Modification of ACT--
Activity of
bacterial-expressed ACT was examined in the presence of diethyl
pyrocarbonate (DEPC). DEPC stock solutions were made up immediately
before use in anhydrous alcohol. All enzyme incubations were carried
out at 25 °C. The affinity-purified enzyme (150 ng) was incubated
with various concentrations of DEPC before dividing into working
aliquots containing 7.5 ng of recombinant ACT. After a 5-min
incubation, excess DEPC was quenched with a corresponding amount of
imidazole before monitoring the ACT activity. For the substrate
protection experiment, ACT was incubated for 5 min with either 10 µM p-coumaroyl-CoA or 0.2 mM
agmatine before the addition of 1 mM DEPC.
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RESULTS |
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Purification of Agmatine Hydroxycinnamoyltransferase-- Shortly after the existence of amine N-hydroxycinnamoyltransferases in plants was demonstrated (1), ACT was partially purified from young etiolated barley seedlings (2). Since then only hydroxycinnamoylagmatine derivatives have been detected and characterized in barley (6, 7, 31, 32). In this study, 3-day-old barley seedlings were chosen as the source for the ACT purification because hordatine accumulation reaches its maximum within the first 3-6 days after germination when plants are grown at 20 °C (4, 33).
Coumaroyl-CoA was used as the hydroxycinnamoyl-CoA substrate to monitor the enzyme activity during purification. It was synthesized using crude extracts of E. coli expressing recombinant tobacco 4-coumarate:coenzyme A ligase (26) and subsequently purified on C-18 solid phase extraction columns (27). The spectrum of the individual purified and concentrated hydroxycinnamoyl-CoAs was as previously reported (28). Additionally, mass spectrometry analysis of purified p-coumaroyl-CoA solution showed a [M + H]+ value of m/z 913.8, the molecular weight of this compound.
Before purification was initiated, crude extracts of a number of commercial barley varieties were tested for ACT activity; however, only minor differences were observed between 10 varieties compared. Seedlings grown in light showed only slightly higher ACT activity; thus, etiolated seedlings were chosen as the enzyme source to avoid the presence of chlorophyll during the purification. Enzyme activity could only be detected in the soluble protein extract; Table I lists the five-step procedure for ACT purification, which includes four different column chromatography steps. The amount of ACT activity in the extracts of young barley seedlings required a concentration of the ACT activity before the first chromatographic step to avoid an excessive dilution. For this purpose, the immobilized reactive dye Cibacron blue F3G-A was used for batch enrichment of the enzyme activity. An 11-fold purification was obtained from the blue Sepharose column after eluting with KCl (Fig. 2, lane 2). It was possible to elute ACT specifically from Cibacron blue F3G-A using the substrate coumaroyl-CoA. However, large quantities of coumaroyl-CoA were needed to efficiently elute the enzyme, and a number of unrelated proteins co-eluted with ACT even when different chromatographic steps were performed before the blue Sepharose step.
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Hydrophobic interaction chromatography was chosen as the third step despite the considerable loss in total ACT activity encountered; this step was found to be essential for successful ACT purification (Fig. 2, lane 3). Active ACT was only recoverable when t-butyl and not phenyl was used as the hydrophobic interaction chromatography ligand. The high KCl concentration (1.25 M) from the second purification step ensured salting-out of the potassium acetate used to generate interactions between the enzyme and the t-butyl media.
The following anion exchange chromatography step separated ACT activity into two peaks, one containing a complex mixture of proteins and the other ~10 proteins as visualized from a silver-stained SDS-PAGE gel; one of them, ~ 48 kDa, was particularly prominent (Fig. 2, lane 4). The two ACT activity peaks were each subjected to Superose 12 HR molecular size exclusion chromatography. In both chromatographic runs, the 48-kDa protein was very prominent, but the first ACT peak from the anion exchange chromatography was still not pure, whereas the second peak provided ACT purified to apparent homogeneity (Fig. 2, lane 5) as judged by silver-stained SDS-PAGE. The purification protocol through four chromatographic steps, thus, provided a 1162-fold purification, with a yield of 3%, the main loss in activity occurring in the hydrophobic interaction chromatography step.
Stability--
Purified ACT stored in Buffer F on ice was stable
for more than 1 month without detectable loss of activity. About 25%
of its enzyme activity was lost after freezing (20 °C).
General Properties of ACT--
The molecular mass of native ACT
was determined by chromatography on a Superose 12 HR 10/30 column
calibrated with molecular mass standards. Native ACT eluted with an
apparent molecular mass of ~40 kDa, suggesting that the native barley
ACT enzyme is a monomer. The isoelectric point of the purified native
enzyme was determined by isoelectric focusing to be pH 5.2 (Fig.
