©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reaction Mechanisms of Homodimeric Plant Polyketide Synthases (Stilbene and Chalcone Synthase)
A SINGLE ACTIVE SITE FOR THE CONDENSING REACTION IS SUFFICIENT FOR SYNTHESIS OF STILBENES, CHALCONES, AND 6`-DEOXYCHALCONES (*)

(Received for publication, November 28, 1994; and in revised form, January 27, 1995)

Susanne Tropf Bärbel Kärcher Gudrun Schröder Joachim Schröder (§)

From the Institut für Biologie II, Universität Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Stilbene (STS) and chalcone (CHS) synthases are homodimeric, related plant-specific polyketide synthases. Both perform a sequential condensation of three acetate units to a starter residue to form a tetraketide intermediate that is folded to the ring systems specific to the different products. Protein cross-linking and site-directed mutagenesis identified a subunit contact site in position 158, close to the active site (Cys). This suggested that the active site pockets may be neighboring, possibly alternating in the condensing reactions rather than acting independently. This was investigated by co-expression of active site mutants with differently mutated, inactive proteins. With both STS and CHS, the heterodimers synthesized the end products, indicating that each subunit performed all three condensations. In co-action with a monomeric reductase, CHS also synthesizes 6`-deoxychalcone, but with the chalcone as second product when using plant preparations. The two enzymes expressed as single species in Escherichia coli synthesized both products, and both were also obtained with a CHS heterodimer containing a single active site. The results showed that 6`-deoxychalcone synthesis required no other plant factor and that the formation of two products may be an intrinsic property of the interaction between dimeric CHS and monomeric reductase.


INTRODUCTION

Stilbene synthases (STS) (^1)occur in a limited number of widely unrelated plants. They synthesize the backbone of the stilbene phytoalexins that have antifungal properties and contribute to the pathogen defenses. Chalcone synthases (CHS) synthesize naringenin chalcone, the precursor for a large number of flavonoids. Both enzymes are polyketide synthases performing complex reactions (Fig. 1). These are most likely identical in the initial part that involves the stepwise condensation of three acetate units from malonyl-CoA to a starter residue to synthesize an enzyme-bound tetraketide intermediate. The reactions correspond to the condensations in fatty acid and other polyketide synthases(1, 2) . The tetraketides are folded to new aromatic ring systems, and these are different in STS and CHS (Fig. 1). The typical STS reaction also includes a decarboxylation that is not performed by CHS (reviewed in (3) and (4) ).


Figure 1: Reactions of STS and CHS. Both enzymes perform with a starter CoA-ester three sequential condensation steps with acetate units from malonyl-CoA. The boxed part shows the postulated enzyme-bound tetraketide intermediates (three acetates added to the 4-coumaroyl residue) that are presumably identical in the STS and CHS reactions. The intermediates are folded to new aromatic ring systems, and these processes are different in the two enzymes. The formation of 6`-deoxychalcone requires the reduction of a specific carbonyl group at the tri- or tetraketide intermediate level. The reaction is performed by a specific NADPH-dependent reductase.



The similarities in the STS and CHS reactions are reflected by extensive similarities in structure and sequences. Both are dimers (5, 6, 7, 8, 9) of identical subunits containing 388-400 amino acids, and the active sites for the condensing reactions are in the same position (Cys, Fig. 2)(10) . The sequences of all known STS and CHS are geq60% identical, and a recent analysis suggested that STS evolved from CHS by a relatively small number of exchanges in key amino acids(11) .


Figure 2: Protein sequences. a, CHS from S. alba; b, STS from A. hypogaea; c, CHS/STS protein hybrid c1an (a protein fusion containing the N-terminal 107 amino acids of the S. alba CHS and 288 amino acids of the A. hypogaea STS; the CHS part in c1an carries three mutations toward the STS sequence, but the Q100E exchange essential for STS activity is lacking). In b and c, only the differences to the CHS are printed; dots indicate sequence identity. The amino acids relevant for this work are in large bold letters, and their positions are indicated. The numbering is based on the CHS from S. alba. Cys is the active site of the condensing reaction.



