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(Last modification: 10. April 2010)
Stilbene Synthase (STS)
The links above lead to
Overviews in this page
Stilbenes and stilbene synthases (STS)
This short overview does not attempt a
comprehensive description; it rather lists some of the publications that in my
opinion (may be biased!) played
key roles in the stilbene synthase story.
If you want to look at the overall reaction: click on
level up (comparison of CHS and STS).
Stilbenes have been of
interest for a long time (Erdtman, 1963;
Billek, 1964;
Billek and Schimpl,
1966), and their possible biosynthesis via a polyketide route was already
discussed in the famous paper proposing that the biosynthesis of chalcones and
stilbenes was with a phenylpropanoid starter unit and three condensations with
acetate units (Birch and
Donovan, 1953).
There are several reviews
and book chapters on the occurrence and biochemistry of stilbenes; to my
knowledge the book of Gorham is the most recent and most comprehensive
(Gorham, 1995).
Stilbenes were also
recognized very early as phytoalexins, i.e. natural products involved in
resistances against pathogen attack, for example in peanuts
(Ingham, 1976;
Keen and Ingham, 1976) and grapes (Langcake
and Pryce, 1977a; 1977b).
As noted above, the
biosynthesis via a polyketide route from a phenylpropanoid starter with three
condensations was proposed very early, but the first demonstrations of the
enzyme activity were much later, around 1980 in the group of H. Kindl
(Rupprich and Kindl, 1978;
Schoeppner and Kindl, 1979;
Fritzemeier and Kindl, 1983), who also wrote a review that was very interesting
at that time (Kindl, 1985).
It was not much later
that the first sequences were published, from peanut (Schröder
et al., 1988; Lanz et al., 1990); and
later from pines (Pinus sylvestris and P. strobus) (Fliegmann
et al., 1992; Raiber et al., 1995;
Schanz et al., 1992), grape (Melchior and Kindl,
1990; Sparvoli et al., 1994;
Wiese et al., 1994), an orchid (Preisig-Müller et
al., 1995), rhubarb (Rheum tartaricum) (Samappito et al., 2003), and
Sorghum bicolor, an important monocot crop plant (Yu et al., 2005).
Functional
investigations by site-directed mutagenesis
The 1990's were a fascinating time to
find out something about the mechanisms of CHS and STS activities, by
site-directed mutagenesis of residues that looked interesting. This was not
quite easy, because no crystal structures were available for supporting ideas and
conclusions, and most of these experiments were based on the ideas that we got
from sequence alignments. Nevertheless, we think that the results were quite
interesting, and all of our conclusions were later confirmed with the
availability of the 3D-structure of the CHS from Medicago sativa
(Ferrer et al., 1999).
The knowledge of the 3D-structure and the wide availability of modelling
facilities of course changed the conditions for such experiments drastically:
the understanding of the reaction mechanism made much better predictions possible. This was then used widely
with all sorts of plant type III PKS, e.g. the acridone synthase
(ACS), benzalacetone synthase (BAS), benzophenone synthase (BPS), and so on. It
should be noted, nevertheless, that modeling itself did not help at all to
understand the functional differences between CHS and STS: that turned out to be
quite tricky: more....
An outline of our experiments at that time and the results:
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Identification of the active site cysteine in CHS and STS
(Lanz
et al., 1991):
All known polyketide synthases (and condensing enzymes in
general) contain a cysteine in the active site; it is absolutely necessary
for enzyme activity because it must covalently bind the starter residue
prior to the condensing reaction. We
tried to identify it in a chalcone and a stilbene synthase, by mutagenizing
all conserved cysteines: fortunately there were only six! Testing of the
mutated proteins clearly showed that only one of the six cysteines was essential: the one in
the position 169, and it was the same position in CHS and STS. This identification was fully confirmed with the later
elucidated crystal structure of the CHS from Medicago sativa.
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Role of histidine (His) and
glutamine (Gln) next to the active site cysteine
(Schröder
and Schröder, 1992):
The CHS and STS known at
that time contained next to the active site cysteine a two-amino-acid motif
of either GlnGln or HisGln or GlnHis. Interestingly, the CHS all contained
GlnGln in CHS, while all STS had GlnHis or HisGln. We speculated that this
might have to do with the type of ring-folding to chalcone or stilbene, and
therefore exchanged the residues by site-directed mutagenesis. The results
with the activities of the mutant proteins were quite interesting, and all
sorts of effects were achieved (have a look at the abstract, or read the
publication), but they also clearly showed that the residues investigated
here had nothing to do with determining the type of
ring-folding to either chalcone or stilbene. Actually, with the huge increase of
type III PKS sequences in the last years: the insights at around 1990 were
based on a too small set of sequences, as we now know in hindsight.
