Eckermann, C., Matthes, B., Nimtz, M., Reiser, V., Lederer, B., Böger, P., and Schröder, J.: Covalent binding of chloroacetamide herbicides to the active site cysteine of plant type III polyketide synthases. Phytochemistry 64, 1045-1054 (2003a)
   Chloroacetamide herbicides inhibit very-long-chain fatty acid elongase, and it has been suggested that covalent binding to the active site cysteine of the condensing enzyme is responsible (Böger et al., 2000, Pest Management Science 56, 497-508), but direct evidence was not available. The proposal implied that other condensing enzymes might also be targets, and therefore we have investigated four purified recombinant type III plant polyketide synthases. Chalcone synthase (CHS) revealed a high sensitivity to the chloroacetamide metazachlor, with 50% inhibition after a 10 min pre-incubation with 1-2 molecules per enzyme subunit, and the inactivation was irreversible. Stilbene synthase (STS) inactivation required 20-fold higher amounts, and 4-coumaroyltriacetic acid synthase and pyrone synthase revealed no response at the highest metazachlor concentrations tested. A similar spectrum of differential responses was detected with other herbicides that also inhibit fatty acid elongase (metolachlor and cafenstrole). The data indicate that type III polyketide synthases are potential targets of these herbicides, but each combination has to be investigated individually. The interaction of metazachlor with CHS was investigated by mass spectrometric peptide mapping, after incubation of the enzymes with the herbicides followed by tryptic digestion. A characteristic mass shift and MS/MS sequencing of the respective peptide showed that metazachlor was covalently bound to the cysteine of the active site, and the same was found with STS. This is the first direct evidence that the active site cysteine in condensing enzymes is the primary common target of these herbicides.
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Eckermann, C., Schröder, G., Eckermann, S., Strack, D., Schmidt, J., Schneider, B., and Schröder, J.: Stilbenecarboxylate biosynthesis: a new function in the family of chalcone synthase-related proteins. Phytochemistry 62, 271-286 (2003b).
   Chalcone (CHS), stilbene (STS) synthases, and related proteins are key enzymes in the biosynthesis of many secondary plant products. Precursor feeding studies and mechanistic rationalization suggest that stilbenecarboxylates might also be synthesized by plant type III polyketide synthases; however, the enzyme activity leading to retention of the carboxyl moiety in a stilbene backbone has not yet been demonstrated. Hydrangea macrophylla L. (Garden Hortensia) contains stilbenecarboxylates (hydrangeic acid and lunularic acid) that are derived from 4-coumaroyl and dihydro-4-coumaroyl starter residues, respectively. We used homology-based techniques to clone CHS-related sequences, and the enzyme functions were investigated with recombinant proteins. Sequences for two proteins were obtained. One was identified as CHS. The other shared 65-70% identity with CHSs and other family members. The purified recombinant protein had stilbenecarboxylate synthase (STCS) activity with dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA or other substrates. We propose that the enzyme is involved in the biosynthesis of lunularic acid. It is the first example of a STS-type reaction that does not lose the terminal carboxyl group during the ring folding to the end product. Comparisons with CHS, STS, and a pyrone synthase showed that it is the only enzyme exerting a tight control over decarboxylation reactions. The protein contains unusual residues in positions highly conserved in other CHS-related proteins, and mutagenesis studies suggest that they are important for the structure or/and the catalytic activity. The formation of the natural products in vivo requires a reducing step, and we discuss the possibility that the absence of a reductase in the in vitro reactions may be responsible for the failure to obtain stilbenecarboxylates from substrates like 4-coumaroyl-CoA.
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Pfeifer, V., Nicholson, G.J., Ries, J., Recktenwald, J., Schefer, A.B., Shawky, R.M., Schröder, J., Wohlleben, W. and Pelzer, S.: A polyketide synthase in glycopeptide biosynthesis: the biosynthesis of the non-proteinogenic amino acid (S)-3,5-dihydroxyphenylglycine. Journal of Biological Chemistry 276, 38370-38377 (2001).
     Balhimycin, a vancomycin-type antibiotic from Amycolatopsis mediterranei contains the unusual amino acid (S)-3,5-dihydroxyphenylglycine (Dpg), with an acetate derived carbon backbone. After sequence analysis of the biosynthetic gene cluster one gene dpgA for a predicted polyketide synthase (PKS) was identified, sharing 20-30% identity with plant chalcone synthases. Inactivation of dpgA resulted in loss of balhimycin production, and restoration was achieved by supplementation with 3,5-dihydroxyphenylacetic acid, which is both a possible product of a PKS reaction and a likely precursor of Dpg. Enzyme assays with the protein expressed in Streptomyces lividans showed that this PKS uses only malonyl-CoA as substrate to synthesize 3,5-dihydroxyphenylacetic acid. The PKS gene is organized in an operon like structure with three downstream genes which are similar to enoyl-CoA-hydratase genes and a dehydrogenase gene. The heterologous co-expression of all four genes led to accumulation of 3,5-dihydroxyphenylglyoxylic acid. Therefore, we now propose a reaction sequence. The final step in the pathway to Dpg is a transamination. A predicted transaminase gene was inactivated, resulting in abolished antibiotic production and accumulation of 3,5-dihydroxyphenylglyoxylic acid. Interestingly, restoration was only possible by simultaneous supplementation with (S)-3,5-dihydroxyphenylglycine and (S)-4-hydroxyphenylglycine, indicating that the transaminase is essential for the formation of both amino acids.