Although three-dimensional structures are not yet available, the similarities in sequence and function suggest a common structure with some differences reflecting the different ring-folding to the end products. We became interested in the subunit interactions because these might well be an integral part of the reactions performed by STS and CHS. The first part of this work deals with the identification of a subunit contact site close to the active site of the condensing reaction, and with the experimental distinction between alternative reaction mechanisms suggested by the finding that the active sites of the two subunits may be close together in the dimer.

The second part analyzes the interaction between CHS and the monomeric reductase that leads to a reduced chalcone (6`-deoxychalcone) by reduction of a specific carbonyl group prior to formation of the chalcone ring system (Fig. 1)(12, 13, 14) . This product is the precursor of the isoflavonoid phytoalexins which are part of the plant defenses against pathogens, and the reductase is the key to their formation. One of the puzzling aspects of that interaction was that plant CHS preparations always produced both 6`-deoxychalcone and chalcone, and this was investigated with both enzymes expressed as single species in Escherichia coli and with CHS heterodimers containing a single active site for the condensing reaction.


EXPERIMENTAL PROCEDURES

Protein Cross-linking

The homobifunctional sulfhydryl-reactive cross-linker BMH (Pierce) containing the spacer arm (CH(2))(6) was used according to previously described principles(15) . The incubations (0.5 ml) contained 50 mM HEPES buffer (pH 7.0), 40 mM NaCl, 10 mM EDTA (pH 7.0), and 3 to 10 µg from extracts prepared as for the enzyme activity assays. They were started by the addition of 25 µl of Me(2)SO (controls) or 25 µl of 10 mM BMH dissolved in Me(2)SO (final 0.5 mM). They were stopped after 60 min at 20 °C with 5 µl of 0.5 M 2-mercaptoethanol and left for 10 min at the same temperature. The proteins were then precipitated at 4 °C by the addition of trichloroacetic acid (final 20%) and washed with cold acetone. The samples were redissolved in 60 µl of SDS-containing sample buffer and analyzed by immunoblotting(10) .

Plasmids for Protein Expression in Escherichia coli

The experiments were performed with the STS from Arachis hypogaea (peanut) and the CHS from Sinapis alba (mustard) cloned for functional expression in E. coli. All plasmids were based on expression vector pQE-6 that provides an optimal promoter-translation-start configuration with protein-coding regions containing a NcoI site in the start AUG(16) . The plasmids and their relevant properties are summarized in Table 1. The prefix pQ/ indicates the vector pQE-6, and the letters and numbers following identify the protein. The positions of the mutated amino acids are marked in Fig. 2.



The plasmids pQ/STS (STS from Pinus sylvestris), pQ/STS (STS from A. hypogaea), pQ/CHS (CHS from S. alba), and pQ/CHS/STS(c1an)[2] had been used before(11, 17) . The mutant protein CHS/STS(c1an)[2] contained the STS from A. hypogaea with the N-terminal first 107 amino acids replaced by the corresponding sequence from the S. alba CHS. The protein had no STS or CHS activity, but the CHS part contained all the mutations necessary to obtain STS activity, except for the Q100E exchange that had been shown to be essential(11) . The first 107 amino acids are shown in Fig. 2. The plasmid pQ/CHS (CHS from P. sylvestris) was provided by Sigrid Raiber in our group.

The pQE-6 expression plasmids for several proteins were constructed from cDNAs cloned in the vectors pTZ19R or pKK233-2 and available from previous work: RED (reductase from Glycine max, soybean)(18) , and STS(C169S)[1], STS(H166Q)[3], CHS(C169S)[1](10) . The CHS(Q100E)[2] mutant in vector pTZ18R was constructed by T. Lanz in our laboratory. The mutants STS(C35S), STS(C158S), CHS(S158C), and pQ/CHS(K180Q)[3] were obtained by site-directed mutagenesis with appropriate custom-synthesized oligonucleotides. The mutagenesis was performed with the cDNAs cloned in vector pTZ19R(19) , and single-stranded DNA was obtained with helper phage M13K07 in E. coli strain RZ1032 (20) . The changes were verified by DNA sequence analysis with the dideoxynucleotide chain termination technique(21, 22) .

