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(Last
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Type III PKS:
Byproducts, Promiscuity, Functional Identification
Essentially all type III PKS enzymes have somewhat annoying properties in vitro, e.g. with recombinant enzymes isolated after
expression in E. coli (or other hosts, like yeasts, insect cells, etcet.):
For
these reasons:
-
3. The functional identification of new members is not always easy:
more....
-
4. Some examples: Physiological
function unknown or uncertain, 'Orphan PKS':
more....
These topics will be discussed briefly below.
The focus will be mostly on chalcone (CHS), stilbene (STS), and
stilbenecarboxylate (STCS) synthases, but the
problems are general for all type III PKS.
1.
Byproducts
Let's have a look at some
representative examples, chalcone synthases (CHS), stilbene synthases (STS), and
stilbenecarboxylate (STCS) synthases. The findings described for these enzymes
can be applied to almost all type III PKS assayed in vitro.
As a rule, all type III enzymes
do not only synthesize the expected products, but also byproducts that can be explained
as derailments from intermediates of incomplete reactions. The figure below shows in the boxes the derailment
product types usually found after in vitro incubations with
CHS, STS, and STCS, with 4-coumaroyl-CoA as representative starter substrate.
They
produce
Benzalacetone (one condensation), arylpyrones (one
condensation), styrylpyrones (two condensations), and
4-Coumaroyltriacetic acid lactone (CTAL, three condensations,
also a pyrone).
Reactions of CHS (chalcone synthase), STS (stilbene
synthase), and STCS (stilbenecarboxylate synthase), and byproducts in vitro.
The reactions are given for the prototype substrate 4-coumaroyl-CoA. The colours
mark the three condensation reactions. The byproducts are boxed. Most abundant
are the pyrone derailment products after two and three condensations (Bisnoryangonin
and 4-Coumaroyltriacetic acid, CTAL).
Benzalacetone,
arylpyrones, and
styrylpyrones as byproducts of CHS reactions had been found already with the first characterization of the
CHS reaction (Kreuzaler and Hahlbrock,
1975a, 1975b;
Hrazdina et al., 1976;
Saleh et al., 1978).
Interestingly, at least benzalacetone and bisnoryangonin are natural products in certain plants. Benzalacetone is the
precursor of the most important aroma component in raspberries, and the
benzalacetone synthase in
raspberry
(Borejsza-Wysocki
and Hrazdina, 1994;
1996) is a type III PKS (Hrazdina and Zheng,
2006; Zheng and Hrazdina,
2008, more...). The benzalacetone synthase is also a type III PKS in
rhubarb (Rheum palmatum,
Abe et al.,
2001; 2003,
more...). Bisnoryangonin is
found in many plants (Beckert
et al., 1997; reviewed in:
Schröder, 1999), but it does not seem proven that it is synthesized by a
type III PKS (more...).
4-Coumaroyltriacetic acid
lactone (CTAL)
was discovered
relatively late (Yamaguchi et al., 1999), and was
also discussed as the physiological product of a type III PKS identified in
Hydrangea macrophylla var. thunbergii (Akiyama
et al., 1999).
The examples described here describe the formation of byproducts
by typical
CHS, STS, and STCS with their physiological substrate 4-coumaroyl-CoA.
Byproducts of the same type (mainly pyrones) can usually found in any in vitro reaction
with any type III PKS and any substrate. That is, if one cares to look for them: they are
easily missed, because extraction of the products at pH higher than 7 with ethyl
acetate, a typical method, will miss essentially all of the pyrones.
