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(Last modification: 10. August 2010)

 

Chalcone Synthase (CHS)

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Overview of Chalcone Synthase

    This short overview does not attempt a comprehensive description; it rather lists some of the publications that played key roles in the chalcone synthase story. If you want to look at the overall reaction: click on level up (comparison of CHS and STS).

    CHS activity was first described in 1972 in extracts of parsley (Petroselinum crispum) in the group of K. Hahlbrock in Freiburg (Kreuzaler and Hahlbrock, 1972). The enzyme was first labelled as 'flavanone synthase', because the chalcone was so quickly converted in a non-enzymatic reaction to the flavanone that it was not detectable as the initial product. That was corrected a few years later with improved techniques (Heller and Hahlbrock, 1980; Sütfeld and Wiermann, 1980), but of course the wrong name was used in the publications up to that time (see citations between 1972 and 1980, in the list below).

Isomerization of Chalcone to Flavanone.
In vitro
, this occurs non-enzymatically at fairly high rates; in vivo, the reaction is carried out by the enzyme chalcone isomerase (CHI). A modern analysis of the reaction is here: Jez et al., 2000d, 2002a, and 2002b.

Nomenclature and numbering: more...

 

    The initial description was followed throughout the 1970's by a series of publications with characterizations of the enzyme properties, mostly again from the group of Hahlbrock in Freiburg (Kreuzaler and Hahlbrock, 1975a; Kreuzaler and Hahlbrock, 1975b; Hrazdina et al., 1976; Kreuzaler et al., 1978; Saleh et al., 1978; Kreuzaler et al., 1979; Schüz et al., 1983). They covered many of the properties becoming more important later, e.g. the substrate promiscuity  and the formation of byproducts: click here for a more detailed discussion. This early work included the development of simple and rapid procedures for the quantification of the enzyme activity (ethylacetate extraction of the products, direct counting of radioactivity, followed by TLC or HPLC for confirmation of the product identity) (Schröder et al., 1979).
    The next leap was the publication of the first cDNA sequence, again of the enzyme from parsley (Petroselinum crispum) and the group of K. Hahlbrock
(Reimold et al., 1983), and this was followed by a burst of sequence publications. CHS is the entry point into flavonoid biosynthesis, and the genes are under complex regulation. The importance of this enzyme was quickly realized, and thus there is up to the present day a huge number of publications using its expression/induction as a marker of all sorts of biological processes.
    An important (and long awaited!) development was the first report on a crystal structure, from the CHS of Medicago sativa, in the group of J. Noel (Ferrer et al., 1999). Its importance cannot be underestimated. Based on it, much more detailed mechanistic studies were possible, see e.g. Jez and Noel, 2000c; Jez et al., 2000b. The precise knowledge of the active site pocket was also a great help in understanding the crystallized pyrone synthase from Gerbera hybrida and the reasons why it preferred much smaller substrates than phenylpropanoid-CoAs and why it carried out only two condensation reactions (Jez et al., 2000a).

The crystal structures were also extremely useful in modeling studies attempting to understand enzymes of the protein family with other functions. It certainly is very useful to study an excellent review published a few years ago (Austin and Noel, 2003).

 

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References

 