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Homepage of Wolfgang Wohlleben 

Morita, H., Noguchi, H., Schröder, J., and Abe, I.: Novel polyketides synthesized with a higher plant stilbene synthase. European Journal of Biochemistry 268, 3759-3766 (2001).
   The physiological function of the stilbene synthase (STS) from groundnut (Arachis hypogaea) is the formation of resveratrol. The enzyme uses 4-coumaroyl-CoA, performs three condensations with malonyl-CoA, and folds the resulting tetraketide into a new aromatic ring system. We investigated the capacity to build novel and unusual polyketides from alternative substrates. Three types of products were obtained: (A) complete reaction (stilbene-type), (B) three condensations without formation of aromatic ring (CTAL-type pyrone derailment), (C) two condensations (BNY-type pyrone derailment). All product types were obtained from 4-fluorocinnamoyl-CoA and analogs in which the coumaroyl moiety was replaced by furan or thiophene. Only type (B) and (C) products were synthesized from other 4-substituted 4-coumaroyl-CoA analogs (-Cl, -Br, -OCH₃). Benzoyl-CoA, phenylacetyl-CoA, and medium chain aliphatic CoA-esters were poor substrates, and the majority of the products was of type (C). The results show that minor modifications can be used to direct the enzyme reaction to form a variety of different and new products. Manipulation of the biosynthesis of polyketides by synthetic analogs could lead to development of a chemical library of pharmaceutically interesting novel polyketides.
-> Similar experiments were previously carried out with chalcone synthase. Take a look at the Japanese homepage (the English version, in case you are not fluent in Japanese):
Homepage of Ikuro Abe
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Zheng, D., Schröder, G., Schröder, J., and Hrazdina, G.: Molecular and biochemical characterization of three aromatic polyketide synthase genes from Rubus idaeus. Plant Mol. Biol. 46, 1-15 (2001).
    Three polyketide synthase (PKS) genes from cell suspension cultures of raspberry (Rubus idaeus L. cv Royalty) were characterized. They showed high similarity in both their nucleotide and deduced amino acid sequences. All three proteins contain the amino acid residues identified in previous work as essential for chalcone synthase (CHS) function. Enzyme activities were investigated after heterologous expression in E. coli. RiPKS1 is a typical naringenin CHS that synthesizes the chalcone as the main reaction product, and p-coumaryltriacetic acid lactone (CTAL) as a minor by-product. RiPKS3 differed from RiPKS1 in four positions (K49R, M64R, P120L, V188A), and the products in vitro were predominantly CTAL and low amounts of chalcone. RiPKS2 had the same four differences to RiPKS1 as RiPKS3, but in addition two further exchanges (R259H, F344L), and the protein had no detectable enzyme activity. Experiments with RiPKS1 containing either 259H or 344L showed that each of the exchanges was sufficient to completely eliminate enzyme activity. These experiments identify amino acid residues in CHS which are important for folding of the tetraketide intermediate to the chalcone (PKS3) and which are in general essential for CHS activity (PKS2). The possible functions of these residues are discussed.
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Jez, J.M., Austin, M.B., Ferrer, J.-L., Bowman, M.E., Schröder, J. and Noel, J.P.: Structural control of polyketide formation in plant-specific polyketide synthases. Chemistry &  Biology 7, 919-930 (2000).
Background: Polyketide synthases (PKSs) generate molecular diversity by utilizing different starter molecules and by controlling the final length of the polyketide. Although exploitation of this mechanistic variability has produced novel polyketides, the structural foundation of this versatility is unclear. Plant-specific PKSs are essential for the biosynthesis of anti-microbial phytoalexins, anthocyanin pigments, and inducers of Rhizobium nodulation genes. 2-Pyrone synthase (2-PS) and chalcone synthase (CHS) are plant-specific PKSs that exhibit 74% amino acid identity. 2-PS forms the triketide methylpyrone from an acetyl-CoA starter molecule and two malonyl-CoAs. CHS forms the tetraketide chalcone using a p-coumaroyl-CoA starter molecule and three malonyl-CoAs. Our goal was to elucidate the molecular basis of starter molecule selectivity and control of polyketide length in this class of PKS.
