T H E JOURNAL OF BIOLOGICAL CHEMlSTRY 0 1984 by The American Society of Biological Chemists, Inc. Vol. 259, No. 11, Iwue of June 10, pp. 6806-6811.1984

Printed in U. S. A.

Purification and Properties of a Stilbene Synthase from Induced Cell Suspension Cultures of Peanut*

(Received for publication, November 21, 1983)

Anne Schoppner and Helmut Kindl From the Department of Chemistry, Philipps-Universitiif Marburg, Hans-Meerwein-Strape, 0-3550 Marburg, Federal Republic of Germany

Stilbene synthase (resveratrol-forming) converts one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA into 3,4',5-trihydroxystiIbene. Fol- lowing selective induction of stilbene synthesis in cell suspension cultures of peanut (Arachis hypogaea), the enzyme was extracted and purified to apparent homo- geneity by chromatography on DEAE-cellulose and hydroxylapatite.

The enzyme was found to be a dimer of estimated M, = 90,000 exhibiting under denaturing conditions a subunit M, of approximately 45,000. The isoelectric point was determined with PI = 4.8.

The enzyme's high selectivity towards p-coumaroyl- CoA (K, = 2 MM) as substrate qualified it as resvera- trol-forming stilbene synthase. Structurally related CoA esters, e.g. dihydro-p-coumaroyl-CoA and cin- namoyl-CoA, were converted less than 1/10 as effi- ciently as p-coumaroyl-CoA. Malonyl-CoA (K, = 10 p ~ ) could not be substituted by acetyl-coA. The puri- fied enzyme was free of chalcone synthase activity. Antibodies raised against stilbene synthase were shown to be monospecific and not to cross-react with chalcone synthase.

Hydroxystilbenes, as examplified by resveratrol (3,4',5- trihydroxystilbene), are constituents of a restricted number of plant species (1-3). Some of the hydroxystilbenes have been shown to function as phytoalexins (4). Their synthesis is induced in plants by the attack of pathogenic fungi (5, 6 ) and their function could be to stop the fungal growth. Young seedlings of peanut (Arachis hypogmu) have been demon- strated to respond to fungal attack by the synthesis of resver- atrol, isopentenyl resveratrol, and cognate compounds (7).

To extend studies on phytoalexin formation to the selec- tively induced synthesis of enzymes and their mRNAs, and thus to the mechanisms underlying the plant resistance, the key enzyme of the biosynthetic sequence, stilbene synthase, needs to be purified and used to prepare monospecific anti- bodies.

Cell cultures of A. hypogaea were presumed to be a valuable model system for studying the induction of stilbene synthase under closely definable conditions, provided they are capable of stilbene synthesis and are susceptible to induction like the intact plant. Callus cultures and cell suspension cultures

* This work was supported by Grant SFB 103 from the Deutsche Forschungemeinschaft and Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ''advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

exhibiting these properties have been established recently

Constitutive stilbene synthases (for overall reaction and structures see Fig. 1) have been detected in plants, e.g. the resveratrol-forming enzyme in rhizomes of Rheum rhaponti- cum (10). A similar but inducible enzyme was implicated in the synthesis of phytoalexins in grape and other Vitaceae (11). Unlike these enzymes a stilbene synthase from Pinus sylvestris was found to be selective in that it catalyzes the formation of pinosylvin (3,5-dihydroxystilbene) (12).

Inducible tissues and inducible cell suspension cultures of A. hypogaea turned out to be optimal sources to obtain stilbene synthase, high specific activity occurring already in the crude extracts. In this paper we describe, for the first time, the purification of a stilbene synthase to apparent homogeneity. Molecular and kinetic properties were examined and com- pared to the data obtained with chalcone synthase, an enzyme which uses the same substrates as stilbene synthase.

(a,g).

EXPERIMENTAL PROCEDURES

Materials [2-"C]Acetyl-CoA (1.5 GBq/mmol), [2-"C]malonyl-CoA (2.0

GBq/mmol), and iod0[2-'~C]acetamide (1.6 GBq/mmol) were pur- chased from The Radiochemical Centre (Amersham, United King- dom). Various cinnamoyl-CoA derivatives and other CoA esters were synthesized via the N-hydroxysuccinimidyl esters according to Refs. 8 and 13. Resveratrol was prepared from Veratrum album (8), 3,4',5- trihydroxybibenzyl was produced by catalytic hydrogenation of the respective stilbene. Pinosylvin was extracted from heartwood of P. sylvestris and purified by column chromatography (12). Other stil- benes and bibenzyls (14) were synthesized by condensation of 3,5- dimethoxybenzaldehyde with substituted esters of phenylacetic acid, decarboxylation of the stilbene carboxylic acid, and demethylation with BBr3.