  • Austin, M. B., Noel, J. P., 2003. The chalcone synthase superfamily of type III polyketide synthases. Natural Product Reports 20, 79-110.
       Covering 1970–2001. This review covers the functionally diverse type III polyketide synthase (PKS) superfamily of plant and bacterial biosynthetic enzymes, from the discovery of chalcone synthase (CHS) in the 1970s through the end of 2001. A broader perspective is achieved by a comparison of these CHS-like enzymes to mechanistically and evolutionarily related families of enzymes, including the type I and type II PKSs, as well as the thiolases and -ketoacyl synthases of fatty acid metabolism. As CHS is both the most frequently occurring and best studied type III PKS, this enzyme's structure and mechanism is examined in detail. The in vivo functions and biological activities of several classes of plant natural products derived from chalcones are also discussed. Evolutionary mechanisms of type III PKS divergence are considered, as are the biological functions and activities of each of the known and functionally divergent type III PKS enzyme families (currently twelve in plants and three in bacteria). A major focus of this review is the integration of information from genetic and biochemical studies with the unique insights gained from protein X-ray crystallography and homology modeling. This structural approach has generated a number of new predictions regarding both the importance and mechanistic role of various amino acid substitutions observed among functionally diverse type III PKS enzymes.
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  • Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., Noel, J. P., 1999. Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. Nature Structural Biology 6, 775-784.
       Chalcone synthase (CHS) is pivotal for the biosynthesis of flavonoid antimicrobial phytoalexins and anthocyanin pigments in plants, It produces chalcone by condensing one p-coumaroyl- and three malonyl-coenzyme A thioesters into a polyketide reaction intermediate that cyclizes, The crystal structures of CHS alone and complexed with substrate and product analogs reveal the active site architecture that defines the sequence and chemistry of multiple decarboxylation and condensation reactions and provides a molecular understanding of the cyclization reaction leading to chalcone synthesis, The structure of CHS complexed with resveratrol also suggests how stilbene synthase, a related enzyme, uses the same substrates and an alternate cyclization pathway to form resveratrol, By using the three-dimensional structure and the large database of CHS-like sequences, we can identify proteins likely to possess novel substrate and product specificity. The structure elucidates the chemical basis of plant polyketide biosynthesis and provides a framework for engineering CHS-like enzymes to produce new products.
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  • Heller, W., Hahlbrock, K., 1980. Highly purified "flavanone synthase" from parsley catalyzes the formation of naringenin chalcone. Archives of Biochemistry and Biophysics 200, 617-619.
       The key reaction of flavonoid biosynthesis, the condensation of the acyl residues from one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA, has previously been assumed to be catalyzed by a “flavanone synthase.” Results are presented here which indicate that not the flavanone but the isomeric chalcone is the immediate product of the synthase reaction. The new term “chalcone synthase” is therefore suggested for the enzyme.
    Note: Another simultaneous report, using a different technique, also showed that the chalcone is the correct product of the polyketide reaction: more...
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  • Hrazdina, G., Kreuzaler, F., Hahlbrock, K., Grisebach, H., 1976. Substrate specificity of flavanone synthase from cell suspension cultures of parsley and structure of release products in vitro. Archives of Biochemistry and Biophysics 175, 392-399.
        The substrate specificity of an extensively purified flavanone synthase from light-induced cell suspension cultures of Petroselinum hortense was investigated. p-Coumaroyl-CoA was found to be the only efficient substrate for flavanone synthesis, producing naringenin (5,7,4'-trihydroxyflavanone). Besides 4-hydroxy-6[4-hydroxystyryl]2-pyrone (F. Kreuzaler and K. Hahlbrock (1975) Arch. Biochem. Biophys. 