The recloning of the cDNAs from vector pTZ19R into expression vector pQE-6 had been described in detail(11) . The steps involved in all cases the introduction of an NcoI site into the start AUG by site-directed mutagenesis and the fusion of the protein-coding region start AUG via the NcoI site to the optimal promoter-translation configuration in the vector(16) .

Co-expression of Two Proteins in E. coli

Comparable expression rates are difficult to obtain if different vectors and promoters are used. We therefore provided both coding regions separately with the same strong promoter (from vector plasmid pQE-6) and inserted the two promoter/coding region units in tandem into one vector (pQE-6). The basis for the constructions were the pQE-6-based expression plasmids (Table 1). The cloning steps for pQ/STS[1 + 2] are shown in Fig. 3A, and the construction of pQ/STS[1 + 3] followed the same principle. Fig. 3B summarizes the steps for pQ/CHS[1 + 2]; in this case, we used a polymerase chain reaction for amplification of the promoter/cDNA unit, and the left primer was designed to create a SacI site in the amplified fragment to facilitate the cloning. The construction of pQ/CHS[1 + 3] is shown in Fig. 3C. Several independent E. coli colonies containing the plasmids were analyzed in all cases, and the constructs were verified by restriction analysis before and after the induction of the bacteria for protein expression.


Figure 3: Construction of plasmids for co-expression of two proteins. The plasmids are drawn in the linearized form (digested with one of the restriction enzymes used in the cloning procedure) to facilitate the presentation of the constructions. Thick lines, STS or CHS cDNA; thin lines, vector sequences. The protein-coding regions of the cDNAs (-x->) are labeled corresponding to the designation in the plasmid names. N, NcoI, restriction site for fusion of the cDNA protein-coding regions to the vector promoter (P> ). >Pri and Pri< in B, position of the primers used for fragment amplification with polymerase chain reaction. Only the restriction sites relevant for the cloning are shown. B, BamHI; S, SmaI; Sa, SalI; Sc, SacI; X, XhoI; Xb, XbaI. -, blunt-end sites; *, site destroyed in the resulting plasmids (ligation of XhoI and SalI). The ligation of SmaI with a filled-in XhoI site regenerated a XhoI site.



STS and CHS Enzyme Extracts, Assays, and Quantification

The standard induction of protein expression in E. coli cells was for 3 h at 28 °C(17) . The CHS(K180Q) mutant was poorly soluble under these conditions, and, therefore, the induction conditions were changed to 25 °C and 1.5 h in all experiments involving that mutant and its co-expression with other proteins. The cells were lysed as described(17) , with the modification that the lysozyme concentration during lysis of the E. coli cells was raised to 2 mg/ml, and the supernatants of a centrifugation for 15 min at 15,000 times g were used as enzyme source. The assays (0.1 ml) contained 10 µM 4-coumaroyl-CoA, 15 µM [2-^14C]malonyl-CoA (60,000 cpm; 0.78 GBq/mmol, Amersham), and 50 mM HEPES buffer adjusted to pH 7 with HCl. The protein amounts and incubation times were adjusted for conditions of linear product formation, in particular for the various mutants. The K(m) determinations were performed with 4-coumaroyl-CoA concentrations between 2 and 20 µM. The CHS products in vitro were a mixture of naringenin chalcone and naringenin flavanone, the latter representing the result of a chemical isomerization of the initial chalcone product to the flavanone during the incubations(23) . To facilitate the evaluation by measuring only one product, the residual chalcone was converted to the flavanone by a second incubation at pH 9 for 10 min at 37 °C(24) . The products were separated by TLC with 20% acetic acid as solvent and quantified with a TLC analyzer(10) . The identity of the products had been previously established by high performance liquid chromatography and gas chromatography-mass spectrometry(10) .