Results: The 2.05 Å resolution crystal structure of 2-PS complexed with the reaction intermediate acetoacetyl-CoA was determined by molecular replacement. 2-PS and CHS share a common three-dimensional fold, a set of conserved catalytic residues, and similar CoA binding sites. However, the active site cavity in 2-PS is approximately one-third the size of that in CHS. Of the twenty-eight residues lining the 2-PS initiation/elongation cavity, four positions are different in CHS. Mutations at three of these positions in CHS (T197L, G256L, and S338I) each altered product formation. Generation of a CHS triple mutant (T197L/G256L/S338I) yielded an enzyme that was functionally identical to 2-PS.
Conclusions: Structural and functional characterization of 2-PS together with generation of a CHS mutant with an initiation/elongation cavity analogous to 2-PS demonstrates that cavity volume governs the choice of starter molecule and controls the final length of the polyketide. These results provide a structural basis for control of polyketide length in other PKSs, and suggest strategies for further increasing the scope of polyketide biosynthetic diversity.
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Home page of Joseph Noel: It is worth a visit!! 

Schröder, J.: The family of chalcone synthase-related proteins: functional diversity and evolution. Recent Advances in Phytochemistry 34, 55-89 (2000).
   (Literature up to November 1999)
   Results in the last few years showed that the well-known chalcone synthase (CHS) is only one example from a family of plant polyketide synthases. Other members of the family which are identified by function and sequences are the stilbene synthases (STS), acridone synthase (ACS), and a pyrone synthase (2PS); all of these proteins share about 65-70% identity with CHS. The properties of several other enzymes suggest that they are members of the protein family, and precursor feeding studies suggest that the number may be much larger than suspected so far. The diversity of functions is based on different substrate specificities, variations in the number of condensation reactions, folding of intermediates to different products, and modification of intermediates by other enzymes.
The recently published first crystal structure of a CHS raises hopes that it will be possible to understand at the protein sequence level the programming of the proteins for the various functions; this then will facilitate the design of enzymes synthesizing new products.
The understanding of the evolution of the protein family is still rudimentary. The available data suggest that the functional diversity known in present-day plants could be the results of fairly recent developments from CHS by gene duplication and mutation. The presence of CHS-related sequences in bacteria indicates that the basic function unit predated the evolution of plants. The recent functional identification of such a protein from Streptomyces griseus suggests that the functional diversity in bacteria may even be larger than in plants.
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Schröder, J.: The chalcone/stilbene synthase-type family of condensing enzymes.  In:  Comprehensive Natural Products Chemistry, Vol. 1: Polyketides and Other Secondary Metabolites Including Fatty Acids and Their Derivatives (ed. Sankawa, U.), Elsevier Science, Oxford, pp. 749-771 (1999).
A review covering the literature up to March 1997. The review also makes some predictions for enzymes likely to be members of this protein superfamily.
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Schröder, J.: Probing plant polyketide synthesis. Nature Structural Biology  6: 714-716 (1999).
    This is a short "News and Views" comment for the paper by the Noel group on the crystal structure of CHS. It discusses some of the functions of enzymes in the protein family and the prospects for the rational design of new and useful enzymes.
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Lukacin, R., Springob, K., Urbanke, C., Ernwein, C., Schröder, G., Schröder, J., Matern, U.: Native acridone synthases I and II from Ruta graveolens L. form  homodimers. FEBS Letters 448, 135-140 (1999).
    Acridone synthase II cDNA was cloned from irradiated cell suspension cultures of Ruta graveolens L. and expressed in Escherichia coli. The translated polypeptide of M_r 42681 revealed a high degree of similarity to heterologous chalcone and stilbene synthases (70-75%), and the sequence was 94% identical to that of acridone synthase I cloned previously from elicited Ruta cells. Highly active recombinant acridone synthases I and II were purified to apparent homogeneity by a four-step purification protocol, and the affinities to N-methylanthraniloyl-CoA and malonyl-CoA were determined. The molecular mass of acridone synthase II was estimated from size exclusion chromatography on a Fractogel EMD BioSEC (S) column at about 45 kDa, as compared to a mass of 44 +/- 3 kDa found for the acridone synthase I on Superdex 75. Nevertheless, the sedimentation analysis by ultracentrifugation revealed molecular masses of 81+/-4 kDa for both acridone synthases. It is proposed, therefore, that the acridone synthases of Ruta graveolens are typical homodimeric plant polyketide synthases.
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Eckermann, S., Schröder, G., Schmidt, J., Strack, D., Edrada, R.A., Helariutta, Y., Elomaa, P., Kotilainen, M., Kilpeläinen, I., Proksch, P., Teeri, T.H. and Schröder, J.: New pathway to polyketides in plants. Nature (London) 396, 387-390 (1998).