169, 84-90) two further release products of the synthase reaction in vitro were identified as 4-hydroxy-5,6-dihydro-6(4-hydroxyphenyl)2-pyrone and p-hydroxybenzalacetone. The apparent Km values for malonyl-CoA and p-coumaroyl-CoA in the reaction leading to naringenin, and for p-coumaroyl-CoA in the reaction leading to the styrylpyrone derivative were 35, 1.6, and 2.6 µM, respectively. With caffeoyl-CoA as substrate only a very small amount of eriodictyol (5,7,3',4'-tetrahydroxyflavanone) was formed besides relatively large amounts of the corresponding styrylpyrone, dihydropyrone, and benzalacetone derivatives. No flavanone formation was observed with feruloyl-CoA as substrate, but again appreciable amounts of the three types of short-chain release products were formed. No reaction at all took place with cinnamoyl-CoA, p-methoxycinnamoyl-CoA, isoferuloyl-CoA, or p-hydroxybenzoyl-CoA. None of the styrylpyrone, dihydropyrone, and benzalacetone derivatives has been detected in the cell cultures in vivo. The present results suggest that naringenin is the only natural product of the synthase reaction and that further substitution in the B-ring of the flavonoids occurs in parsley at or after the flavanone stage. The nature of the smaller release products is consistent with the assumption of a stepwise addition of acetate units from malonyl-CoA to the acyl moiety of the starter molecule, p-coumaroyl-CoA.
    Note: The enzyme was first labelled as 'flavanone synthase', because the chalcone was so quickly converted in a non-enzymatic reaction to the flavanone that it was not detectable as the initial product. That was corrected a few years later with improved techniques (see reference below), but the wrong name was used in the publications up to that time:  more...
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  • Jez, J. M., Austin, M. B., Ferrer, J.-L., Bowman, M. E., Schröder, J., Noel, J. P., 2000a. Structural control of polyketide formation in plant-specific polyketide synthases. Chemistry & Biology 7, 919-930.
    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 Rhizob_ium 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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  • Jez, J. M., Ferrer, J.-L., Bowman, M. E., Dixon, R. A., Noel, J. P., 2000b. Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. Biochemistry 39, 890-902.
        Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jet, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 Angstrom resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.
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  • Jez, J. M., Noel, J. P., 2000c. Mechanism of chalcone synthase - pKa of the catalytic cysteine and the role of the conserved histidine in a plant polyketide synthase. Journal of Biological Chemistry 275, 39640-39646.
       Polyketide synthases (PHS) assemble structurally diverse natural products using a common mechanistic strategy that relies on a cysteine residue to anchor the polyketide during a series of decarboxylative condensation reactions that build the final reaction product. Crystallographic and functional studies of chalcone synthase (CHS), a plant-specific PKS, indicate that a cysteine-histidine pair (Cys(164)His(303)) forms part of the catalytic machinery. Thiol-specific inactivation and the pH dependence of the malonyl-CoA decarboxylation reaction were used to evaluate the potential interaction between these two residues. Inactivation of CHS by iodoacetamide and iodoacetic acid targets Cys(164) in a pH-dependent manner (pK(a) = 5.50). The acidic pK(a) of Cys(164) suggests that an ionic interaction with His(303) stabilizes the thiolate anion. Consistent with this assertion, substitution of a glutamine for His(303) maintains catalytic activity but shifts the pK(a) of the thiol to 6.61. Although the H303A mutant was catalytically inactive, the pH- dependent incorporation of [C-14]iodoacetamide into this mutant exhibits a pK(a) = 7.62. Subsequent analysis of the pH dependence of the malonyl-CoA decarboxylation reaction catalyzed by wild- type CHS and the H303Q and C164A. mutants also supports the presence of an ion pair at the CHS active site. Structural and sequence conservation of a cysteine-histidine pair in the active sites of other PKS implies that a thiolate-imidazolium ion pair plays a central role in polyketide biosynthesis.
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  • Jez, J. M., Bowman, M. E., Dixon, R. A., Noel, J. P., 2000d. Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase. Nature Structural Biology 7, 786-791.
    Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcone synthesized by chalcone synthase (CHS) into (2S)-naringenin, an essential compound in the biosynthesis of anthocyanin pigments, inducers of Rhizobium nodulation genes, and antimicrobial phytoalexins. The 1.85 Angstrom resolution crystal structure of alfalfa CHI in complex with (2S)-naringenin reveals a novel open-faced beta-sandwich fold. Currently, proteins with homologous primary sequences are found only in higher plants. The topology of the active site cleft defines the stereochemistry of the cyclization reaction. The structure and mutational analysis suggest a mechanism in which shape complementarity of the binding cleft locks the substrate into a constrained conformation that allows the reaction to proceed with a second-order rate constant approaching the diffusion controlled limit. This structure raises questions about the evolutionary history of this structurally unique plant enzyme.
    Zurück