Cell Suspension Cultures Cell cultures of peanut (A. hypogaea) were started from hypocotyls

of 4-day-old seedlings and were further propagated as callus cultures (8). Suspension cultures were established and maintained under white light a t 26 "C for more than 40 transfers. Cells were transferred to new medium every 12 days. Properties of these suspension cultures and the mode of induction of enzymes responsible for the formation of stilbenes were determined by methods described elsewhere (8).

Enzyme Preparation and Assays Extracts from Cell Suspension Cultures-After transfer to fresh

medium, peanut cultures were grown for 12 days and then, at this late stage of stationary phase, induced by dilution with fresh medium. Following an induction period of 12 h, cells were harvested by filtra- tion and washed once with 100 mM potassium phosphate (pH 8.0) containing5 mM mercaptoethanol (buffer A). The cell material (about 200 g, fresh weight) was resuspended in 20 ml of buffer A and homogenized in a Waring Blendor at 0 "C. Cell walls and unbroken cells were sedimented by centrifugation at 20,000 X g for 15 min, and soluble enzymes were recovered in the supernatant. Immediately after

6806

Stilbene Synthase of Peanut Cell Cultures 6807

on

FIG. 1. Overall reaction catalyzed by stilbene synthases. Thep-hydroxy derivative of cinnamic acid ester ( M H ) is converted into resveratrol (3,4',5-trihydroxystilbene). The unsubstituted cin- namic acid ester ( b H ) gives rise to pinosylvin (3,5-dihydroxystil- bene). By this way, the carboxyl group of the CsC3 compound and the 3 C2 units originating from malonyl-CoA form a new aromatic ring.

centrifugation, the pooled supernatants were treated with Dowex 1- X4 (5 g/100 ml of fluid) previously equilibrated in buffer A. After stirring for 30 min, the anion exchanger was filtered off. The crude enzyme solution was brought to 65% saturation with ammonium sulfate at pH 8.0 and stirred for 2 h. The precipitate was removed by centrifugation at 10,OOO X g for 30 min. Stilbene synthase was recovered from the supernatant by adding ammonium sulfate to 90% saturation and collecting the protein by centrifugation (10,000 X g, 30 min), The precipitate was dissolved in 12 ml of 10 mM potassium phosphate (pH 8.0) containing 1 mM mercaptoethanol (buffer B) and the solution desalted by passing through a column (1.8 X 55 cm) with Sephadex G-50.

DEAE-cellulose Chromntography-Following fractional ammo- nium sulfate precipitation, the enzyme solution (25 ml) was applied to a DEAE-cellulose column (Whatman DE52) (3 X 10 cm) previously equilibrated in buffer B. Protein was washed on the column with 150 ml of equilibration buffer, and bound enzymes were eluted by a linear salt gradient (10-200 mM potassium phosphate, pH 8.0). Fractions of 7 ml were collected at controlled temperature (4 "C). Salt concentra- tion was determined conductometrically. Elution of stilbene synthase was measured with the standard assay using 40 pMp-coumaroyl-CoA, and peak fractions (47-83 mM phosphate) were pooled. The protein was concentrated by filtration under pressure (Amicon Corp.) and the solution was adjusted to 15 mM potassium phosphate and 1 mM mercaptoethanol.

Hydroxyylopatite Chromatography-The enzyme was loaded on a hydroxylapatite column (Bio-Gel HTP) (1.6 X 14 cm) and washed with 60 ml of buffer B. A linear phosphate gradient (10-100 mM) was applied to elute the stilbene synthase. Fractions of 4.5 ml were collected, and protein was concentrated.

Ultrogel AcA 44 Chromtography-Fractions with stilbene syn- thase activity were loaded on an Ultrogel AcA 44 column (1.8 X 60 cm) for molecular sieving. This column was equilibrated previously with buffer B. Fractions of 2.2 ml were collected and assayed for protein and enzyme activity. The column was equilibrated with the following M, standards: catalase (250,000), citrate synthase (lOO,OOO), malate dehydrogenase (70,000), and peroxidase (40,000). The void volume was determined with blue dextran.