3). Others found optimal ACT activity
after a 30-min incubation (2). We also observed that ACT activity in
the crude extract increased during incubation up to 30 min; however,
the purified protein did not require preincubation. In contrast, a 20%
loss in enzyme activity was found after 15 min of incubation at
25 °C. The addition of a thiol was found to be essential to retain
ACT activity during purification. At least 1 mM was
required for optimal activity, and there was no difference between
2-mercaptoethanol and -monothioglycerol at a 10 mM
concentration; however, activity was reduced by 30% in the presence of
dithiothreitol. Thus 2-mercaptoethanol and
-monothioglycerol were
chosen for purification and activity assays, respectively. A narrow pH
optimum at pH 7.5 with half-maximal activity at ±0.6 pH unit was
detected, in agreement with the optimum previously reported (2).
Optimal activity was achieved using Tris buffer in the range 50-100
mM. The purified ACT was not affected by up to 10 mM MgCl2 or CaCl2 at pH 7.5;
however, similar concentrations of MnCl2,
CuSO4, or ZnSO4 reduced ACT activity by 29, 85, and 99%, respectively. Up to 10% (v/v) ethanol did not affect ACT activity. The maximum ACT activity rate was measured at temperatures between 20 and 50 °C, and the highest rate was found at 40 °C. From the linear area of an Arrhenius plot the apparent energy of
activation was calculated to be 55 kJ mol
1.
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N-terminal Sequencing of the Purified Barley ACT-- Approximately 20 pmol (1 µg) of the ACT purified to apparent homogeneity was subjected to SDS-PAGE and subsequently electroblotted to a polyvinylidene fluoride membrane. The 48-kDa protein visualized by Coomassie staining was cut out and sequenced by Edman degradation. Only a few pmol of phenylthiohydantoin derivatives were detectable in the first cycle, indicating that the protein could be partially N-terminal-blocked. The first 5 amino acids were deduced from this Edman degradation, and a second trial was performed using 0.2 nmol (10 µg) of ACT protein. Again the protein appeared to be partially N-terminal-blocked, but the first 15 amino acids were deducible, MKITVHSSKAVKPEY.
Detection of Three ACT Isoforms--
After N-terminal sequencing
we found that replacing the anion exchange chromatography media
RESOURCE Q with MonoBeads Q resolved ACT activity into three peaks
(Fig. 4). After Superose 12 HR molecular exclusion, all three ACT isoforms showed apparent molecular masses of
~40 kDa, and in the order of elution the peaks showed specific activities at 9.9, 86.0, and 30.7 nanokatal × mg1
protein, respectively. Using these preparations the three ACT isoforms were compared by kinetic studies (Table
II). The two later activity peaks (Fig.
4) correspond to the single activity peak of the protein purified to
homogeneity by RESOURCE Q chromatography (Table I).
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Cloning of ACT cDNAs from Wheat and Barley--
The N-terminal
peptide sequence from purified barley ACT was found to be identical or
highly similar to predicted N-terminal sequences from six barley and
wheat EST clones (accession numbers BF259608, BI959297, BF628198,
BI955449, BF619699, and BM137380). Two nested primers were designed to
amplify the complete coding region and used with phage primers for
PCR on phage cDNA libraries from Blumeria
graminis-infected leaves from barley and wheat. Two distinct bands
of 1115 base pairs and 1187 base pairs were amplified by nested 3'-RACE
from the barley library. Sequencing of the cloned products in pHV-ACT5
and pHV-ACT6, respectively, showed that they differed in the 3'
non-coding region, i.e. they represented two distinct,
partial mRNAs, but both encoded 353-residue-long C-terminal
polypeptides that were nearly identical (98%). One-sided nested
amplification using the primer corresponding to the peptide sequence
and the two vector primers 3' to the cDNA yielded a 1508-base pair
product (pHV-ACT5-28-6) that contained the complete coding region of
ACT. This clone has 99.8% nucleotide sequence identity to pHV-ACT5 and
93% sequence identity to pHV-ACT6 in the 3' region. The full-length
cDNA predicts a protein of 439 amino acid residues with a
calculated molecular mass of 47584 Da and an isoelectric point at pH
5.04, which is consistent with the properties determined for native
barley ACT. Comparing the deduced amino acid sequence of pHV-ACT5-28-6
and EST sequences with the N-terminal sequence of the native barley
protein, the cDNA-encoded ACT lacks an N-terminal signal peptide,
suggesting a cytosolic location in accordance with purification data
from this study. A partial sequence was obtained from wheat
(pTA-W3) coding for amino acid residues 87-439 in barley ACT
indicating a nearly identical protein (95%).