  • Jez, J. M., Noel, J. P., 2002a. Reaction mechanism of chalcone isomerase - pH dependence, diffusion control, and product binding differences. Journal of Biological Chemistry 277, 1361-1369.
    Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of bicyclic chalcones into tricyclic (S)- flavanones. The activity of CHI is essential for the biosynthesis of flavanone precursors of floral pigments and phenylpropanoid plant defense compounds. We have examined the spontaneous and CHI-catalyzed cyclization reactions of 4,2',4',6'-tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, 2',4'-dihydroxychalcone, and 4,2'-dihydroxychalcone into the corresponding flavanones. The pH dependence of flavanone formation indicates that both the non-enzymatic and enzymatic reactions first require the bulk phase ionization of the substrate 2'-hydroxyl group and subsequently on the reactivity of the newly formed 2'-oxyanion during C-ring formation. Solvent viscosity experiments demonstrate that at pH 7.5 the CHI-catalyzed cyclization reactions of 4,2',4',6'tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, and 2',4'-dihydroxychalcone are similar to 90% diffusion-controlled, whereas cyclization of 4,2'-dihydroxychalcone is limited by a chemical step that likely reflects the higher pK(alpha) of the 2'-hydroxyl group. At pH 6.0, the reactions with 4,2',4',6'tetrahydroxychalcone and 4,2',4'-trihydroxychalcone are similar to 50% diffusion-limited, whereas the reactions of both dihydroxychalcones are limited by chemical steps. Comparisons of the 2.1-2.3 Angstrom resolution crystal structures of CHI complexed with the products 7,4'-dihydroxyflavanone, 7-hydroxyflavanone, and 4'-hydroxyflavanone show that the 7-hydroxyflavanones all share a common binding mode, whereas 4'-hydroxyflavanone binds in an altered orientation at the active site. Our functional and structural studies support the proposal that CHI accelerates the stereochemically defined intramolecular cyclization of chalcones into biologically active (2S)-flavanones by selectively binding an ionized chalcone in a conformation conducive to ring closure in a diffusion-controlled reaction.
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  • Jez, J. M., Bowman, M. E., Noel, J. P., 2002b. Role of hydrogen bonds in the reaction mechanism of chalcone isomerase. Biochemistry 41, 5168-5176.
    In flavonoid, isoflavonoid, and anthocyanin biosynthesis, chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into (S)-flavanones with a second-order rate constant that approaches the diffusion-controlled limit. The three-dimensional structures of alfalfa CHI complexed with different flavanones indicate that two sets of hydrogen bonds may possess critical roles in catalysis. The first set of interactions includes two conserved amino acids (Thr48 and Tyr106) that mediate a hydrogen bond network with two active site water molecules. The second set of hydrogen bonds occurs between the flavanone 7-hydroxyl group and two active site residues (Asn113 and Thr190). Comparison of the steady-state kinetic parameters of wild-type and mutant CHIs demonstrates that efficient cyclization of various chalcones into their respective flavanones requires both sets of contacts. For example, the T48A, T48S, Y106F, N113A, and T190A mutants exhibit 1550-, 3-, 30-, 7-, and 6-fold reductions in k(cat) and 2-3-fold changes in K-m with 4,2',4'-trihydroxychalcone as a substrate. Kinetic comparisons of the pH-dependence of the reactions catalyzed by wild-type and mutant enzymes indicate that the active site hydrogen bonds contributed by these four residues do not significantly alter the pK(a) of the intramolecular cyclization reaction. Determinations of solvent kinetic isotope and solvent viscosity effects for wild-type and mutant enzymes reveal a change from a diffusion-controlled reaction to one limited by chemistry in the T48A and Y106F mutants. The X-ray crystal structures of the T48A and Y106F mutants support the assertion that the observed kinetic effects result from the loss of key hydrogen bonds at the CHI active site. Our results are consistent with a reaction mechanism for CHI in which Thr48 polarizes the ketone of the substrate and Tyr106 stabilizes a key catalytic water molecule. Hydrogen bonds contributed by Asn113 and Thr190 provide additional stabilization in the transition state. Conservation of these residues in CFIs from other plant species implies a common reaction mechanism for enzyme-catalyzed flavanone formation in all plants.