    The repertoire of secondary metabolism (involving the production of compounds not essential for growth) in the plant kingdom is enormous, but the genetic and functional basis for this diversity is hard to analyse as many of the biosynthetic enzymes are unknown. We have now identified a key enzyme in the ornamental plant Gerbera hybrida (Asteraceae) that participates in the biosynthesis of compounds that contribute to insect and pathogen resistance. Plants transformed with an antisense construct of gchs2, a complementary DNA encoding a previously unknown function, completely lack the pyrone derivatives gerberin and parasorboside. The recombinant plant protein catalyses the principal reaction in the biosynthesis of these derivatives: GCHS2 is a polyketide synthase that uses acetyl-CoA and two condensation reactions with malonyl-CoA to form the pyrone backbone of the natural products. The enzyme also accepts benzoyl-CoA to synthesize the backbone of substances that have become of interest as inhibitors of the HIV-1 protease. GCHS2 is related to chalcone synthase (CHS) and its properties define a new class of function in the protein superfamily. It appears that CHS-related enzymes are involved in the biosynthesis of a much larger range of plant products than was previously realized.
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Schröder, J., Raiber, S., Berger, T., Schmidt, A., Schmidt, J., Soares-Sello, A.M., Bardshiri, E., Strack, D., Simpson, T.J., Veit, M. and Schröder, G.: Plant polyketide synthases: a chalcone synthase-type enzyme which performs a condensation reaction with methylmalonyl-CoA in the biosynthesis of C-methylated chalcones. Biochemistry 37, 8417-8425 (1998).
    Heterologous screening of a cDNA library from Pinus strobus seedlings identified clones for two chalcone synthase (CHS) related proteins (PStrCHS1 and PStrCHS2, 87.6% identity). Heterologous expression in Escherichia coli showed that PStrCHS1 performed the typical CHS reaction, that it used starter CoA-esters from the phenylpropanoid pathway, and that it performed three condensation reactions with malonyl-CoA, followed by the ring closure to the chalcone. PstrCHS2 was completely inactive with these starters and also with linear CoA-esters. Activity was detected only with a diketide derivative (N-acetylcysteamine thioester of 3-oxo-5-phenylpent-4-enoic acid) that corresponded to the CHS reaction intermediate postulated after the first condensation reaction. PstrCHS2 performed only one condensation, with 6-styryl-4-hydroxy-2-pyrone derivatives as release products. The enzyme preferred methylmalonyl-CoA against malonyl-CoA, if only methylmalonyl-CoA was available. These properties and a comparison with the CHS from Pinus sylvestris suggested for PstrCHS2 a special function in the biosynthesis of secondary products. In contrast to P. sylvestris, P. strobus contains C-methylated chalcone derivatives, and the methyl group is at the position predicted from a chain extension with methylmalonyl-CoA in the second condensation of the biosynthetic reaction sequence. We propose that PstrCHS2 specifically contributes the condensing reaction with methylmalonyl-CoA to yield a methylated triketide intermediate. We discuss a model that the biosynthesis of C-methylated chalcones represents the simplest example of a modular polyketide synthase.
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Zuurbier, K.W.M., Leser, J., Berger, T., Hofte, A.J.P., Schröder, G., Verpoorte, R. and Schröder, J.: 4-Hydroxy-2-pyrone formation by chalcone and stilbene synthase with nonphysiological substrates. Phytochemistry 49, 1945-1951 (1998).
    Valerophenone synthase (VPS) is a polyketide synthase that catalyzes the formation of the phloroglucinol derivatives in the synthesis of the bitter acids in hop (Humulus lupulus). The reaction uses isovaleryl-CoA or isobutyryl-CoA, but otherwise it is identical to that of the chalcone synthase in flavonoid biosynthesis. Our study showed that chalcone synthase can perform the function of VPS, but not perfectly, because the majority of the reactions terminated after two condensation reactions (products: 4-hydroxy-2-pyrone derivatives). The same experiments with stilbene synthase yielded exclusively the 4-hydroxy-2-pyrone derivatives, not the products expected from three condensation reactions. The results are discussed in the context of the functional diversity and evolution in the family of CHS-related polyketide synthases.
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Christensen, A.B., Gregersen, P.L., Schröder, J. and Collinge, D.B.: A chalcone synthase with an unusual substrate preference is expressed in barley leaves in response to UV-light and pathogen attack. Plant Mol. Biol. 37, 849-857(1998).