The relative enzyme activities were not based on total protein in the extracts because the rate of protein expression varied between the mutants. Therefore, each enzyme extract (soluble proteins after a centrifugation of 15 min at 15,000 times g) was analyzed by immunoblotting with STS or CHS antiserum, and the relative enzyme activity was based on the quantity of the immunodecorated protein(10) .

Solubility of the Cloned Proteins as Measure for the Amount of Correctly Folded Dimers and Misfolded Aggregates

The E. coli lysates were centrifuged for 15 min at 15,000 times g as for preparation of the enzyme extracts. The pellets and supernatants were treated with SDS-containing sample buffer, analyzed by immunoblotting after SDS-gel electrophoretic separation of the proteins, and the percentage of soluble (supernatant) to total immunoreactive protein (supernatant plus pellet) was calculated. Previous comparisons with gel filtration experiments (11) had shown that this evaluation was a useful tool to distinguish between misfolded protein aggregates (insoluble, no enzyme activity) and dimers (soluble, active; monomers were never detected either with CHS or STS). The parent STS or CHS typically yielded values of 50-60%. Like in many other cases (see (25) for review), this reflected that a certain percentage of the proteins expressed in the heterologous host were not folded correctly, but formed insoluble aggregates. Similar techniques have been used by others as diagnostic tools for correct protein folding(26) .

Co-action of CHS with Reductase in the Formation of 6`-Deoxychalcone

The CHS sources were E. coli extracts containing CHS (CHS S. alba) or the heterodimers from CHS(C169S)[1] plus CHS(K180Q)[3] (plasmid pQ/CHS[1 + 3]). The reductase was provided by extracts containing RED (plasmid pQ/RED, Table 1), and the control was an extract from E. coli cells containing the vector pQE-6. The enzyme preparation was carried out as for the standard CHS assays, except that 0.1 M potassium phosphate (pH 7.0) was used instead of HEPES buffer (pH 7.0).

The incubations (0.1 ml) contained the components of the standard CHS assays and, in addition, 0.5 mM NADPH, 10 mM glucose 6-phosphate, and 0.35 unit of glucose-6-phosphate dehydrogenase (NADPH regenerating system, Boehringer Mannheim). The pH 6 required for the co-action of the reductase with CHS (12) was obtained by using 50 mM potassium phosphate (pH 6.0) as buffer. The protein extracts were added in such amounts that less than 25% of the radioactive substrate (malonyl-CoA) was converted into the products (linear assay conditions). The amounts established in a series of trial experiments were 5 µg of protein for CHS, 20 µg for CHS heterodimers from co-expressed subunits, and 20 µg for RED.

The incubations were stopped after 30 min at 30 °C, and the products were extracted into ethyl acetate without prior treatment at pH 9 (no conversion of the chalcone into flavanone, see CHS assay). Naringenin chalcone, naringenin flavanone, and 6`-deoxychalcone were separated by TLC and quantified with a TLC analyzer. The separation of the three substances was achieved by a two-solvent system. The plates were developed for 4 cm with 15% acetic acid, dried, and then further developed in chloroform/glacial acetic acid/H(2)O (10:9:1, v/v/v)(12) . Chalcone synthesis was defined as the sum of radioactivity in naringenin chalcone and flavanone.


RESULTS

Subunit Contact Site Close to the Active Site in STS and CHS

Experiments with the homobifunctional sulfhydryl group cross-linker BMH showed that the subunits of the STS from A. hypogaea were cross-linked by the agent (Fig. 4, lane 1+). The apparent size of the protein (about 120 kDa) was larger than expected from a homodimer of two 43-kDa subunits, but such anomalous migration of cross-linked proteins in denaturing gels was often observed(27, 28) . Aggregation with E. coli protein(s) appeared unlikely because the native enzyme migrated to about 86 kDa in gel filtration experiments, and because the size of the cross-linked protein was the same in several variations of the assay conditions, including NaCl concentrations up to 1 M (not shown).