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  • Kreuzaler, F., Hahlbrock, K., 1972. Enzymic synthesis of aromatic compounds in higher plants: formation of naringenin (5,7,4'-trihydroxy-flavanone) from p-coumaroyl-coenzyme A and malonyl-coenzyme A. FEBS Letters 28, 69-72.
        In this communication, we report for the first time the cell-free formation of a flavonoid (.5,7,4'-trihydroxyflavanone) from p-coumaroyl CoA and malonyl-CoA by an enzyme preparation from illuminated parsley cell suspension cultures. Evidence is presented that the aromatic "ring A" of the flavanone is derived from malonate while "ring B" originates from the phenyl ring of p-coumarate (cf. fig. 1).
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  • Kreuzaler, F., Hahlbrock, K., 1975a. Enzymatic synthesis of aromatic compounds in higher plants. Formation of bis-noryangonin (4-hydroxy-6[4-hydroxystyryl]2-pyrone) from p-coumaroyl-CoA and malonyl-CoA. Archives of Biochemistry and Biophysics 169, 84-90.
       Cell-free extracts from light-induced cell suspension cultures of Petroselinum hortense catalyzed, in the presence of mercaptoethanol or dithioerythritol, the formation of bisnoryangonin from p-coumaroyl-CoA and malonyl-CoA. Radioactivity from the 3H- and 14C-labeled acyl moieties of p-coumaroyl-CoA and malonyl-CoA, respectively, was incorporated into the product at a molar ratio of 1:2. This result supports earlier conclusions from experiments in vivo favoring a mechanism of synthesis for the pyrone ring of bisnoryangonin according to the “acetate rule.” Bis-noryangonin could not be detected in cultured Petroselinum hortense cells in vivo. Our present results suggest that the styrylpyrone derivative formed in vitro is an artificial product of the first enzyme of the flavonoid pathway, flavanone synthetase. In the course of a 300-fold purification of this enzyme, the bis-noryangonin-synthesizing activity was always associated with the flavanone synthetase activity. The concentration of certain thiol reagents, such as mercaptoethanol or dithioerythritol, the ionic strength of the buffer, and the degree of purity of the enzyme preparation had a pronounced, differential effect on the amounts of flavanone and styrylpyrone formed by the flavanone synthetase. A possible explanation for the mechanism of formation of the artificial product, bis-noryangonin, is discussed.
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  • Kreuzaler, F., Hahlbrock, K., 1975b. Enzymic synthesis of an aromatic ring from acetate units. Partial purification and properties of flavanone synthase from cell-suspension cultures of Petroselinum hortense. European Journal of Biochemistry 56, 205-213.
       Flavanone synthase was isolated and purified about 300-fold from fermenter-grown, light-induced cell suspension cultures of Petroselinum hortense. The enzyme catalyzed the formation of the flavanone naringenin from p-coumaroyl-CoA and malonyl-CoA. Trapping experiments with an enzyme preparation, which was free of chalcone isomerase activity, revealed that in fact the flavanone and not the isomeric chalcone was the immediate product of the synthase reaction. Thus the enzyme is not a chalcone synthase as previously assumed. No cofactors were required for flavanone synthase activity. The enzyme was strongly inhibited by the two reaction products naringenin and CoASH, by the antibiotic cerulenin, by acetyl-CoA, and by several compounds reacting with sulfhydryl groups. Optimal enzyme activity was found at pH 8.0, at 30°C, and at an ionic strength of 0.1–0.3 M potassium phosphate. EDTA, Mg2+, Ca2+, or Fe2+ at concentrations of about 0.7 μM did not affect the enzyme activity. Apparent molecular weights of approx. 120000, 50000, and 70000, respectively, were determined for flavanone synthase and two metabolically related enzymes, chalcone isomerase and malonyl-CoA : flavonoid glycoside malonyl transferase. The partially purified flavanone synthase efficiently catalyzed the formation of malonyl pantetheine from malonyl-CoA and pantetheine. This malonyl transferase activity, and a general similarity with the condensation steps involved in the mechanisms of fatty acid and 6-methyl-salicylic acid synthesis from "acetate units", are the basis for a hypothetical scheme which is proposed for the sequence of reactions catalyzed by the multifunctional flavanone synthase.
    Note: The enzyme was first labelled as 'flavanone synthase', because the chalcone was so quickly converted in a non-enzymatic reaction to the flavanone that it was not detectable as the initial product. That was corrected a few years later with improved techniques (see reference below), but the wrong name was used in the publications up to that time:  more...
