(Last modification: 25.
February
2010)
Diese Datei
enthält unsere Publikationen über die Familie
der
Typ III PKS Enzyme
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Cook, D., Rimando, A. M., Clemente, T. E.,
Schröder, J., Dayan, F. E., Nanayakkara, N. P. D., Pan, Z., Noonan, B.
P., Fishbein, M., Abe, I., Duke, S. O., Baerson, S. R., 2010: Alkylresorcinol
synthases from Sorghum bicolor involved in the biosynthesis of
the allelopathic benzoquinone sorgoleone.
Plant Cell 22, 867-878.
Sorghum bicolor
is considered to be an allelopathic crop species, producing phytotoxins
such as the lipid benzoquinone sorgoleone, which likely accounts for
many of the allelopathic properties of Sorghum spp. Current
evidence suggests that sorgoleone biosynthesis occurs exclusively in
root hair cells and involves the production of an alkylresorcinolic
intermediate (5-[(Z,Z)-8',11',14'-pentadecatrienyl]resorcinol) derived
from an unusual 16:3{Delta}9,12,15 fatty acyl-CoA starter unit. This led
to the suggestion of the involvement of one or more alkylresorcinol
synthases (ARSs), type III polyketide synthases (PKSs) that produce
5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter
units via iterative condensations with malonyl-CoA. In an effort to
characterize the enzymes responsible for the biosynthesis of the
pentadecyl resorcinol intermediate, a previously described expressed
sequence tag database prepared from isolated S. bicolor (genotype
BTx623) root hairs was first mined for all PKS-like sequences.
Quantitative real-time RT-PCR analyses revealed that three of these
sequences were preferentially expressed in root hairs, two of which (designated
ARS1 and ARS2) were found to encode ARS enzymes capable of accepting a
variety of fatty acyl-CoA starter units in recombinant enzyme studies.
Furthermore, RNA interference experiments directed against ARS1 and ARS2
resulted in the generation of multiple independent transformant events
exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and
ARS2 are likely to participate in the biosynthesis of sorgoleone in
planta. The sequences of ARS1 and ARS2 were also used to identify
several rice (Oryza sativa) genes encoding ARSs, which are likely
involved in the production of defense-related alkylresorcinols.
Reprint request
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Brand, S.,
Hölscher, D., Schierhorn, A.,
Svato, A., Schröder, J., and Schneider, B:
A type III polyketide synthase from Wachendorfia thyrsiflora and its role in diarylheptanoid and
phenylphenalenone biosynthesis.
Planta 224, 413-428 (2006), available online: 23.02.2006
Chalcone synthase (CHS) related type III plant polyketide synthases (PKSs) are
likely to be involved in the biosynthesis of diarylheptanoids (e.g. curcumin and
polycyclic phenylphenalenones), but no such activity has been reported. Root
cultures from Wachendorfia thyrsiflora (Haemodoraceae) are a suitable
source to search for such enzymes because they synthesize large amounts of
phenylphenalenones, but no other products that are known to require CHSs or
related enzymes (e.g. flavonoids or stilbenes). A homology-based RT-PCR strategy
led to the identification of cDNAs for a type III PKS sharing only approximately
60% identity with typical CHSs. It was named WtPKS1 (W. thyrsiflora
polyketide synthase 1). The purified recombinant protein accepted a large
variety of aromatic and aliphatic starter CoA esters, including phenylpropionyl-
and side-chain unsaturated phenylpropanoid-CoAs. The simplest model for the
initial reaction in diarylheptanoid biosynthesis predicts a phenylpropanoid-CoA
as starter and a single condensation reaction to a diketide. Benzalacetones, the
expected release products, were observed only with unsaturated
phenylpropanoid-CoAs, and the best results were obtained with 4-coumaroyl-CoA
(80% of the products). With all other substrates, WtPKS1 performed two
condensation reactions and released pyrones. We propose that WtPKS1 catalyses
the first step in diarylheptanoid biosynthesis and that the observed pyrones are
derailment products in the absence of downstream processing proteins.
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Austin,M.B.;
Bowman,M.E.; Ferrer,J.-L.; Schröder,J.; Noel,J.P.:
An aldol switch discovered in stilbene synthases mediates cyclization
specificity of type III polyketide synthases.
Chemistry & Biology 11, 1179-1194 (2004)
Stilbene synthase (STS) and chalcone synthase (CHS) each catalyze the
formation of a tetraketide intermediate from a CoA-tethered phenylpropanoid
starter and three molecules of malonyl-CoA, but use different cyclization
mechanisms to produce distinct chemical scaffolds for a variety of plant
natural products. Here we present the first STS crystal structure, and
identify, by mutagenic conversion of alfalfa CHS into a functional stilbene
synthase, the structural basis for the evolution of STS cyclization
specificity in type III polyketide synthase (PKS) enzymes. Additional
mutagenesis and enzymatic characterization confirms that electronic effects
rather than steric factors balance competing cyclization specificities in CHS
and STS. Finally, we discuss the problematic in vitro reconstitution of
plant stilbenecarboxylate pathways, using insights from existing biomimetic
polyketide cyclization studies to generate a novel mechanistic hypothesis to
explain stilbenecarboxylate biosynthesis.
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Joseph Noel; die meiste Arbeit stammte aus seinem Labor!!
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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 (2003)
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 (2003).
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.
Mehr...
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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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Typ III PKS in Bakterien
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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.
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-> 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 Molecular Biology 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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Geza Hrazdina
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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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Titelbild von Chemistry &
Biology, Dezember
2000.
Zur Home page
von Joseph P. Noel: Ein Besuch lohnt sich!.
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Schröder,
J.: The family of chalcone synthase-related proteins: functional diversity and
evolution. Recent Advances in Phytochemistry 34, 55-89 (2000).
(Verarbeitete Literatur: Bis 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).
Ein Übersichtsartikel, der die Literatur bis März 1997 diskutiert. Es werden
auch Vorhersagen gemacht, welche Enzymaktivitäten vermutlich durch Enzyme
der Übersicht katalysiert werden. Viele der Vorhersagen wurden inzwischen
bestätigt!
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Schröder, J.:
Probing plant polyketide synthesis.
Nature Structural Biology 6: 714-716 (1999).
Dies ist ein kurzer "News and Views" Comment für
die Publikation der Noel-Gruppe über die erste Kristall-Struktur einer
CHS. Diskutiert und gezeigt werden einige der enzymatischen Funktionen von
Proteinen der Übersicht, und die Aussichten für das mechanistische
Verständnis, die sich aus der 3D-Struktur ergeben.
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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 Mr
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 Molecular Biology 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 in Plant Science 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 Molecular
Biology
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 Km for the
substrate 4-coumaroyl-CoA but a lower Vmax 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 Molecular Biology 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 Km and a 3-fold reduction
of Vmax, 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.
Archives in Biochemistry and Biophysics 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 5oC 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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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).
European Journal of Biochemistry 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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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.
Zeitschrift für Naturforschung 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 prioder zur 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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