Figure 4: Immunoblots of STS and CHS after incubations without(-) or with (+) BMH cross-linker. Lanes: 1, STS (STS A. hypogaea); 2, mutant STS(C35S); 3, mutant STS(C158S); 4, CHS (CHS S. alba); 5, mutant CHS(S158C). The sizes of the protein standard markers are indicated at the left side.



No such cross-link was detected with the CHS from S. alba (Fig. 4, lane 4+), and the same negative results were obtained with the CHS and STS from P. sylvestris (not shown). This indicated that a cysteine specific for the STS from A. hypogaea was responsible. The protein contained eight cysteines, but six are conserved in all CHS and STS, and this left the residues in position 35 and 158 as candidates (Fig. 2, only the sequences for the A. hypogaea STS and S. alba CHS are shown because these were used for the experiments). The two cysteines were mutagenized separately to serine, resulting in the mutants STS(C35S) and STS(C158S). Both possessed high enzyme activity (80 ± 5% of the parent protein), indicating that these residues played no essential or important role in the STS reaction. The cross-linking experiments showed that Cys158 was responsible for the subunit linking of the STS, because STS(C35S) revealed the cross-linked protein (Fig. 4, lane 2+), but STS(C158S) did not (Fig. 4, lane 3+).

The six conserved cysteines in the A. hypogaea STS had been mutagenized in previous work(10) , and experiments with those mutants revealed that the subunits were cross-linked by BMH in all cases (not shown). Taken together, these experiments investigated all cysteines in the STS from A. hypogaea. The modification of Cys was the only one leading to the loss of cross-linking, indicating that no other cysteine was involved, and that the two cysteines in this position were cross-linked. We concluded that this amino acid was at or very close to the interface of the two subunits.

The CHS from S. alba contained a serine in position 158 (Fig. 2). It was mutagenized to cysteine (CHS(S158C)) to test whether the positions in STS and CHS were equivalent with respect to the closeness of the subunits or not. The mutant protein was active (90 ± 8% of the unmodified enzyme), and the subunits could now be cross-linked by BMH (Fig. 4, lane 5+). The result indicated that the amino acids in position 158 were at or close to the subunit interface in both STS and CHS.

Other experiments with site-directed mutagenesis (^2)indicated that Lys, a residue strictly conserved in all STS and CHS, was important not only for activity of the enzymes (one example to be discussed later), but also for the structure of both proteins. A direct proof for a role in subunit interaction, however, was not possible because most mutations of that amino acid led to insoluble protein aggregates, and, therefore, no distinction could be made between a role in the initial folding of the subunits or in dimerization. Nevertheless, the close neighborhood of Cys to positions 158 and 180 (Fig. 2) suggested that the two active site pockets in the dimer may be close to each other. One of the intriguing possibilities suggested by this hypothesis was that the two active sites did not operate independently, but cooperated during the three condensing reactions.

Independent or Cooperative Action of the Active Sites in STS and CHS: Models and Experimental Strategy

Fig. 5summarizes the essential points of two alternative models that are based on (i) the basic similarity of the condensing reactions of CHS and STS with those of other polyketide synthases, (ii) the evidence for CHS that no acyl-carrier protein is involved in the reactions(23, 29, 30, 31) , and (iii) the finding that both STS and CHS contain a single cysteine essential for enzyme activity(10) . It should be noted that the models include no assumptions on other subunit interactions in the reaction, e.g. the stabilization of the enzyme-bound intermediates or the ring-folding to stilbenes or chalcones.


Figure 5: Models for independent (1) or cooperative (2) action of the two subunits in the three sequential condensing reactions performed by STS and CHS. Each subunit (A and B) contains one active site (thiol group of Cys). The starter residue (X) or the intermediates with one or two acetate units (1-X or 2-1-X) are bound to the thiol group prior to the condensation with acetate units from malonyl-CoA. 3-2-1-X corresponds to the tetraketide intermediate shown in Fig. 1. The intermediates are not modified in the standard STS or CHS reaction. The only known exception is the biosynthesis of 6`-deoxychalcone that involves a reduction of a specific carbonyl group prior to the CHS ring closure (Fig. 1).