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  • Kreuzaler, F., Light, R. J., Hahlbrock, K., 1978. Flavanone synthase catalyzes CO2 exchange and decarboxylation of malonyl-CoA. FEBS Letters 94, 175-178.
    Note: The enzyme was first labelled as 'flavanone synthase', because the chalcone was so quickly converted in a non-enzymatic reaction to the flavanone that it was not detectable as the initial product. That was corrected a few years later with improved techniques (see reference below), but the wrong name was used in the publications up to that time:  more...
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  • Kreuzaler, F., Ragg, H., Heller, W., Tesch, R., Witt, I., Hammer, D., Hahlbrock, K., 1979. Flavanone synthase from Petroselinum hortense. Molecular weight, subunit composition, size of messenger RNA, and absence of pantetheinyl residue. European Journal of Biochemistry 99, 89-96.
       Flavanone synthase from irradiated cell suspension cultures of parsley was purified to apparent homogeneity. Molecular weights of about 77 000 for the enzyme and about 42 000 for the subunits were determined respectively by sedimentation-equilibrium measurements and disc-gel electrophoresis in the presence of dodecyl sulfate. A specific antiserum was prepared for the enzyme and was used in an assay for flavanone synthase mRNA activity in partially purified RNA preparations. The apparent molecular size of flavanone synthase mRNA was estimated by sucrose gradient centrifugation and gel electrophoresis under partially denaturing conditions. Values of about 17 S and Mr = 0.62 X 10(6) were obtained. The fractionation patterns suggested that flavanone synthase mRNA was homogeneous in size. All together, the results support the idea that the enzyme is composed of two subunits which are probably identical. Amino acid analysis and a microbial assay were carried out to test the possible occurrence of cysteamine, beta-alanine, and pantothenate in the enzyme. The results were negative, indicating the absence of pantetheine or a similar residue. The possible similarity in mechanism between flavanone synthase and 3-oxoacyl-(acyl carrier protein) synthase is discussed.
    Note:
    The enzyme was first labelled as 'flavanone synthase', because the chalcone was so quickly converted in a non-enzymatic reaction to the flavanone that it was not detectable as the initial product. That was corrected a few years later with improved techniques (see reference below), but the wrong name was used in the publications up to that time:  more...
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  • Reimold, U., Kröger, M., Kreuzaler, F., Hahlbrock, K., 1983. Coding and 3' noncoding nucleotide sequence of chalcone synthase messenger RNA and assignment of amino acid sequence of the enzyme. EMBO Journal 2, 1801-1805.
       The nucleotide sequence of an almost complete c(complementary)DNA copy of chalcone synthase mRNA from cultured parsley cells (Petroselinum hortense) was determined. The cDNA copy comprised the complete coding sequence for chalcone synthase, a short A-rich stretch of the 5' non-coding region and the complete 3' non-coding region including a poly(A) tail. The amino acid sequence deduced from the nucleotide sequence of the cDNA is consistent with a partial N-terminal sequence analysis, the total amino acid composition, the cyanogen bromide cleavage pattern, and the apparent MW of the subunit of the purified enzyme.
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  • Saleh, N. A. M., Fritsch, H., Kreuzaler, F., Grisebach, H., 1978. Flavanone synthase from cell suspension cultures of Haplopappus gracilis and comparison with the synthase from parsley. Phytochemistry 17, 183-186.
       Flavanone synthase was isolated and purified ca 62-fold from cell suspension cultures of Haplopappus gracilis. The enzyme preparation catalysed the formation of naringenin from 4-coumaryl-CoA and malonyl-CoA with a pH optimum of ca 8. The same enzyme was also capable of synthesizing eriodictyol from caffeyl-CoA and malonyl-CoA; in this case the pH optimum lay between 6.5 and 7. The homogeneous flavanone synthase from cell suspension cultures of parsley showed the same dependence of the pH optimum on the nature of the cinnamyl-CoA. It can be concluded that both naringenin and eriodictyol are natural products of the synthase reaction.