Assays for Stilbene Synthase Actiuity-The standard assay was carried out with p-coumaroyl-CoA. 10 pl of 170 pM [2-"C]malonyl- CoA (4 kBq) were incubated with 10 p1 of 1 mM p-coumaroyl-CoA, and 80 pl of enzyme preparation containing 1 mM dithiothreitol. The reaction proceeded for 30 min at 30 "C and was then stopped by adding 100 pl of 0.01 N HCI and 10 pl of resveratrol(2 mg/ml of ethyl acetate). The products were extracted with ethyl acetate and isolated by thin layer chromatography. The activity of 1 pkat' was defined as incorporation of 1 pmol of p-coumaroyl-CoA into resveratrol/s a t 30 "C.

Analysis of the Radioactive Products Formed in Stilbene Synthase Incubations-Three different solvent systems were used to character- ize stilbenes and bibenzyls by thin layer chromatography (Kieselgel 60 Fm, Merck). In addition, recrystallization to constant specific activity was performed in many cases to provide the proof of identity. Routinely, the ethyl acetate extract was chromatographed in system I which allows the stilbene to be separated from the respective chalcone, flavanone as well as from contaminating traces of malonic acid and malonyl-CoA. System I contains CHC1,:ethyl acetate: HCOOH (5:41). System I1 contains n-hexane:ethyl acetate:methanol (50:50:1). System I11 contains CHC&:acetic acid (9:l).

RF values for resveratrol are 0.71 in I, 0.36 in 11, 0.24 in 111. RF values for naringenin are 0.82 in I, 0.57 in 11, 0.38 in 111. Other

' The abbreviations used are: kat, katal; SDS, sodium dodecyl sulfate.

stilbenes and bibenzyls were analyzed according to Ref. 14. Electrophoretic Procedures-Portions of stilbene synthase prepa-

rations were prepared for electrophoresis by precipitation with ice- cold 10% (w/v) trichloroacetic acid. The pellet obtained after centrif- ugation was redissolved in about 50 pl of sample buffer (10% sucrose, 5% mercaptoethanol, 2% SDS). A discontinuous system according to Laemmli (15) was used in a slab gel (20 X 30 cm). Polyacrylamide gel electrophoresis under non-denaturing conditions was performed at pH 7.5 in gel system 6 as described by Maurer (16). Isoelectric focusing was on slab gels (Servalyt Precotes, 3-10). Focusing was carried out for 2 h at 2 "C at 1200 V. Immunoelectrophoresis was performed on an agarose gel (1% w/v) in 50 mM Tris-HC1 (pH 7.5), applying 1.5 pg of the enzyme to the gel (8 cm) at the cathodal end. Electrophoresis was carried out for 2 h at 200 V.

Sedimentation Velocity Centrifugation

The molecular weight of enzymatically active enzyme was esti- mated according to Martin and Ames (17). We used a linear sucrose gradient ranging from 15 to 35% (w/v) sucrose in buffer B and placed in tubes of a SW-40 rotor (Beckman). Centrifugation was at 40,000 rpm and 5 'C for 40 h; authentic proteins were run in parallel.

Other Determinations

Double immunodiffusion was carried out in agarose gel (1% w/v, in 0.14 M NaCl containing 20mM sodium phosphate buffer, pH 7.2), cast to a depth of 4 mm. Diffusion was allowed to take place for 40 h in humidity chambers at 5 "C. Radioactivity (18) and protein (19) were measured according to established methods.

RESULTS

Purification of Stilbene Synthase-Cells of A. hypogwa grown in suspension reached the stationary phase at day 11- 12. If the cultures were then diluted with fresh medium, the level of stilbene synthase activity increased markedly as com- pared to the level of this enzyme during the logarithmic phase of growth or at the stationary phase without induction, re- spectively. Also in terms of specific activity (picokatal per mg of total extractable protein), stilbene synthase activity was found to be induced due to this treatment, oiz. by a factor of 20. Therefore, cell suspension cultures subjected to this rather selective induction and reaching maximal activities of stilbene synthase 12 h later were used as starting material for the preparation. After extracting the cells and immediately ab- sorbing phenols on polystryol resin, purification was carried out by ammonium sulfate fractionation and by chromatogra- phy on DEAE-cellulose (Fig. 2). The latter procedure yielded a 13-fold increase in specific activity. Stilbene synthase was then separated from contaminating proteins by chromatog- raphy on hydroxylapatite (Fig. 3). A rather narrow fraction was taken (fractions 30-45) and analyzed by molecular sieving (Fig. 4 ).