The features common to both models are that the starter group (X, e.g. a 4-coumaroyl-residue) is bound to the active site (thiol group of Cys) prior to the condensing reaction, that malonyl-CoA serves directly as donor of the acetate unit, and that the product (1 - X) is released as CoA-ester. Two repetitions of the steps led to the tetraketide intermediate which is then folded either to a chalcone or a stilbene (see also Fig. 1). Model 1 assumes that each subunit performs all three condensation reactions and that two end products are synthesized per dimer and reaction cycle. Model 2 considers the possibility that the subunits alternate in the condensing reactions and that one product is synthesized per dimer. A close neighborhood of the active sites also permits a model 2 variant (not shown), in which the starter residue is bound to Cys in subunit A and the malonyl residue is transferred to Cys in subunit B prior to the condensing reaction. A role for other cysteines in malonyl residue binding was excluded by the finding that Cys was the only cysteine essential for enzyme activity ( (10) and this work).

The two models could be distinguished with dimers containing only one active site. In that case, model 1 would predict an active enzyme (possibly with about 50% of the parent activity), while Model 2 would predict that such protein was either inactive or would at most release a product containing only one acetate unit added to the 4-coumaroyl residue. Interestingly, a substance synthesized via such single acetate addition (4-hydroxyphenylbutan-2-one) is known in nature(32) , and similar substances were also observed as by-products of CHS reactions under nonoptimal assay conditions(33) .

It has not been possible so far to reassociate STS or CHS subunits to active enzyme, and that precluded a biochemical approach. The techniques of molecular biology, however, allowed a different strategy. It used mutants in the active site cysteine (Cys), co-expression with another mutant, and the functional analysis of the resulting heterodimers containing a single active site.

STS: a Single Active Site Is Sufficient for Enzyme Activity

The first combination tested was the co-expression of the proteins STS(C169S)[1] and CHS/STS(c1an)[2] from plasmid pQ/STS[1 + 2] (Table 1, Fig. 3A). The first was inactive because of the active site mutation, and the second had no activity because it lacked the exchange of glutamine to glutamic acid in position 100 that is essential for STS activity(11) . As measured by the solubility of the proteins (Table 2), both mutants revealed no obvious difficulties in dimer formation and thus were suitable candidates for complementation experiments. Table 2shows that the co-expression led to appreciable STS activity when compared to the parent enzyme STS. This would seem possible only if the subunits formed heterodimers in which the defect in CHS/STS(c1an)[2] was complemented by the correct sequence in STS(C169S)[1]. The heterodimers could have contained only one active site, and the result therefore clearly argued in favor of model 1. It also indicated that the acetate unit was taken directly from malonyl-CoA without prior binding of the residue to the enzyme. The fact of functional complementation moreover indicated that the two subunits cooperated in the formation of the products. No large differences between parent and heterodimer were observed in the K(m) for 4-coumaroyl-CoA (Table 2), suggesting that the affinity to the starter CoA-ester was not greatly influenced by large cooperative effects of the two active sites in the parent enzyme.



The success of the complementation between the amino acids in positions 169 and 100 led us to attempt a complementation between the much closer amino acids in positions 169 and 166 by co-expression of the mutants STS(C169S)[1] and STS(H166Q)[3] from plasmid pQ/STS[1 + 3]. The H166Q protein was a previously used mutant (10, 34) of low STS activity (Table 2); the only difference to the parent enzyme was the exchange of the histidine in position 166 to glutamine (Fig. 2), a residue that is strictly conserved in all CHS. The results of the co-expression revealed the same activities as with STS(H166Q)[3] alone (Table 2), and, therefore, the interpretation was not as unambiguous as in the case discussed before. Nevertheless, the data could be interpreted as complementation, because all other possibilities (no or inactive heterodimers) should have led to a significant reduction of the apparent activity because of the presence of inactive homodimers.