Alignments of the ACT5-28-6 amino acid sequence in a protein family
data base (34) reveals that ACT belongs to a highly diverse transferase
superfamily responsible for CoA-dependent acyl transfer.
Several plant transferases within this family have been characterized,
including the HCBT from Dianthus caryophyllus (15), the
deacetylvindoline 4-O-acetyltransferase (EC 2.3.1.107) from
Catharanthus roseus (35), and the anthocyanin 5-aromatic acyltransferase (EC 2.3.1.153) from Gentiana triflora (36). The barley ACT has a histidine-containing motif, HIVSD, starting at
residue His-152. This is identical to the highly conserved motif
HXXXD found in transferases belonging to this family.
Generally, there is a low sequence identity to ACT among homologous
plant transferases. A second consensus sequence, the DFGWGXP
motif (35), can be found in barley ACT starting at residue Asp-385, but
this motif seems to be less conserved in monocotyledonous species. Alignments of the barley ACT5-28-6, the amino acid sequences of the
five highest scoring sequences obtained in a TblastN data base search
(30), the wheat pTA-W3, and the derived amino acid sequence from a
wheat EST clone (accession number BM137380) are shown in Fig
5.
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A phylogenetic analysis of biochemically characterized plant
acyltransferases indicated the existence of four evolutionary groups
(37). Group A includes acyltransferases that transfer hydroxycinnamoyl
groups to acceptors from the shikimate pathway (Fig.
6). Acyltransferases of group B and C are
involved in taxol or anthocyanidin biosynthesis, respectively. Group D
comprises acyltransferases that esterify a hydroxyl moiety of metabolic unrelated molecules. When the barley ACT and the five sequences with
highest score from the TblastN search (Fig. 5) were included in a
corresponding phylogenetic analysis, a fifth evolutionary group (E)
seemed to be defined. This group might define acyltransferases transferring hydroxycinnamoyl groups to acceptors derived from the
polyamine pathway. Hydroxycinnamic acid amides of di- and polyamines
are widely distributed in the plant kingdom (38). The amino acid
sequence of pHV-ACT5-28-6 shows 75% (AL606646) and 38% (AC114474)
identity to the two rice proteins that ACT congregates with in Fig. 6.
Proteins in group A show 27-29% identity and the
transferases of the other groups (B-E) show less than 20%
identity.
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Expression of ACT in E. coli--
The construct pET-ACT contains
the barley ACT5-28-6 cDNA in the expression vector pET15b and
encodes ACT with additional 20 amino acids including a six-histidine
affinity purification tag and a thrombin protease cleavage site at the
N terminus of ACT. The expression construct and the empty vector pET15b
were transformed into BL21 CodonPlus (DE3) RIL cells, and expression of
soluble ACT was first tested at the standard induction temperature of 37 °C for 3 h. Only a moderate level of ACT activity was found in the soluble pET-ACT extract. SDS-PAGE analysis of total cellular protein did show the accumulation of an ~50-kDa protein in the induced pET-ACT and not in the un-induced pET-ACT or in the control culture (not shown). This size corresponds to the calculated molecular mass of 49635 Da for the recombinant ACT including the His tag and the
protease site. Reducing the temperature to 28 °C during induction by
isopropyl-1-thio--D-galactopyranoside increased the
amount of soluble recombinant enzyme produced. The recombinant ACT was
affinity-purified (Fig. 7) and used for
kinetic comparison to ACT purified from barley.
|
Substrate Specificity and Kinetics of Native and Recombinant ACT-- The three ACT isoforms purified from barley seedlings had very similar affinities (Km) and relative substrate specificities (V/Km) toward the individually tested hydroxycinnamoyl-CoAs, all showing the highest specificity for coumaroyl-CoA. Only ACT3 differed slightly in that the relative specificity for feruloyl-CoA and caffeoyl-CoA was more than double that detected for ACT1 and ACT2. The affinity for the acyl acceptor was also very similar between the native isoforms and highly specific for agmatine. In addition to agmatine, tyramine and putrescine were tested as acceptors for coumaroyl-CoA, but no activity could be detected. These kinetic data are in agreement to those found for the partially purified ACT from barley (2).