    A cDNA clone was isolated by differential hybridization from a library prepared from barley leaves inoculated with the fungus Blumeria graminis f.sp. hordei (Bgh). The open reading frame of the insert (designated HvCHS2) encoded a polypeptide with 72-79% identity to chalcone synthases (CHS) and 65-68% identity to stilbene synthases. Alignments of the amino acid sequence of HvCHS2 with the consensus sequence of naringenin-CHS (EC 2.3.1.74) reveals significant differences between HvCHS2 and naringenin-CHS. HvCHS2 transcripts accumulate strongly in barley leaves in response to inoculation with Bgh, whereas only insignificant accumulation of barley naringenin-CHS (CHS1) transcripts is seen upon the inoculation. The accumulation of HvCHS2 transcripts is also elicited by UV light. To compare the activity of HvCHS2 with the activity of CHS1, the two enzymes were expressed in Escherichia coli. Both HvCHS2 and CHS1 catalyse the formation of chalcones. However, HvCHS2 and CHS1 differ in their substrate requirements. CHS1 uses cinnamoyl-CoA and 4-coumaroyl-CoA at comparable rates whereas feruloyl-CoA is a poor substrate for this enzyme. In contrast, HvCHS2 converts feruloyl- CoA and caffeoyl-CoA at the highest rates whereas cinnamoyl-CoA is a poor substrate. Thus, HvCHS2 is a novel pathogen and UV light induced homoeriodictyol/eriodictyol CHS involved in the direct production of flavonoids possessing multi-substituted B-rings.
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Schröder J.: A family of plant-specific polyketide synthases: facts and predictions. Trends Plant Sci. 2: 373-378 (1997).
    The enzymes synthesizing chalcones, stilbenes, and acridones are closely related plant-specific polyketide synthases. Recent results suggest that they belong to a family of condensing enzymes that catalyze the initial key reactions in the biosynthesis of several biologically and pharmaceutically interesting substances. The product range is even more extended by modification of reaction intermediates. Recent analysis has revealed that related sequences occur in bacteria, suggesting that the protein family is much older than previously assumed. The publication also makes some predictions for reactions in which CHS-type proteins may be involved.
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Helariutta, Y., Elomaa, P., Kotilainen, M., Griesbach, R.J., Schröder, J. and Teeri, T.H.: Chalcone synthase-like genes active during corolla development are differentially expressed and encode enzymes with different catalytic properties in Gerbera hybrida (Asteraceae). Plant Mol. Biol. 28, 47-60 (1995).
    Recent studies on chalcone synthase (CHS) and the related stilbene synthase (STS) suggest that the structure of chs-like genes in plants has evolved into different forms, whose members have both different regulation and capacity to code for different but related enzymatic activities. We have studied the diversity of chs-like genes by analysing the structure, expression patterns and catalytic properties of the corresponding enzymes of three genes that are active during corolla development in Gerbera hybrida. The expression patterns demonstrate that chs-like genes are representatives of three distinct genetic programmes that are active during organ differentiation in Gerbera. Gchs1 and gchs3 code for typical CHS enzymes, and their gene expression pattern temporally correlates with flavonol (gchs1, gchs3) and anthocyanin (gchs1) synthesis during corolla development. Gchs2 is different. The expression pattern does not correlate with the pigmentation pattern, the amino acid sequence deviates considerably from the consensus of typical CHSs, and the catalytic properties are different. The data indicate that it represents a new member in the large superfamily of CHS and CHS-related genes.
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Raiber, S., Schröder, G. and Schröder, J.: Molecular and enzymatic characterization of two stilbene synthases from Eastern white pine (Pinus strobus): a single Arg/His difference determines the activity and the pH dependence of the enzymes. FEBS Letters 361, 299-302 (1995).
    Pinus strobus (Eastern white pine) contains stilbenes biosynthetically derived from cinnamoyl-CoA (pinosylvin) or dihydrocinnamoyl-CoA (dihydropinosylvin). We screened a P. strobus cDNA library with a stilbene synthase (STS) probe from Pinus sylvestris. The eight isolated cDNAs represented two closely related STS genes with five amino acid differences in the proteins. The enzyme properties were investigated after heterologous expression in Escherichia coli. Both proteins preferred cinnamoyl-CoA against dihydrocinnamoyl-CoA and thus represented pinosylvin synthases. Otherwise they revealed large differences. STS1 had only 3-5% of the activity of STS2, its pH optimum was shifted to lower values (pH 6), and it synthesized with cinnamoyl-CoA a second unknown product. Site-directed mutagenesis demonstrated that a single Arg-to-His exchange in STS1 was responsible for all of the differences. The proton acceptor properties of His are discussed as the reason for the properties of STS1.
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Tropf, S., Kärcher, B., Schröder, G. and Schröder, J.: 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. Journal of  Biological Chemistry 270, 7922-7928 (1995).
    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-169). This suggested that the active site pockets may be neighboring, possibly alternating in the condensing reactions rather than acting independently. This was investigated by coexpression 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 E. 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.
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Tropf, S., Lanz, T., Rensing, S.A., Schröder, J. and Schröder, G.: Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution. Journal of Molecular Evolution 38, 610-618 (1994).