The situation gets even worse if one uses non-physiological
substrates (see also substrate promiscuity below): typically, both CHS and STS
can use many other substrates quite well. However, in most cases the products
are not the expected ones (chalcone-type or stilbene-type ring-closure) because
the reactions are terminated prematurely. CHS, for example, most
often carries out only two condensations and releases a pyrone (->
bisnoryangonin type). Or it manages three condensations, but fails to complete
the CHS-type ring-folding, with the result that a pyrone of the CTAL-type is
released. Note that the same byproducts will also be obtained with STS and STCS, see
the figure above! Therefore, if you find such pyrones with a new type III PKS:
are these 'real' products, or just byproducts of CHS, STS, or STCS under
non-optimal in vitro assay conditions? Another question: are you sure
this was the 'right' substrate? The answer is often not easy, see
below.
And also consider this: Not even the specificity with
respect to CHS- and STS-type ring folding is absolute: a few percent of the CHS
products were found to be stilbenes, and conversely a few percent of the STS
products were identified as chalcones (Yamaguchi et al.,
1999; Suh et al., 2000).
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2.
Substrate
Promiscuity
All of these
enzymes are not very substrate-specific. CHS and STS, for example, will accept
any CoA-ester which roughly resembles the size of the physiological substrate
4-coumaroyl-CoA. This has also been discovered rather early, and one of the good
early examples is the work of
Schüz et al. (1983), which demonstrated that benzoyl-CoA,
hexanoyl-CoA, and other aliphatic CoA-esters are good substrates of CHS. This
substrate promiscuity was confirmed in all other studies investigating the
properties of type III enzymes.
In most cases the reaction with non-physiological substrates does not lead to the expected CHS- or
STS-type product: Usually the reactions terminate prematurely or the
ring-folding to the proper products is not possible, leading to pyrones as
derailment products (see above).
Sometimes it gets rather puzzling. For example, the
physiological substrate of the
pentaketide and
octaketide synthases from
Aloe arborescens is thought to be malonyl-CoA (or acetyl-CoA). However,
in vitro they readily accept much larger substrates, e.g. long-chain fatty
acid CoA-esters, and even the CHS/STS prototype substrate 4-coumaroyl-CoA. The
products are the pyrones from two or three condensations. With 4-coumaroyl-CoA,
there was no evidence for
chalcone or stilbene products. One would think that in the long run
one would like some additional evidence for the proposed physiological functions
of these proteins (some possibilities for such experiments are discussed below).
And it is not only starter substrate promiscuity: many
of the enzymes are essentially not very choosy about the chain extenders. The
typical physiological extender is malonyl-CoA, but methylmalonyl-CoA has also
been implicated in at least one case as physiological chain extender (Schröder
et al., 1998; more...), and several other enzymes have been shown in vitro to make some
products with methylmalonyl-CoA (Abe
et al., 2002; Abe et al.,
2003; Abe et al., 2006).
An interesting aspect of this promiscuity is that it
can be used to produce novel, unusual products by employing totally unnatural
substrates, with sometimes unexpected
products (Morita
et al., 2000; Morita
et al., 2001).
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3.
Consequences for the
Functional Identification of New Type III
Enzymes
The two
properties discussed above have some significant consequences if you are interested
in identifying the physiological functions of new members of the protein family.
Most new members are obtained by homology-based cloning (e.g. by cross-hybridization
or by RT-PCR with degenerate primers), or more recently by genome mining
(homology searches in genome databases). This will usually pick up the members of
the protein family in a given species. The next step is then the identification
of the function.
The standard procedure is nowadays: expression of a recombinant
protein in E. coli (or another host, like yeast), purification of the
protein, and tests with various substrates. The first choice of these will be
guided by the expected starter substrate(s), but a reasonably careful study will
also investigate others (e.g. those similar to the predicted substrate).
Certainly the prototype substrates of CHS and STS (4-coumaroyl-CoA and a few
others from the phenylpropanoid pathway) should be tested, because at least CHS
is ubiquitous in plants, and usually it will be picked up in the screening
procedure. The choice of other substrates will depend on the natural products
known from the plant (if there is some information!).