Chromatography on hydroxylapatite did not lead to an increase in specific activity although electrophoretic analysis demonstrated that several proteins were separated from the stilbene synthase during this chromatography. The main in- terest was to obtain a homogeneous protein rather than to improve the specific activity of the preparation. A typical purification run (Table I) yielded a more than 100-fold in- crease in specific activity.

In most cases, the stilbene synthase eluted from the hydrox- ylapatite column was homogeneous as judged by SDS-gel electrophoresis. In cases when it was necessary to remove trace amounts of contaminating proteins by chromatography on Ultrogel AcA 44, this procedure consistently resulted in a significant decrease of the enzyme's specific activity (approx- imately 30%). We failed to prepare stilbene synthase with higher specific activity by utilizing additional purification steps, and including agents to enhance stability. When a purified enzyme was concentrated up t o 100 pg of protein/ml

6808 Stilbene Synthase of Peanut Cell Cultures a n 1.0 100

0 1 0 m . o . o

FRACTION N U I I C R

FIG. 2. Elution pattern of stilbene synthase from DEAE- cellulose. Following ammonium sulfate fractionation, 12 ml of en- zyme solution were desalted by passing through a column with Seph- adex G-50. This preparation was then purified on Whatman DE52 cellulose as described under “Experimental Procedures.” Dashed tine shows the concentration of phosphate determined in the eluate con- ductometrically. The burs indicate stilbene synthase activity. -, protein. Three sets of fractions were pooled and concentrated.

20 40 60 00

FRACTION NUMBER

FIG. 3. Purification of a stilbene synthase by hydroxylapa- tite chromatography. A partially purified preparation after anion exchange chromatography (see Fig. 2) was concentrated and equili- brated with 10 mM phosphate (pH 8.0) containing 1 mM mercapto- ethanol. The enzyme was loaded onto a Bio-Gel HTP column (14 X 1.6 cm) equilibrated with buffer B. The column was washed with 2 ut (ut: total volume of the column) of buffer B and then eluted with increasing concentrations of phosphate. Fractions of 4.5 ml were collected and assayed for stilbene synthase. Symbols as described in the legend to Fig. 2.

and adjusted to 1 mM dithiothreitol and 10% sucrose, the preparation lost 30% of its activity during 3 weeks at -20 “C.

Molecular Properties of Sti&ene Synthase-The molecular weight of the active enzyme estimated either by molecular sieving (Fig. 4) or by sedimentation velocity centrifugation in a sucrose gradient (data not shown) was 90,000. In both cases, the activity range of stilbene synthase was between the one of pig heart citrate synthase (Mr = 100,000) and malate dehydrogenase (Mr = 70,000). Electrophoresis (Fig. 5) under denaturing conditions in the presence of SDS revealed a subunit molecular weight of M, = 45,000. The active enzyme is, therefore, a dimer. During purification, a second band at

I CS YDH

. . . 10 30 50

CIIACTION *UNDER

FIG. 4. Analysis of purified stilbene synthase and estima- tion of its molecular weight by molecular sieving on Ultrogel AcA 44. A sample (1.0 ml, total volume) containing 5 pkat of stilbene synthase purified through step 5 was chromatographed on a 150-ml column and compared with standard proteins run in parallel (see “Experimental Procedures”). The position of malate dehydrogenase (MDH, M, = 70,000) and citrate synthase (CS, M, = 10,000) are indicated by arrows.

TABLE I Purification of stilbene synthase activity

Cells (220 g, net weight) of induced cell cultures were used. Activity is expressed as picomole of p-coumaroyl-CoA converted into resver- atrol/s at 30 “C (1 pkat).

Purification ShP protein units activitv

Total Total Specific Recovery Purifi- cation

mg p h t p h t l k g % -fold crude extract 915 384 0.4 100 1.0 Dowex 1 treatment 870 410 0.4 106 1.1 Ammonium sulfate 98 340 3.5 88 8.3

fractionation (including Sephadex G-50 sieving)

DE52 cellulose eluate 7.5 340 45.3 88 108 Hydroxylapatite eluate 1.4 82 58.5 21 139

subunit M, = 42,000 appeared which is probably a proteolytic artifact. If stilbene synthase was precipitated from crude extracts with antiserum almost none of this accompanying band was observed on gels. The isoelectric point of the en- zyme, PI 4.8, was determined by electrofocussing on a slab gel (Fig. 6).