Complementation of CHS Subunits

In view of the similarities and differences in STS and CHS, it was an interesting question whether a complementation of the amino acids in positions 100 and 169 was also possible with CHS. We used in these experiments the plasmid pQ/CHS[1 + 2](Table 1, Fig. 3B), i.e. the co-expression of CHS(C169S)[1] (active site destroyed) and CHS(Q100E)[2]. The latter carried the exchange of Gln to Glu as only mutation, and the protein possessed about 14% of the parent activity. The co-expression led to a relative activity of about 28% (Table 2), based on the amount of total immunodecorated protein. Taking into account the presence of the inactive (CHS(C169S)[1]) and the partially active (CHS(Q100E)[2]) homodimers, the increase in relative activity after co-expression indicated heterodimer formation and functional complementation.

This conclusion also implied that a single active site in the heterodimers was sufficient for CHS activity, but other explanations were not rigorously excluded because of the residual activity of CHS(Q100E)[2]. We investigated that point by co-expression of CHS(C169S)[1] and CHS(K180Q)[3] from plasmid pQ/CHS[1 + 3] (Table 2, Fig. 3C). As noted before, mutations of Lys led in most cases to insoluble protein aggregates. The K180Q exchange was one of the few resulting in an appreciable amount of soluble protein (Table 2), but that required the lowering of the temperature from 28 °C to 25 °C during protein expression. The mutant revealed no significant enzyme activity. Table 2shows that the co-expression of the two inactive proteins led to CHS activity, indicating not only that the defect in position 180 was complemented by the other subunit in the heterodimers, but also that a single active site in a dimer was sufficient for CHS activity. Like with STS, the K(m) for 4-coumaroyl-CoA revealed no significant differences between heterodimer and parent (Table 2).

Co-action of Reductase and CHS in 6`-Deoxychalcone Formation: No Plant Factor Is Required, and a Single Active Site in CHS Is Sufficient to Synthesize Two Products

A specific, NADPH-dependent, monomeric reductase can co-act with CHS to produce 6`-deoxychalcone (Fig. 1), and this is so far the only known case that an intermediate of the CHS reaction is modified prior to formation of the new ring system. Although the basics of that co-action had been elucidated(12) , there were several aspects that remained puzzling. One was that the 6`-deoxychalcone represented at most 35-50% of the products; the other product was always the nonreduced chalcone synthesized in the typical CHS reaction. That was not only observed with crude extracts (13, 14, 35) , but in the most extensively investigated soybean system also with purified enzymes and an excess of reductase(12) . One of the reasons could be an as yet undiscovered plant factor not present in sufficient quantities in vitro. Another possibility was that only certain CHS variants could co-act with reductase (both enzymes are encoded in gene families for slightly different proteins(18, 36, 37, 38, 39, 40, 41) ). Finally, it was possible that the monomeric reductase interacted for sterical reasons with CHS in such way that it could reduce only one of the two intermediates synthesized by the two CHS subunits.

We addressed these points in two types of experiments. (i) We employed cloned proteins both for CHS and the reductase, thus eliminating the heterogeneities in both enzymes and also the possible effects of other plant factors; these experiments took advantage of the fact that the reductase co-acts with CHS from all plants tested so far ((12) )(^3); (ii) we used a CHS heterodimer that possessed only one active site und therefore could produce only one product per reaction cycle.

The data from representative experiments are summarized in Table 3. The soybean reductase (RED) and the unmodified S. alba CHS (CHS) co-acted in the formation of 6`-deoxychalcone, but the nonreduced chalcone was also produced. This showed for the first time that no other plant factor was necessary for the formation of the reduced chalcone, and that a single CHS species synthesized both products. The experiments with the CHS heterodimer possessing a single active site were performed with pQ/CHS[1 + 3]. The co-incubation with the reductase also led to the formation of both products, and the ratio was not largely different from that observed with the unmodified CHS. A percentage of more than 35-45% 6`-deoxychalcone was also never obtained in the experiments that investigated the optimal ratios between reductase and CHS or its heterodimer. Those incubations tested widely different ratios of the two enzymes, including a large excess of reductase.