If you are
lucky, you will get the expected result: the new type III PKS is not simply
another CHS, but uses another substrate to synthesize a natural product (or a
predicted precursor) known from the plant. Or it uses a well-known substrate,
but carries out a novel reaction to a novel product. Then you are well-off, even
if related substrates are also accepted: theses results are a nice addition to
the identification, and just show again the promiscuity of these enzymes.
Unfortunately it is often not that easy, and
there are many reasons for complications. A trivial one is: the protein is not
soluble in the heterologous host, and thus it is not even possible to do
functional studies. There are many tricks and attempts to cure
that, but sometimes nothing really helps, and then one does not even get the
chance to test the various possible substrates. Or the protein does accept the
predicted substrate, but for some reason fails to complete the reaction to the
expected product, or/and other substrates are also accepted, and thus this
promiscuity hinders the identification of the 'right' substrate. Sometimes one
does get the expected reaction, but it is so similar to that of CHS that a
functional distinction in vitro is not possible. One example is the
valerophenone synthase (VPS) from hop (more...). Even now it does not seem clear which
of the many cloned cDNAs is the 'real' VPS or CHS: some of the proteins seem to
have both activities. Also take notice that there are several other type III PKS
that carry out
CHS-type ring-foldings (more...), but with other substrates, and the same can be said
for the
STS-type ring-folding (more...).
One of the not so trivial reasons seems to be in
several cases that the isolated type III PKS by itself cannot complete the
reaction to the expected product because something is missing. This is most
likely the case in some cases that are discussed in other chapters of this
website: either a postulated reductase is missing (more...),
or another reaction (more...).
In those cases it gets difficult. Then you must
try to gather corroborating evidence, something that should be done in the long
run anyway. Examples (the list is certainly not complete!):
-
Look at the protein sequence:
-
If it is 90% or more identical to typical CHS, the chances are high that you
cloned another CHS. Enzymes from the superfamily with other functions usually
share only 70% identity or less with standard CHS.
-
Look at the residues/motifs that were identified to be important for
substrate and product size, and try to get a model based on known crystal
structures (easily possible via Internet, e.g. with
Swissmodel). It must be noted, however, that most of the interesting
insights came from enzymes whose physiological role was known already.
-
Can one predict functions of new enzymes from
such gazing at motifs? The chances do not look so good.
In most cases such attempts had only limited value. Take one typical example:
even nowadays it is hardly possible to distinguish CHS, STS, and STCS simply
by looking at the sequences, or at models. And even completed 3D-structure
might not be sufficient (more...)
-
I
will elaborate on this a bit more, based on thoughts recently published in a
review (Jiang et al., 2008)
on the possibilities to make deductions/predictions from protein sequences (phylogenetic/relationship
analysis):
-
Such predictions will hardly be possible for
mosses, ferns, and gymnosperm enzymes because CHS and non-CHS are
clustering so close together that a distinction is not possible.
-
It is a bit more complicated with the
angiosperms. A protein clustering with CHS is not necessarily a CHS, but
can be anything else: examples are the Fabales (legumes) where CHS and STS
are in the same large cluster: gazing at sequences or motifs will not give
you a good answer. On the other hand, if an angiosperm protein clusters far
away from CHS, but with other non-CHS enzymes, the chances are good that the
enzyme is not a CHS. However, please note: this will not tell you the
function; that group is very heterogenous if it comes to that!
-
It is also a bit complicated with
monocotyledons.
The proteins tend to be in one large cluster, but that may contain CHS and
non-CHS: CHS8 from Sorghum bicolor, for example is actually a STS (Yu
et al., 2005), but clusters together with CHS from Sorghum and other
monocots. On the other hand, proteins clustering far away from known CHS are
likely to be non-CHS, just like with the angiosperms. So far, this is only
based on one enzyme, the curcuminoid synthase (CUS) which is discussed
somewhere else in this website (more...),
and it will be interesting to see whether this can be shown for other
monocot type III PKS.
New in March 2010: the Alkylresorcinol Synthases (ARS) from
sorghum (Sorghum bicolor) and rice (Oryza sativa) are
actually in a large group that phylogenetically is far removed from the
CHS/STS from monocoteledons:
more..