Stilbene synthases have been found to be very sensitive towards mercurials and other compounds modifying-SH groups. Following inactivation with mercuribenzoate, full reactivation of the enzyme was achieved by excess of dithio- threitol. Also during purification, dithiothreitol or mercapto- ethanol were required to keep the stilbene synthase in the catalytically active form. The involvement of an exposed ”SH group in the catalytic process was further established by rapid labeling of purified stilbene synthase with iodo[2- 14C]acetamide (data not shown).

Monospecific Antibodies-Antibodies against purified stil- bene synthase were raised in rabbits. They were monospecific as judged by the single precipitation line observed on doubIe immunodiffusion. Antibodies were assayed against the puri- fied enzyme and crude preparations of stilbene synthase from A. hypogaea (Fig. 7). Anti-chalcone synthase antibodies, raised against the enzyme from parsley cell cultures, did not cross-react with purified synthase. But anti-stilbene synthase antibodies precipitated resveratrol-forming stilbene synthases from other sources (data not shown).

Stilbene Synthase of Peanut Cell Cultures 6809

A B

60000-

30000*

a b c d e FIG. 5. Subunit molecular weight of stilbene synthase as

assayed on SDS-polyacrylamide slab gel. Enzyme samples a t different stages of purification were subjected to electrophoresis in 8% gels. The lanes contained: a, phosphorylase, catalase, fumarase, citrate synthase, lactate dehydrogenase, and carbonicanhydrase, as markers; 6, enzyme preparation after ammonium sulfate fractiona- tion; c DEAE-cellulose peak fraction; d, purified enzyme after chro- matography on hydroxylapatite; e, fumarase (subunit M, = 49,500) and citrate synthase (subunit M, according to amino acid sequence 48,969, behaves on gels as M, = 46,000).

PHI- -

+

FIG. 6. Isoelectric focusing and non-denaturing electropho- resis of native stilbene synthase on slab gels (in the absence of SDS). A, determination of the isoelectric point by focusing on slab gels. Stilbene synthase purified through step 5 was applied to the gel in the range of pH 8. B, electrophoresis of the purified enzyme at pH 7.5 in the absence of SDS. Stilbene synthase migrates as an anion.

Specificity of Stilbene Synthase-Purified stilbene synthase from A. hypogaea was assayed with malonyl-CoA and various CoA esters of C6C3 carboxylic acids (Table 11). The high specific radioactivity of one of the two substrates and the exact identification of each product enabled us to measure also less pronounced conversions and thus to evaluate the structural requirements to be met by the substrates. In this respect, the enzyme from A. hypogwa cell cultures was char- acterized by its high selectivity towards the p-hydroxy com-

3

1 2 4

FIG. 7. Immunodiffusion and immunoelectrophoresis of stilbene synthase preparations. A, double immunodiffusion on agar. The wells contained crude preparation of stilbene synthase ( I ) , anti-stilbene synthase antiserum (2), purified stilbene synthase (31, anti-chalcone synthase antiserum (4 ) . B, immunoelectrophoresis on 1% agar at pH 7.5. The well at the cathodal side received crude synthase enriched by ammonium sulfate fractionation, the step prior to anion exchange chromatography.

pound. Cinnamoyl-CoA was less than 1/10 as efficient as p- coumaroyl-CoA. This is in contrast to the properties of the stilbene synthase from P. sylvestris which catalyzes the for- mation of pinosylvin from cinnamoyl-CoA with three times higher rates than the conversion of the respective p-hydroxy compound into resveratrol. Among the other CoA esters tested as possible substrates, the dihydro derivative of p-coumaroyl- CoA, p-hydroxyphenylpropionyl-CoA, can be regarded as sub- strate of this stilbene synthase, although with comparingly low velocity.

With respect to the second substrate, the experiments une- quivocally proved that malonyl-CoA cannot be replaced by a related compound, e.g. acetyl-coA.