    Note
    :
    The enzyme was first labelled as 'flavanone synthase', because the chalcone was so quickly converted in a non-enzymatic reaction to the flavanone that it was not detectable as the initial product. That was corrected a few years later with improved techniques (see reference below), but the wrong name was used in the publications up to that time:  more...
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  • Schröder, J., Heller, W., Hahlbrock, K., 1979. Flavanone synthase: simple and rapid assay for the key enzyme of flavonoid biosynthesis. Plant Science Letters 14, 281-286.
       It is suggested that flavanone synthase activity should be measured when the key reaction of flavonoid biosynthesis is to be tested. A simple and rapid procedure for the determination of flavanone synthase activity, based on extraction of the 14C -labelled product(s) into ethylacetate, is described. The enzyme can be stored under appropriate conditions for several weeks without significant loss of activity. Results obtained with cell suspension cultures of parsley indicate that the activity of flavanone synthase is regulated differently from the activity of phenylalanine ammonia-lyase, an enzyme frequently referred to as a key enzyme of flavonoid biosynthesis.
    Note
    :
    The enzyme was first labelled as 'flavanone synthase', because the chalcone was so quickly converted in a non-enzymatic reaction to the flavanone that it was not detectable as the initial product. That was corrected a few years later with improved techniques (see reference below), but the wrong name was used in the publications up to that time:  more...
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  • Schüz, R., Heller, W., Hahlbrock, K., 1983. Substrate specificity of chalcone synthase from Petroselinum hortense. Formation of phloroglucinol derivatives from aliphatic substrates. Journal of Biological Chemistry 258, 6730-6734.
        The substrate specificity of chalcone synthase, the key enzyme of flavonoid biosynthesis, was investigated. A purified enzyme preparation from cell suspension cultures of parsley (P. hortense) catalyzed chain elongations with acetate units from malonyl-CoA, using various aromatic and aliphatic CoA esters as starter molecules. Malonyl-CoA could not be replaced by malonyl acyl carrier protein in the standard chalcone synthase assay. Butyryl-CoA, hexanoyl-CoA and benzoyl-CoA served as substrates for the condensation reaction with similar efficiency as 4- coumaroyl-CoA, the natural substrate of the enzyme. Acetyl-CoA and octanoyl-CoA were relatively poor substrates. Among the products formed with the 2 most efficient aliphatic substrates tested, butyryl-CoA and hexanoyl-CoA, were the respective chalcone analogs, phlorobutyrophenone and phlorocaprophenone. Chalcone synthase and the corresponding enzyme of fatty acid synthesis in higher plants, beta-ketoacyl-acyl carrier protein synthase, may have a common evolutionary origin.
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  • Sütfeld, R., Wiermann, R., 1980. Chalcone synthesis with enzyme extracts from tulip anther tapetum using a biphasic enzyme assay. Archives of Biochemistry and Biophysics 201, 64-72.
       The in vitro synthesis of chalcones has been demonstrated using a special biphasic enzyme assay. The highly viscous lower phase in this assay stems from a tapetum fraction of anthers of Tulipa cv. “Apeldoorn” which has been used an enzyme source. The upper phase of this system consists of a reaction mixture of the normal “flavanone synthase” assay. It is suggested that chalcone synthesis occurs at the boundary layer between the two phases. To prevent spontaneous as well as enzymatic cyclization of the chalcones formed (phloroglucinyl type), the pH of the upper phase must not be allowed to exceed pH 4.0. Under these pH conditions, chalcone formation by a reverse reaction of chalcone-flavanone isomerase can be excluded. The measured substrate specificity of the “chalcone synthase” corresponds to the conditions of chalcone formation in the natural system. Using p-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA, respectively, as substrates, the enzyme system forms the correspondingly substituted chalcones which are also accumulated in the loculus of tulip anthers. It is suggested that this chalcone synthase is identical to the previously described “flavanone synthase”. The results can be further explained as follows. (i) Not flavanones, but rather chalcones are the first C15 intermediates of flavonoid biosynthesis in tulip anthers. (ii) In this Tulipa system, the substitution pattern of three different hydroxycinnamic acids can be transferred unchanged into the flavonoid C15 stage. (iii) The role of chalcone-flavanone isomerase is to cyclize chalcones to flavanones on the direct biosynthetic pathway to the further accumulated flavonol glycosides. (iv) The sensitivity of the reaction with regard to chalcone production points to the localization of chalcone synthase in a most unstable and, up to now, unknown tapetal compartment. Since purification of the enzyme results in exclusive production of flavanones, it is suggested that certain “chalcone stabilizing factors” must occur in the natural system. (v) The phenomenon of chalcone accumulation in tulip anthers, however, must be caused by a complex system, distinguished by cooperation of certain biochemical and physiological conditions, and, finally, by special compartmentation of the enzymes which are responsible for the biosynthesis of flavonoids.
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File History:

  • 10.08.2010: Figure for isomerization of chalcone to flavanone, a few citations for modern analyses

  • 19. Nov. 2008: text, addition of Abstracts to citations

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