The kinetic properties of the recombinant ACT5-28-6 were very similar
to the native ACT isoforms, in particular compared with those of ACT2
(Table II). The specific activity (189 nanokatal × mg1 protein) of the affinity-purified recombinant enzyme
was, however, 6-fold higher than that detected for the native ACT
purified to apparent homogeneity (Table I) but only 2-fold higher
compared with ACT2. The product of an activity assay containing the
recombinant ACT was analyzed by reversed phase purification and mass
spectrometry. The products consisted of only one component showing a
[M + H]+ value of m/z 277.3 (data not shown),
identical to the molecular weight of coumaroylagmatine. The tandem mass
spectrometry fragmentation pattern of the 277.3 m/z compound (data not shown) was identical to a
previous study of authentic p-coumaroylagmatine (7), thus verifying the identity of the purified protein.
Chemical Modification of ACT--
DEPC is known to be a reasonably
selective reagent for histidine residues, thus causing inactivation of
enzymes involving histidine in the active site. Activity of the
recombinant ACT decreased about 90% upon a 5-min incubation with 1 mM DEPC. Loss of activity could be a matter of
denaturation, but the decrease in ACT activity was substantially
reduced if ACT was preincubated for 5 min with one of the substrates
before incubation with DEPC (Fig. 8),
additionally suggesting the presence of DEPC-sensitive residues in the
active site of ACT.
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DISCUSSION |
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ACT catalyzes the first step in the synthesis of hydroxycinnamoylagmatine derivatives. This step combines the polyamine and the phenylpropanoid pathway, resulting in metabolites that seem to be involved in the broad-spectrum resistance of barley to fungal attack, the major reason to study the biosynthesis of these low molecular weight compounds. Previously, ACT was partially purified by ammonium sulfate fractionation, size exclusion, and affinity chromatography using agmatine as the ligand (2). In this study, a new approach was developed. Using a four-column purification procedure we succeeded in purifying an ACT isoform to apparent homogeneity and demonstrated the existence of at least three ACT isoforms in young barley seedlings. The kinetic properties of the purified enzyme were in good agreement with the data previously detected for partially purified barley ACT (2). The molecular native size of ~40 kDa was as earlier reported. The three characterized ACT isoforms all showed the highest specificity for p-coumaroyl-CoA as the acyl donor, and no activity was detectable using acyl acceptors other than agmatine. This specificity for the acyl acceptor has also been reported for the purified carnation HCBT, which only has activity in the presence of anthranilate (15). In contrast, purified putrescine N-hydroxycinnamoyltransferase and THT have been reported to be much broader in substrate specificity with respect to acyl acceptors (12, 14, 39).
Analyzing the dynamics of barley soluble phenolics involved in
broad-spectrum resistance to the powdery mildew fungus, a 2.5-fold increase in p-coumaroylhydroxyagmatine was detected in the
leaves of a resistant barley line (2). It will be very interesting to
see if one of the purified ACTs can use the -hydroxyagmatine as a
substrate. Previously no ACT activity could be detected using homoagmatine or N-carbamoylputrescine as acyl acceptors
(2).
The recombinant ACT had a 6-fold higher specific activity than that of
the native protein. This could indicate that ACT loses some activity
during purification, the purified protein is contaminated, or that ACT
is posttranslationally modified. The specific activity of previously
purified N-hydroxycinnamoyltransferases was in the range
45-210 nanokatal × mg1 protein (11-15), which is
in close proximity to our values for both native and recombinant barley
ACT.
The activity of potato THT was strongly stimulated by Ca2+ or Mg2+ (39), and tobacco THT activity was enhanced by ethanol in the incubation mixture (12). Neither of these additions affected ACT activity. However, the presence of Mn2+, Cu2+, or Zn2+ considerably reduced the enzyme activity, perhaps because the requisite thiol is readily oxidized in the presence of these metals.
Microsequencing of ACT indicated that the electroblotted protein band was partially N-terminal-blocked. A 15-amino acid sequence of the N terminus was obtained, but based on the yield of the amino acid residues it is possible to estimate that more than 98% of the molecules were blocked to Edman degradation. In a data base search, several barley EST clones isolated from etiolated barley seedlings showed exact matches to the deduced N-terminal sequence of barley ACT. This prompted cloning of the ACT using the N-terminal sequence obtained. The cloning of ACT also indicated the presence of several isoforms. Two distinct partial clones were obtained, pHV-ACT5 and pHV-ACT6, encoding highly homologous C-terminal polypeptides. The high nucleotide sequence identity between the full-length pHV-ACT5-28-6 and the partial pHV-ACT5 strongly suggests that they represent identical mRNAs. The expression of the pHV-ACT5-28-6 clone in E. coli resulted in synthesis of highly active ACT protein, strongly suggesting that pHV-ACT6 also encodes an ACT isoform.