    Chalcone (CHS) and stilbene (STS) synthases are related plant- specific polyketide synthases that are key enzymes in the biosynthesis of flavonoids and of stilbene phytoalexins, respectively. A phylogenetic tree constructed from 34 CHS and four STS sequences revealed that the STS formed no separate cluster but grouped with CHS from the same or related plants. This suggested that STS evolved from CHS several times independently. We attempted to simulate this by site-directed mutagenesis of an interfamily CHS/STS hybrid, which contained 107 amino acids of a CHS from Sinapis alba (N-terminal) and 287 amino acids of a STS from Arachis hypogaea. The hybrid had no enzyme activity. Three amino acid exchanges in the CHS part (Gln-100 to Glu, Val-103 to Met, Val-105 to Arg) were sufficient to obtain low STS activity, and one additional exchange (Gly-23 to Thr) resulted in 20-25% of the parent STS activity. A kinetic analysis indicated (1) that the hybrids had the same K_m for the substrate 4-coumaroyl-CoA but a lower V_max than the parent STS, and (2) that they had a different substrate preference than the parent STS and CHS. Most of the other mutations and their combinations led to enzymatically inactive protein aggregates, suggesting that the subunit folding and/or the dimerization was disturbed. We propose that STS evolved from CHS by a limited number of amino acid exchanges, and that the advantage gained by this enzyme function favored the selection of plants with improved STS activity.
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Schanz, S., Schröder, G. and Schröder, J.: Stilbene synthase from Scots pine (Pinus sylvestris).  FEBS Letters 313, 71-74 (1992).
    Stilbene synthases are named according to their substrate preferences. By this definition, enzymes preferring cinnamoyl-CoA are pinosylvin synthases, and proteins with a preference for phenylpropionyl-CoA are dihydropinosylvin synthases. We investigated the assignment of a stilbene synthase cloned from Scots pine (Pinus sylvestris) as dihydropinosylvin synthase and the proposal of an additional pinosylvin synthase (1992, Plant Mol. Biol. 18, 489-503). The results show that the previous interpretation was misled by several unexpected factors. Firstly, we found that the substrate preference and the activity of the plant-specific protein expressed in Escherichia coli was influenced by bacterial factors. This was reduced by improvement of the expression system, and the subsequent kinetic analysis revealed that cinnamoyl-CoA rather than phenylpropionyl-CoA is the preferred substrate of the cloned stilbene synthase. Secondly, mixing experiments showed that extracts from P. sylvestris contain factor(s) which selectively influenced the substrate preference, i.e. the activity was reduced with phenylpropionyl- CoA, but not with cinnamoyl-CoA. This explained the apparent differences between plant extracts and the cloned enzyme expressed in E. coli. Taken together, the results indicate that the cloned enzyme is a pinosylvin synthase, and there is no evidence for a second stilbene synthase. This study cautions that factors in the natural and in new hosts may complicate the functional identification of cloned sequences.
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Fliegmann, J., Schröder, G., Schanz, S., Britsch, L. and Schröder, J.: Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants. Plant Mol. Biol. 18, 489-503 (1992).
    Chalcone synthase (CHS) and stilbene synthase (STS) are closely related polyketide synthases which are key enzymes in the biosynthesis of flavonoids and stilbenes. Scots pine (Pinus sylvestris) is an interesting plant for a direct comparison of the enzymes. It not only contains the usual flavonoids, but also an unusual chalcone derivative (pinocembrin), and it synthesizes stilbenes of the pinosylvin type. We analysed a CHS and a STS by molecular cloning and functional expression in Escherichia coli. The CHS was active not only with 4-coumaroyl-CoA (to naringenin chalcone), but also with cinnamoyl-CoA (leading to pinocembrin). The STS was identified as dihydropinosylvin synthase, because it preferred dihydrocinnamoyl-CoA to cinnamoyl-CoA. The protein deviated in 47 positions from the CHS consensus. It had 73.2% identity with the CHS from P. sylvestris and only 65.3% with a STS from peanut (Arachis hypogaea). We also investigated the regulation of both enzyme types in P. sylvestris plantlets exposed to stress. CHS was present in non-stressed plantlets, and induction led to a transient increase with a peak after 16 h. STS1 type activities were regulated differently and were absent in non-stressed plantlets. Increases were observed after a lag period of at least 6 h, and highest activities were obtained after 30 h. The analysis of the reactions in the plant extracts and the substrate specificity of the cloned STS suggest that the plants contain at least two different types of STS: the cloned dihydropinosylvin synthase and a pinosylvin synthase which preferentially utilizes cinnamoyl-CoA as substrate.
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Schröder, G. and Schröder, J.: A single change of histidine to glutamine alters the substrate preferences of a stilbene synthase. Journal of Biological Chemistry 267, 20558-20560 (1992).
