(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). Return to top
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...
Return
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
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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.
Zurück
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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.
Zurück
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...
Return
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...
Return
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...
Return
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...
Return
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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