Barley ACT defines a new class of proteins belonging to a superfamily of acyltransferases with very diverse biological functions. Several plant transferases have been characterized within this superfamily, and two consensus sequences seem to be highly conserved within them. The common motif HXXXD can be found throughout this superfamily (Fig. 5). Single-site mutations of the histidine motif indicates that it is a part of the active site of the dihydrolipoamide S-acyltransferase (40). Chemical modification of barley ACT using the histidine-reactive DEPC suggested that a histidine is important for the catalytic mechanism of ACT, which is further emphasized by the substrate preincubation of ACT that considerably decreases the effect of DEPC. The second consensus sequence, DFGWGXP, is located in the C termini of the transferases, but no function has yet been assigned to this motif. Considering substrates, it is notable that tryptophan in the motif is substituted by more polar residues in the family subgroup E (Fig. 6), e.g. Gly-388 in ACT (Fig. 5), the only group that apparently does not to use aromatic acyl acceptors.
The biological function of the hydroxycinnamoylagmatine derivatives is not yet established, and ACT activity has hitherto only been detected in the young seedlings of barley. Thus, it is of major interest that the ACT clone (pHV-ACT5-28-6) and putative ACT EST clones were from mRNA isolated from plants infected with fungal pathogens. The ACT clones obtained in this study were from a cDNA library constructed from barley leaves 12 h after inoculation with an incompatible isolate of B. graminis forma specialis hordei. One EST clone (accession number BI955449) was isolated from libraries of 7-day-old green leaves of Mla6-resistant barley challenged with an avirulent isolate (5874) of B. graminis f. sp. hordei. Another clone (accession number BM137380) was isolated from wheat spikes sprayed at anthesis with Fusarium graminearum. These results suggest that ACT is involved in stress or defense responses of cereals. Putative barley ACT EST clones have also been isolated from roots (accession number BF259608) and rachises (accession number BI959297), indicating that ACT is widely distributed in the barley plant.
The hordatines have long been known to be antifungal compounds in the young barley seedling, and they seem to be restricted to the genus Hordeum (4, 5). The oxidative phenol coupling mechanism involved in the synthesis of the hordatines is not known (Fig. 1), but it has been demonstrated that the process can be catalyzed by hydrogen peroxide in the presence of catalytic amounts of horseradish peroxidase, giving a racemic form of the otherwise optical active metabolites (41). If the hydroxycinnamoylagmatines are peroxidase substrates, one could expect to find hordatine in other cereals now that hydroxycinnamoylagmatine derivatives have been detected in wheat (8) and a clone almost identical to barley ACT has been isolated from wheat (Fig. 6). The availability of the ACT cDNA clones provides excellent tools to study the regulation of ACT grown under natural conditions and in response to a pathogen attack(s). In particular, it should also be possible to detect ACT expression in other cereals if a correspondingly high similarity exists as between barley and wheat.
Histochemical examinations of leaf epidermis indicate that
guanidine-containing compounds are highly abundant in the papilla of
several cereals after powdery mildew infection (9). The papilla is a
local cell wall reinforcement of cytoplasmic produced materials
polymerized in the cell wall adjacent to the attacking pathogen. It
will be particularly interesting to study the composition of papilla by
modulating the synthesis of hydroxycinnamoylagmatine derivatives using
transgenic barley harboring a RNA interference or antisense-ACT
construct. Additionally, recombinant expression of the ACT now allows
sufficient recovery of protein to obtain antibodies and enable
immunochemical studies.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. S. Scott-Craig and Dr. P. Schweizer for providing cDNA libraries, Dr. C. J. Douglas for providing the E. coli strain expressing the recombinant tobacco 4-coumarate:coenzyme A ligase, Dr. S. Jacobsen for assistance in interpretation of the Edman degradation results, Dr. H. Egsgaard for assisting the mass spectroscopy, to Dr. T. H. Roberts for critical 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) AY228552 and AY234333.
Supported by the Danish Research Academy.
§ To whom correspondence should be addressed. Tel.: 45-46-77-41-21; Fax: 45-46-77-41-22; E-mail: soren.rasmussen@risoe.dk.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M213041200
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ABBREVIATIONS |
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The abbreviations used are: ACT, agmatine coumaroyltransferase; CV, column volumes; DEPC, diethyl pyrocarbonate; EST, expressed sequence tag; HCBT, N-hydroxycinnamoyl/benzoyltransferase; RACE, rapid amplification of cDNA ends; THT, tyramine N-hydroxycinnamoyltransferase; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; contig, group of overlapping clones.
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