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Evolution in vitro: conversion of a CHS into a STS (Tropf
et al., 1994):
A
phylogenetic tree developed with the protein sequences available at that time
suggested that STS developed from CHS not only once, but several times
independently in various plant families. We attempted to simulate that: we
created a CHS/STS hybrid that was totally inactive;
it
contained 107 amino acids of the CHS from Sinapis alba (N-terminal)
and 287 amino acids of the STS from Arachis hypogaea
(C-terminal). Then
we investigated by mutagenesis which changes would be necessary to obtain an enzyme with STS activity.
As it turned out, only three changes in the CHS part were sufficient to
obtain a basic STS activity, and just one more mutation raised the activity
to about 20-25% of the parent STS. We proposed that the
advantage gained by this enzyme function (aquisiton of a new phytoalexin!) would
strongly favor the selection of plants with
improved STS activity.
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Evidence that CHS and STS must be homodimers (Tropf
et al., 1995):
There
were a number of publications claiming that CHS and STS are probably monomers.
We developed a strategy to test that. First we showed by cross-linking and
mutagenesis of the identified amino acids that there is a subunit contact
site at position 158: this argued strongly for homodimers.
The position was pretty close to the active site
cysteine (Cys169), suggesting that the active site pockets of the two
monomers might be close neighbours. In that case: was it possible that the
two subunits did not act independently, but cooperated in the formation of
the products? This was investigated as follows: we made a co-expression of
two proteins which were both inactive (based on different mechanismen!),
and tested whether the co-expression led to active proteins. This would be
possible only if the inactive subunits formed heterodimers in which they
complemented each other's deficiencies. Actually, it worked: we obtained
enzyme activity, with CHS as well as with STS (both were investigated).
Another interesting aspect of the suspected close
neighbourhood of the active sites: was it possible that the three
condensations are carried out in a sort of 'ping-pong', i.e. alternating
between the subunits? We could test this because one of the two mutants was
planned just for that purpose: the cysteine in the active site was
inactivated by mutagenesis. The important question then was: how many
condensations could be carried out by the active heterodimer? According to
the question asked above there would be two possibilities: a) according to
the 'ping-pong' mechanism, only one condensation would be possible because
only one of the subunits had an active site cysteine; b) three
condensations were possible only if each subunit could carry out all three
condensations. The result was clear-cut: the CHS made chalcones, and the
STS synthesized stilbenes. Conclusion: both subunits can carry out all
three condensation reactions independently. This is fully consistent with
the conclusions from the later 3D-structures.
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Orphan STS in
Psilotum nudum
Interestingly, cDNAs for
proteins with stilbene synthase activity were also obtained from
Psilotum nudum (Whisk
Fern, a primitive vascular plant) (Yamazaki et al., 2001).
The identification was based on studies with recombinant proteins after
expression in E. coli (substrate preference, type of products synthesized
in vitro). The functional identification was clear-cut, but it remained
puzzling that stilbenes or their derivatives are not known from that plant. It
therefore remains an interesting question: does this simply reflect our
ignorance of the compounds in the plants? Right now this protein should be
classified as 'orphan
PKS'. It is always surprising to realize how
little we know on natural products in many plants. Maybe they do contain
substances synthesized via STS-type reactions, but with other substrates. These are valid
concerns: all type III PKS are
notoriously promiscuous with respect to substrates, and it is often very
difficult to deduce a physiological role from in vitro experiments:
more....
Interesting question: do these presumed
STS enzymes contain the aldol switch found in the Pinus sylvestris STS?
Click here for more...
Links
to other examples of orphan type III PKS
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References
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Schröder,
G., Brown, J. W. S., Schröder, J., 1988.
Molecular
analysis of resveratrol synthase: cDNA, genomic clones and relationship with
chalcone synthase. European Journal of Biochemistry 172, 161-169.
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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Lanz,
T., Schröder, G., Schröder, J., 1990.
Differential regulation of genes for resveratrol synthase in cell cultures of
Arachis hypogaea. Planta 181, 169-175.
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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Fliegmann,
J., Schröder, G., Schanz, S., Britsch, L., Schröder, J., 1992.
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.
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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Schanz,
S., Schröder, G. and Schröder, J., 1992.
Stilbene synthase from Scots pine (Pinus sylvestris).
FEBS Letters 313, 71-74.
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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Raiber,
S., Schröder, G., Schröder, J., 1995. 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.
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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Billek, G., 1964.
Stilbene im Pflanzenreich. In: Zechmeister, L. (Ed.), Fortschritte der Chemie
Organischer Naturstoffe, Vol. 22.
Springer-Verlag, Vienna,
pp. 115-152.
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Billek, G., Schimpl,
A., 1966. Biosynthesis of plant stilbenes. In: Billek, G. (Ed.), Biosynthesis
of Aromatic Compounds, Pergamon Press, Oxford, pp. 37-44.
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Birch, A. J.,
Donovan, F. W., 1953.
Studies in relation to biosynthesis.
I. Some possible routes to derivatives of orcinol and phloroglucinol.
Australian Journal of Chemistry 6, 360-368.
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Erdtman, H., 1963. Some aspects of chemotaxonomy. In: Swain, T. (Ed.),
Chemical Plant Taxonomy, Academic Press, London, pp. 89-125.