-
Can you measure the activity
in
vitro, and is it inducible?
- Then you can gather supporting evidence by showing that the mRNA increase correlates with
increases in activity.
-
Is your plant amenable to genetic
techniques, e.g. gene transfer, incl. RNAi approaches?
- Try to get a knock-out
of the function, either in the gene itself or by RNAi, or overexpress the
protein in its natural host: correlate the absence of increased presence of
the activity with the changes you did. Unfortunately, however, it is a fact
that many plants with the most interesting products are not amenable to these
techniques.
-
One
good possibility could be: express the isolated gene/cDNA in a plant that is
nicely amenable to genetic manipulation, e.g. Arabidopsis thaliana,
tobacco, etcet.
- If you are lucky, you'll find new natural products that
correspond to the expectations. If you are unlucky, you'll find nothing at
all. That can happen, for example, if the new host does not provide the
necessary substrate. Or a worse variation of bad luck: the 'right' substrate is
missing, but there are substitutes in that plant that are also acceptable to the enzyme: in
that case the promiscuity of these proteins might lead to products that have nothing to do with the 'real' function in the original plant.
If everything fails to get an unambiguous identification, but you still want to
publish your findings: pray for a reviewer who understands and accepts the
problems. However, you should be able to demonstrate that you tried whatever was
possible for you.
A final remark. Genome projects nowadays provide lots of sequences, and in many
cases also some that look like type III PKS. That can be very tempting because
it can provide chances for novel findings. However, it probably is useful to
exercise some caution before one goes into that, if you don't have a reasonable
idea what the physiological function might be, i.e. if there is no natural
product (or its precursor) that might be synthesized via such PKS
reaction. The substrate promiscuity will certainly lead to the result that the
enzyme accepts some substrate and produces something with. But how do you
interpret the results? One should have a simultaneous careful investigation of
the natural products, but how many labs do have the facilities, and, more
importantly, the motivation if nothing is known in the first place?
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4.
Type
III PKS: Physiological function
unknown or uncertain: 'Orphan PKS'
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References
-
Abe, I., Sano, Y., Takahashi,
Y., Noguchi, H., 2003. Site-directed mutagenesis of
benzalacetone synthase: the role of Phe215 in plant type III polyketide
synthases. Journal of Biological Chemistry 278, 25218-25226.
Benzalacetone synthase (BAS) and chalcone synthase (CHS) are plant-specific
type III polyketide synthases (PKSs) that share [~]70% amino acid sequence
identity. BAS catalyzes a one-step decarboxylative condensation of
4-coumaroyl-CoA with malonyl-CoA to produce a diketide benzalacetone,
whereas CHS performs sequential condensations with three malonyl-CoA to
generate a tetraketide chalcone. A homology model suggested that BAS has the
same overall fold as CHS with cavity volume almost as large as that of CHS.
One of the most characteristic features is that Rheum palmatum BAS lacks
active site Phe-215; the residues 214LF conserved in type III PKSs are
uniquely replaced by IL. Our observation that the BAS I214L/L215F mutant
exhibited chalcone-forming activity in a pH-dependent manner supported a
hypothesis that the absence of Phe-215 in BAS accounts for the interruption
of the polyketide chain elongation at the diketide stage. On the other hand,
Phe-215 mutants of Scutellaria baicalensis CHS (L214I/F215L, F215W, F215Y,
F215S, F215A, F215H, and F215C) afforded increased levels of truncated
products; however, none of them generated benzalacetone. These results
confirmed the critical role of Phe-215 in the polyketide formation reactions
and provided structural basis for understanding the structure-function
relationship of the plant type III PKSs.
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Abe,
I., Takahashi, Y., Lou, W., Noguchi, H., 2003. Enzymatic formation of
unnatural novel polyketides from alternate starter and nonphysiological
extension substrate by chalcone synthase. Organic Letters 5, 1277-1280.