Apparent K, values were determined for p-coumaroyl-CoA and malonyl-CoA. In the range of 0.2-5 K, the enzyme exhibited typical Michaelis-Menten kinetics. High affinities of p-coumaroyl-CoA (K,,, = 2. lo4 M) and malonyl-CoA (K, = 10. lo-' M) towards the enzyme suggest that the enzyme can function in vivo at rather low concentrations of CoA esters.

With p-coumaroyl-CoA and malonyl-CoA as substrates, resveratrol was the sole product of the enzyme-catalyzed reaction. Neither naringenin nor its chalcone nor p-hydrox- ystyrylpyrone (or p-hydroxyphenyl pyrone) were formed. Pu- rified stilbene synthase does, therefore, not contain chalcone synthase activity. This held true over then pH range of 6.5-9 at which range stilbene synthase is active. Optimal activity was measured between pH 7.5 and 8.5. Under these condi- tions, we were unable to detect any stilbene 2-carboxylic acid as intermediary product.

Coenzyme A, one of the reaction products, does not signif- icantly inhibit the enzyme at the conditions used in the assay. It may, however, influence the reaction rate under physiolog- ical conditions when the concentration of coenzyme A exceeds the value of 100 p ~ . In vitro reduced rates (43%) were observed at 0.1 mM coenzyme A while 0.5 mM coenzyme A led to 80% inhibition.

DISCUSSION

The studies describe the purification of an enzyme catalyz- ing the conversion of a CoA ester of an aromatic C&3 acid and malonyl-CoA into a hydroxystilbene as outlined in Fig. 1. Resveratrol (Fig. 1, R = OH) was demonstrated to be the sole aromatic compound being released from the enzyme. Neither polyketo derivatives nor a stilbene 2-carboxylic acid were detectable in the reaction mixture. It is, therefore, sug- gested that all intermediates to be postulated (Fig. 8) remain at the enzyme surface.

Another striking feature of the enzyme's specificity con- cerning the products formed is the lack of even trace amounts

6810 Stilbene Synthase of Peanut Cell Cultures

TABLE I1 Substrate specificity of stilbene synthase

The purified enzyme (0.1 pkat) was assayed with radioactive malonyl-CoA and various aromatic CoA esters. The final concentrations were 17 and 100 p ~ , respectively. Products were identified by chromatography and quantified on the basis of radioactivity. Results were related to the formation of resveratrol, by setting its formation at 100%.

First substrate s $ ~ ~ r ~ ~ R, R?

Malonyl-CoA H H H

OH R2 OH OH

Acetyl-coA Malonyl-CoA

OH OCHB OH H OH H

0 H OH

Trivial name

Cinnamoyl-CoA

p-Coumaroyl-CoA

rn-Coumaroyl-CoA Caffeoyl-CoA

Feruoyl-CoA p-Coumaroyl-CoA Dihydro-p-coumaroyl-CoA

Product identified

3.5-Dihydroxystilbene (Pinosylvin)

3,4’,5-Trihydroxystilbene (resveratrol) 3,3’,5-Trihydroxystilbene 3,3’,4’,5-Tetrahydroxystilbene

3,4’,5-Trihydroxy-3’-methoxystiIbene (3,4’,5-Trihydroxystilbene) 3,4’,5-Trihydroxybibenzyl

Relative amounts

9.5 100

9 8 6

<1 13

Dihydro-rn-coumaroyl-CoA 3,3’,5-Trihydroxybibenzyl 5

co2 XSH

1-6 ” Chalcone - Flavonoid. OH

FIG. 8. Probable mechanism of enzymatic stilbene synthesis indicating some of the possible intermediates and products. For sake of clearness the p-hydroxystyryl group is given as R and CoA is denoted by X. Following decarboxylation of malonyl-CoA, a nucleophilic displacement can take place at the ester group (first row) resulting in the formation of a fi-keto ester. Two further condensa- tions with malonyl-CoA should eventually lead to a triketo acid derivative (CoA ester of a 3,5,7-triketo acid; second row) which may be the branching point giving rise either to a stilbene carboxylic acid by aldol reaction (C-2-C-7) shown as 2+7 or affording a chalcone by ester condensation ( C l - C - 6 ) shown as 1 4 .

of chalcone, which, in principle, could arise from the same substrates (Fig. 8). The pathway either to chalcones or to stilbenes is, therefore, under pure topological control. This reinforces the view of repeatedly malonyl-CoA condensing enzymes (14, 20-22) as being matrices which allow the po- lyketide chain to be folded in a defined, ring-like manner prior to the cyclization reaction.