    Stilbene and chalcone synthases are related polyketide synthases which use the same substrates but from different products. The environment of the condensing active site cysteine is highly conserved, except for the positions -2 and -3. All chalcone synthases contain Gln-Gln and prefer 4-coumaroyl-CoA as starter CoA ester, while the two known stilbene synthases contain Gln-His or His-Gln (preference phenylpropionyl-CoA and 4-coumaroyl-CoA, respectively). We investigated whether the presence and/or position of the histidine influences the substrate preference and the product specificity (stilbene or chalcone). The two amino acid motifs in the chalcone synthase from Pinus sylvestris (Gln-Gln) and in the stilbene synthases from P. sylvestris (Gln-His) and Arachis hypogaea (His-Gln) were changed by site-directed mutagenesis into all sequence combinations as found in the natural enzymes. Assays with the mutant proteins showed that the histidine does not determine the product specificity. With the chalcone and the stilbene synthase from P. sylvestris, any sequence deviation reduced the activity without marked effects on the substrate preference. The stilbene synthase from A. hypogaea was different. The change from His-Gln to Gln-His abolished enzyme activity almost completely with all three substrates. The change to Gln-Gln selectively reduced the activity with 4-coumaroyl-CoA, and the kinetic analysis indicated a slight increase in K_m and a 3-fold reduction of V_max, when compared with the parent enzyme. This converted the enzyme from a resveratrol-forming into a dihydropinosylvin-forming stilbene synthase.
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Welle, R. and Schröder, J.: Expression cloning in Escherichia coli and preparative isolation of the reductase coacting with chalcone synthase during the key step in the biosynthesis of soybean phytoalexins. Arch. Biochem. Biophys. 293, 377-381 (1992).
    The cDNA for the reductase involved in the biosynthesis of 6'-deoxychalcone (4,2',4'-trihydroxychalcone), the first specific intermediate in the pathway to soybean phytoalexins, was cloned into the expression vector pKK233-2 and transformed into E. coli. Using this source, about 5 mg of homogeneous reductase was isolated from 45 g of cells. The protein purification protocol differs completely from the scheme applied to soybean cell cultures. Size, N-terminal and specific enzyme activities were identical for the plant and E. coli protein. The pure protein is fairly stable, retaining 70% of initial activity after storage at 5^oC during 4 weeks. This protein is used for crystallization and in the study of its protein-protein interaction with chalcone synthase.
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Link to Polyketide Reductase in 6'-Deoxychalcone Biosynthesis  

Lanz, T., Tropf, S., Marner, F.-J., Schröder, J. and Schröder, G.: The role of cysteines in polyketide synthases: site-directed mutagenesis of resveratrol and chalcone synthases, two key enzymes in different plant-specific pathways. Journal of Biological Chemistry 266, 9971-9976 (1991) .
    Resveratrol and chalcone synthases are related plant-specific polyketide synthases that are key enzymes in the biosynthesis of stilbenes and flavonoids, respectively. The stepwise condensing reactions correspond to those in other polyketide and fatty-acid synthases. This predicts that the two proteins also contain cysteines that are essential for enzyme activity because they bind the substrates. We exchanged, in both enzymes, all of the 6 conserved cysteines into alanine by site-directed mutagenesis and tested the mutants after expression of the proteins in the Escherichia coli heterologous system. Only cysteine 169 was essential in both enzymes, and inhibitor studies suggest that it is the main target of cerulenin, an antibiotic reacting with the cysteine in the active center of condensing enzymes. Most of the other exchanges led to reduced activities. In two cases, the enzymes responded differently, suggesting that the cysteines at positions 135 and 195 may be involved in the different product specificity of the two enzymes. The sequences surrounding the essential cysteine 169 revealed no similarity to the active sites of condensing enzymes in other polyketide synthases and in fatty acid biosynthesis. The available data indicate that resveratrol and chalcone synthases represent a group of enzymes that evolved independently of other condensing enzymes.
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Welle, R., Schröder, G., Schiltz, E., Grisebach, H. and Schröder, J.:  Induced plant responses to pathogen attack: analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean (Glycine max L. Merr.cv. Harosoy 63). Eur. J. Biochem. 196, 423-430 (1991).
    In soybean (Glycine max L.) pathogen attack induces the formation of glyceollin-type phytoalexins. The biosynthetic key enzyme is a reductase which synthesizes a 4,2',4'-trihydroxychalcone in co-action with chalcone synthase. Screening of a soybean cDNA library from elicitor-induced RNA in lambda gt11 yielded two classes of reductase-specific clones. The deduced proteins match to 100% and 95%, respectively, with 229 amino acids sequenced in the purified plant protein. Four clones of class A were expressed in E. coli and the proteins were tested for enzyme activity in extracts supplemented with chalcone synthase. All were active in 4,2',4'-trihydroxychalcone formation, and the quantification showed that shorter lengths of the cDNAs at the 5' end correlated with progressively decreasing enzyme activities. Genomic blots with DNA from plants capable of 4,2',4'-trihydroxychalcone synthesis revealed related sequences in bean (Phaseolus vulgaris L.) and peanut (Arachis hypogaea L.), but not in pea (Pisum sativum L.). No hybridization was observed with parsley (Petroselinum crispum) and carrot (Daucus carota) which synthesize other phytoalexins. The reductase protein contains a leucine-zipper motif and reveals a marked similarity with other oxidoreductases most of which are involved in carbohydrate metabolism.