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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.
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Fritzemeier, K.-H., Kindl, H., 1983.
S9,10-Dihydrophenanthrenes as phytoalexins of Orchidaceae. Biosynthetic
studies in vitro and in vivo proving the route from
L-phenylalanine to dihydro-m-coumaric acid, dihydrostilbene and
dihydrophenanthrenes. European Journal of Biochemistry 133, 545-550.
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Gorham, J., 1995. The Biochemistry of the Stilbenoids. Chapman &
Hall, London.
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Ingham, J. L., 1976. 3,5,4'-trihydroxystilbene as a phytoalexin from
groundnuts (Arachis hypogaea). Phytochemistry 15, 1791-1793.
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Keen,
N. T., Ingham, J. L., 1976. New stilbene phytoalexins from American cultivars
of Arachis hypogaea. Phytochemistry 15, 1794-1795.
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Kindl, H., 1985. Biosynthesis of stilbenes. In: Higuchi, T. (Ed.),
Biosynthesis and Biodegradation of Wood Components, Academic Press, New York,
pp. 349-377.
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Langcake, P., Pryce, R. J., 1977a. A new class of phytoalexins from
grapevines. Experientia 33, 151-152.
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Langcake, P., Pryce, R.
J., 1977b. The production of resveratrol and the viniferins by grapevines in
response to ultraviolet irradiation. Phytochemistry 16, 1193-1196.
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Melchior, F., Kindl, H., 1990. Grapevine stilbene synthase cDNA only
slightly differing from chalcone synthase cDNA is expressed in Escherichia
coli into a catalytically active enzyme. FEBS Letters 268, 17-20.
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Preisig-Müller, R., Gnau, P., Kindl, H., 1995. The inducible
9,10-dihydrophenanthrene pathway: characterization and expression of bibenzyl
synthase and S-adenosylhomocysteine hydrolase. Archives of Biochemistry
and Biophysics 317, 201-207.
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Rupprich, N., Kindl, H., 1978. Stilbene synthases and stilbenecarboxylate
synthases. I. Enzymatic synthesis of 3,5,4'-trihydroxystilbene from
p-coumaroyl coenzyme A and malonyl coenzyme A. Hoppe-Seyler's Zeitschrift für
Physiologische Chemie 359, 165-172.
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Samappito, S., Page, J. E., Schmidt, J.,
De-Eknamkul, W., Kutchan, T. M., 2003. Aromatic and pyrone polyketides
synthesized by a stilbene synthase from Rheum tataricum.
Phytochemistry 62, 313-323.
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Schoeppner, A., Kindl, H., 1979. Stilbene
synthase (pinosylvine synthase) and its induction by ultraviolet light.
FEBS Letters 108, 349-352.
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Sparvoli, F., Martin, C., Scienza, A., Gavazzi, G., Tonelli, C., 1994.
Cloning and molecular analysis of structural genes involved in flavonoid and
stilbene biosynthesis in grape (Vitis vinifera L.).
Plant
Molecular Biology 24, 743-755.
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Wiese, W., Vornam,
B., Krause, E., Kindl, H., 1994.
Structural organization
and differential expression of three stilbene synthase genes located on a 13
kb grapevine DNA fragment. Plant Molecular Biology 26, 667-677.
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Yamazaki, Y., Suh, D.-Y., Sitthithaworn, W., Ishiguro, K., Kobayashi, Y.,
Shibuya, M., Ebizuka, Y., Sankawa, U., 2001. Diverse chalcone synthase
superfamily enzymes from the most primitive vascular plant, Psilotum nudum.
Planta 214, 75-84.
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Yu,
C. K. Y., Springob, K., Schmidt, J., Nicholson, R. L., Chu, I. K., Yip, W. K.,
Lo, C., 2005. A stilbene synthase gene (SbSTS1) is involved in host and
non-host defense responses in Sorghum.
Plant Physiology 138, 393-401.
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Functional investigations by site-directed
mutagenesis
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Lanz,
T., Tropf, S., Marner, F.-J., Schröder, J., Schröder, G., 1991. 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.
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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Schröder,
G., Schröder, J., 1992. A single change of histidine to glutamine
alters the substrate preferences of a stilbene synthase. Journal of Biological
Chemistry 267, 20558-20560.
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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Tropf,
S., Lanz, T., Rensing, S. A., Schröder, J., Schröder, G., 1994.
Evidence that stilbene synthases have developed from chalcone synthases
several times in the course of evolution.
Journal of
Molecular Evolution 38, 610-618.
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 V-max than the parent STS,
and (2) that they had a different substrate preference than the parent STS and
CHS. Most of the other mutations and their combinations led to enzymatically
inactive protein aggregates, suggesting that the subunit folding and/or the
dimerization was disturbed. We propose that STS evolved from CHS by a limited
number of amino acid exchanges, and that the advantage gained by this enzyme
function favored the selection of plants with improved STS activity.
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Tropf,
S., Kärcher, B., Schröder, G., Schröder, J., 1995. 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.
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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