In the
chalcone synthase (CHS) enzyme reaction, both the starter molecule and the
extension unit of the poyketide chain elongation reaction were
simultaneously replaced with nonphysiological substrates. When incubated
with benzoyl-CoA and methylmalonyl-CoA as substrates, recombinant CHS from
Scutellaria baicalensis afforded an unnatural novel triketide,
4-hydroxy-3,5-dimethyl-6-phenyl-pyran-2-one, along with a tetraketide,
4-hydroxy-3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-pyran-2-one. On the
other hand, the enzyme also accepted hexanoyl-CoA and methylmalonyl-CoA as
substrates to produce an unnatural novel triketide,
4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one.
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Abe,
I., Takahashi, Y., Noguchi, H., 2002. Enzymatic formation of an unnatural C6-C5
aromatic polyketide by plant type III polyketide synthases. Organic Letters 4,
3623-3626.
Substrate specificities of plant polyketide synthases (PKSs) were
investigated using analogues of malonyl-CoA, the extension unit of the
polyketide chain elongation reactions. When incubated with methylmalonyl-CoA
and 4-coumaroyl-CoA, plant PKSs (chalcone synthase from Scutellaria
baicalensis, stilbene synthase from Arachis hypogaea, and
benzalacetone synthase from Rheum palmatum) afforded an unnatural
C(6)-C(5) aromatic polyketide, 1-(4-hydroxyphenyl)pent-1-en-3-one, formed by
one-step decarboxylative condensation of the two substrates. In contrast,
succinyl-CoA was not accepted as a substrate by the enzymes.
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Abe,
T., Noma, H., Noguchi, H., Abe, I., 2006. Enzymatic formation of an unnatural
methylated triketide by plant type III polyketide synthases.
Tetrahedron Letters 47, 8727-8730.
Octaketide synthase, a novel plant-specific type III polyketide
synthase from Aloe arborescens, efficiently accepted (2RS)-methylmalonyl-CoA
as a sole substrate to produce 6-ethyl-4-hydroxy-3,5-dimethyl-2-pyrone. On
the other hand, a tetraketide-producing chalcone synthase from Scutellaria
baicalensis and a diketide-producing benzalacetone synthase from Rheum
palmatum also yielded the unnatural methylated C9 triketide pyrone as a
single product by sequential decarboxylative condensations of three
molecules of (2RS)-methylmalonyl-CoA.
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Abe, I.,
Takahashi, Y., Morita, H., Noguchi, H., 2001. Benzalacetone synthase: a novel
polyketide synthase that plays a crucial role in the biosynthesis of
phenylbutanones in Rheum palmatum. European Journal of Biochemistry
268, 3354-3359.
Benzalacetone synthase (BAS) is a novel plant-specific polyketide synthase
that catalyzes a one step decarboxylative condensation of 4-coumaroyl-CoA
with malonyl-CoA to produce the C 6 -C 4 skeleton of phenylbutanoids in
higher plants. A cDNA encoding BAS was for the first time cloned and
sequenced from rhubarb (Rheum palmatum), a medicinal plant rich in
phenylbutanoids including pharmaceutically important phenylbutanone
glucoside, lindleyin (anti-inflammatory action in extracts, Fig. 4:
derivative of raspberry ketone, by attaching a sugar+phenyl-derivative to
hydroxy group of coumaroyl starter residue). The cDNA encoded a 42 kDa
protein that shares 60-75% amino acid sequence identity with other members
of the CHS-superfamily enzymes. Interestingly, R. palmatum BAS lacks
the active-site Phe215 residue (numbering in CHS) which has been proposed to
help orient substrates and intermediates during the sequential condensation
of 4-coumaroyl-CoA with malonyl-CoA in CHS. On the other hand, the catalytic
cysteine-histidine dyad (Cys164 - His303) in CHS is well conserved in BAS. A
recombinant enzyme expressed in E. coli efficiently afforded
benzalacetone as a single product from 4-coumaroyl-CoA and malonyl-CoA.