DISCUSSION

Polyketide synthases are usually complexes of several different subunits or large proteins with domains for the different functions(1, 2) . STS and CHS appear to be exceptional because the complex reactions are performed by a single protein consisting of two comparably small, identical subunits, and the lack of protein sequence homology with other polyketide synthases indicated that they represent independent developments(10) . The results presented in this work demonstrated that each subunit was capable of performing all three condensation steps, and they provided the first definite evidence that malonyl-CoA was the direct donor of the acetate units. The complementation of defective subunits in the heterodimers indicated that the subunits cooperate in the formation of the products, but it is not possible yet to distinguish whether this is in stabilization of the enzyme-bound intermediates or in the ring closure or both.

The relative activities obtained by the co-expression of inactive subunits were 22 ± 6% (STS[1 + 2]) and 15 ± 3% (CHS[1 + 3]) when compared with the fully active enzymes (Table 2). The determinations were based on the amount of immunoreactive protein in Western blots, and, in the co-expression experiments, these included the inactive homodimers. The mutants were expressed from identical promoter-translation start configurations (Fig. 3A). It therefore seems a reasonable assumption that they were synthesized at comparable rates and available at about equimolar concentrations for dimerization. Assuming no strong preference for either hetero- or homodimer formation, the ratio of AA + BB and AB + BA dimers could have been about 1:1, suggesting a correction factor of 2 for the specific activity of the heterodimer. This would increase the activity in the (STS[1 + 2]) co-expression to about 44% of the parent enzyme, and that came reasonably close to the 50% expected for a dimer possessing only one active site, but otherwise being fully functional. The values obtained with (CHS[1 + 3]) were lower, and that was likely a consequence of the reduced capability of the CHS(K180Q) mutant to form dimers (Table 2).

No basic differences between STS and CHS were detected in all of these experiments, and it was therefore an interesting question whether it would be possible to create a CHSbulletSTS heterodimer functional in both activities. This was tested by co-expression of CHS(K180Q) and CHS/STS(c1an) carrying an additional C169S mutation, but no activity was detectable. A second attempt by co-expression of CHS/STS(c1an) with CHS(Q100E) showed the residual CHS activity from the mutated CHS, but no stilbene formation was observed. These experiments covered only a few combinations, but it may well be possible that the protein conformations guiding the ring closures to stilbenes and chalcones are incompatible either with heterodimer formation or with the enzymic function of such a dimer, in particular if the ring closure requires a cooperation of the subunits. A mutual exclusion of CHS and STS activity also seems to be suggested by previous results from a large number of other mutagenesis experiments that failed to detect a protein with both activities(11) .

The mechanisms of the complex interaction between the dimeric CHS and the monomeric reductase remain unexplained. The results with the CHS heterodimer containing only one active site offered no support for the hypothesis that the reductase reduced only one of the two intermediates synthesized by dimeric CHS, because in that case one might have expected that the 6`-deoxychalcone would represent a more dominant product than with the parent CHS. The formation of two products may be an intrinsic property of the interaction, and a more detailed analysis would require the three-dimensional protein structures.


FOOTNOTES

*
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 206 and by 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 by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 49-761-203-2691; Fax: 49-761-203-2601; jschroe{at}sun2.ruf.uni-freiburg.de.

(^1)
The abbreviations used are: STS, stilbene synthase(s); 6`-deoxychalcone, 4,2`,4`-trihydroxychalcone; BMH, bismaleimidohexane; chalcone, 4,2`,4`,6`-tetrahydroxychalcone; CHS, chalcone synthase(s); RED, reductase co-acting with CHS in 6`-deoxychalcone formation.

(^2)
S. Tropf, unpublished work.

(^3)
B. Kärcher, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to S. Raiber from our group for providing the pQE-6 plasmids for expression of the CHS and the STS from P. sylvestris and to T. Lanz for construction of the mutant CHS(Q100E).


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