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Link to Polyketide Reductase in 6'-Deoxychalcone Biosynthesis  

Lanz, T., Schröder, G. and Schröder, J.: Differential regulation of genes for resveratrol synthase in cell cultures of Arachis hypogaea. Planta 181, 169-175 (1990).
    Resveratrol synthase (RS; EC 2.1.1.-) catalyzes the formation of the phytoalexin resveratrol from 4-coumaroyl-CoA and malonyl-CoA. We present the characterization of new genomic RS sequences (RS3, RS4), and describe studies with gene-specific oligonucleotides on the expression of four different RS sequences (RScDNA, RS1, RS2, RS3) during growth of a cell culture from Arachis hypogaea L. and after application of various inducers (elicitor from Phytophthora megasperma, yeast extract, and dilution of the cultures). Transcripts from RScDNA were induced by all of the factors tested, and they represented the majority of all identified RS RNAs. Expression from RS1 and RS3 was much lower than from RScDNA, and transcripts from RS2 were never detected. Both RS1 and RS3 were induced by elicitor, but they reacted differently from the other inducers: RS1 was induced by yeast extract, but RS3 was not, and RS3 was induced by dilution of the cultures, but RS1 was not. The results indicate that the RS genes in A. hypogaea represent a gene family, and that some of the members are regulated by different signals. The quantitative data also show that the sum of the transcripts identified with gene-specific oligonucleotides was lower than the total amount of RS-specific transcripts, indicating that the cells contain active genes which have not yet been identified.
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Schröder, J. and Schröder, G.: Stilbene and chalcone synthases: related enzymes with key functions in plant-specific pathways. Z. Naturforsch. 45c, 1-8 (1990).
    Several years of extensive research using the new powerful techniques of molecular biology have enabled the direct comparison of functionally or evolutionarily related genes and their products at the nucleotide and amino acid sequence levels. Two types of synthase with similar functions are discussed as an interesting example. Stilbene synthases, e.g. resveratrol synthase, produce the stilbene backbone as a key reaction in the biosynthesis of stilbene-type phytoalexins. Chalcone synthase is a key enzyme in the biosynthesis of flavonoids, including certain phytoalexins derived from a 6'-deoxychalcone which is synthesized by cooperation of chalcone synthase with a reductase. Resveratrol and chalcone synthases utilize the same substrates (4-coumaroyl-CoA and 3 molecules of malonyl-CoA) and catalyze the same condensing type of enzyme reaction (resulting in sequential addition of acetate units via malonyl-CoA), but the products differ in the newly formed ring systems (resveratrol and naringenin chalcone). A comparative analysis of cloned DNA sequences and of the reaction mechanisms indicates that the two enzymes are closely related. It seems likely that the proteins possess a common scaffold for substrate recognition and for the condensing reaction, and that the different folding of an enzyme-bound intermediate prior to closure of the new aromatic ring is responsible for the formation of the different products. The same type of condensing reaction is utilized by the 2-ketoacyl-ACP synthases of fatty-acid biosynthesis. However, the available data indicate that these enzymes share little overall homology with either resveratrol or chalcone synthase. One exception may be a short amino acid sequence which corresponds to the active center of the condensing reaction in 2-ketoacyl-ACP synthases. 

Schröder, G., Brown, J.W.S. and Schröder, J.: Molecular analysis of resveratrol synthase: cDNA, genomic clones and relationship with chalcone synthase. European Journal of Biochemistry 172, 161-169 (1988).
    Resveratrol synthase (RS), a key enzyme in biosynthesis of stilbene-type phytoalexins, catalyzes the formation of resveratrol from coumaroyl-CoA and malonyl-CoA. Two cDNA clones, pGSC1 and pGSC2, have been isolated from cDNA libraries established with poly(A)-rich RNA from peanut (Arachis hypogaea) cell cultures specifically induced for RS. These cDNAs were used to identify two genomic clones (pGSG10 and pGSG11). Sequence analysis shows that the two clones overlap in a large stretch of nearly identical sequences, and that pGSG10 contains the 5' and pGSG11 the 3' end of RS genes. The sequences reveal a single intron, and the size of the predicted protein is 42.7 kDa, in close agreement with that observed in polyacrylamide gels (43 kDa). Chalcone synthase (CHS), a key enzyme of flavonoid biosynthesis, utilizes the same substrates as RS, but the product is different (naringenin chalcone). Comparison of RS with CHS consensus sequences shows that the two genes are related. Homology extends throughout the coding region, and the intron in RS is at the same position as a conserved intron in CHS. However, RS reveals a substantial number of amino acid differences to CHS in positions highly conserved in all CHS enzymes. It is proposed that the two proteins possess a commmon scaffold necessary for binding of the substrates and the type of enzyme reaction, and that the differences are responsible for the formation of different products.
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