Following proper folding of the carbon chain, cyc!ization can take place by nucleophilic attack of a carbanion (at C-2 of the polyketo acid ester) on the carbonyl group at C-7. This attack by C-2 is indicated by the symbol 2 + 7 and is displayed in Fig. 8. The aldol reaction furnishes a derivative of 2,4- dihydroxy-6-alkylbenzoic acid and leads to a stilbene (Fig. 8, R = HO-CsH,CH=CH-). If however, the carbon chain is arranged in another manner, cyclization can proceed by

ester condensation between C-1 and C - 6 (designated 1 3

6). It gives rise to a l-acy1-2,4,6-trihydroxybenzene (the class of chalcones) and then to flavanoids.

Condensation of one molecule of p-coumaroyl-CoA with only one or two molecules of malonyl-CoA may yield @-keto acids giving rise to side reactions as discussed in Ref. 22. These products of side reactions, i.e. a-pyrones, have not been observed in the reaction catalyzed by stilbene synthase.

Stilbene Synthases Grouped According to Their Substrate Specificity-A number of CoA esters of aromatic carboxylic acids were tested as substrates of the purified enzyme. It was demonstrated that the stilbene synthase from peanut is spe- cific as far as malonyl-CoA is concerned and is highly selective with respect to the second substrate. These data are most consistent with previous findings indicating that stilbene synthases use either cinnamoyl-CoA or p-coumaroyl-CoA as substrates, but never both with a similar efficiency. It is evident, also from other data in Table 11, that the stilbene synthase selects its substrate by probing the substitution pattern at the aromatic ring. Probably, this region of the substrate has first to be bound to the active center of the enzyme leaving the thio ester group free and thus accessible for the nucleophilic attack by the second substrate.

We also tested cy,fl-dihydro derivatives of cinnamoyl-CoA, uiz. derivatives of phenylpropionyl-CoA. Probing the catalytic activity of stilbene synthase towards these compounds was required as recently another type of stilbene synthase (biben- zyl synthase) was discovered which selectively utilized dihy- dro-m-coumaroyl-CoA (m-hydroxyphenylpropionyl-CoA) as substrate (14). For the peanut enzyme, however, the dihydro derivatives were clearly not the preferred substrates.

Comparison of Stilbene Synthase with Chalcone Synthase- The results presented herein are of particular interest as they rule out the requirement that chalcones accompany stilbenes as products. Both stilbene synthase and chalcone synthase function with the same substrates, malonyl-CoA and p-cou- maroyl-CoA. In addition, it could have been assumed that stilbene-forming activity is rather a side reaction catalyzed by a modulated form of chalcone synthase.

Together with our immunological studies, clear evidence is provided that the two activities are attributable to two differ-

Stilbene Synthase of Peanut Cell Cultures 681 1

ent proteins. Although clearly distinguishable by their prod- ucts and their immunological properties, chalcone synthase and stilbene synthase bear many similar properties, e.g. both enzymes exhibit similar M , and subunit Mr.

Chalcone synthase was discernible in crude extracts from A. hypogaea cell cultures. Whether this activity reflected a small amount of a chalcone synthase present in the cell cultures or was an intrinsic property of stilbene synthase prior to further purification could not be decided in the case of the Arachis enzyme; the chalcone synthase activity was too low. However, with enzyme preparations from other sources, e.g. cell cultures of Picea excelsa, we were able to separate stilbene synthase and chalcone synthase activities. While anti-stilbene synthase antibodies against the peanut enzyme cross-react with resveratrol-forming stilbene synthase from P. excelsa, they did not precipitate chalcone synthase from P. excelsa.2 Furthermore, anti-chalcone synthase antibodies from Petro- selinum hortense cell cultures were unable to precipitate the purified stilbene synthase from peanut (Fig. 7). This rules out the possibility that the two catalytic activities are located at the same enzyme whose catalytic properties are being modu- lated by low molecular weight effectors.

The function of stilbene synthases has important implica- tions for the synthesis of stilbenes and their derivatives which are the main phytoalexins in some economically relevant plant genera. DNA sequences coding for the stilbene synthase protein may increasingly become focuses of research if genes of plant resistance are looked for.

Acknowledgment-We thank Dr. K. Hahlbrock, Cologne, for pro- viding us antibodies against chalcone synthase.

* C.-H. Rolfs and H. Kindl, unpublished data,

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