Further, in contrast with CHS that showed broad substrate specificity toward
aliphatic CoA esters, BAS did not accept hexanoyl-CoA, isobutyryl-CoA,
isovaleryl-CoA, and acetyl-CoA as a substrate. Finally, besides the
phenylbutanones in rhubarb, BAS has been proposed to play a crucial role for
the construction of the C 6 -C 4 moiety of a variety of natural products
such as medicinally important gingerols in ginger plant.
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Akiyama,
T., Shibuya, M., Liu, H. M., Ebizuka, Y., 1999. p-Coumaroyltriacetic
acid synthase, a new homologue of chalcone synthase, from Hydrangea
macrophylla var. thunbergii. European Journal of Biochemistry 263,
834-839.
Chalcone synthase and stilbene synthase are plant-specific polyketide
synthases. They catalyze three common consecutive decarboxylative
condensations and specific cyclization reactions. They are highly homologous
to each other, and are likely to fall into a family of polyketide synthases
along with acridone synthase and bibenzyl synthase. Two cDNA clones (named
HmC and HmS), both of which show high homology to the known chalcone
synthases, were obtained from leaves of Hydrangea macrophylla
var. thunbergii. They were expressed in Escherichia coli in
order to determine their enzyme functions. Detection of chalcone formation
clearly indicated that HmC encoded chalcone synthase, while HmS protein
catalyzed the formation of neither chalcone nor stilbene. However, a novel
pyrone, a lactonization product of a linear tetraketide was detected in
reaction products of HmS protein. This proves that HmS encodes a novel
polyketide synthase that catalyzes only chain elongation without cyclization.
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Beckert,
C., Horn, C., Schnitzler, J.-P., Lehning, A., Heller, W., Veit, M., 1997.
Styrylpyrone biosynthesis in Equisetum arvense L. Phytochemistry
44, 275-283.
Styrylpyrone synthase was detected in cell free extracts from gametophytes
of Equisetum arvense. This new enzyme catalyses the formation of
styrylpyrones from malonyl-CoA and hydroxycinnamoyl- CoA precursors. A
standard enzyme assay was established. The enzyme activity was characterized
in partially purified protein extracts. p-Coumaroyl-CoA was accepted as
substrate at pH 6.0-8.5 in various buffer systems with the formation of
bisnoryangonin, and optimum enzyme activity was observed in potassium
phosphate buffer at pH 7.5. Caffeoyl-CoA was accepted as substrate only in
potassium phosphate buffer at pH 6.0-7.5 with formation of hispidin; optimum
enzyme activity was observed at pH 7.0. The apparent K-m values were 220 µM
for caffeoyl-CoA and 230 µM for p-coumaroyl-CoA. The temperature optimum of
the enzyme activity was 37 degree for bisnoryangonin and 30 degree for
hispidin formation. Molecular weight determination by FPLC indicated that
this protein has a native molecular weight of ca 56-77 kDa. Styrylpyrones
accumulate in rhizomes of sporophytes and gametophytes of E. arvense
as major constitutive metabolites. In these organs no flavonoids could be
detected. In green sprouts, styrylpyrone accumulation is only detected as a
local response to mechanical wounding or microbial attack, and flavonoids
are accumulated as major polyketide metabolites. Thus, chalcone synthase is
active in the sporophytes and might have developed in the course of
evolution from styrylpyrone synthase present in the more primitive
gametophytes.
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Borejsza-Wysocki,
W., Hrazdina, G., 1994. Biosynthesis of p-hydroxyphenylbutan-2-one in
raspberry fruits and tissue cultures. Phytochemistry 35, 623-628.
p-Hydroxyphenylbutan-2-one (pHPB), the raspberry ketone, is responsible for
the characteristic aroma of raspberries. The compound accumulates rapidly
during the later maturation stages of the berries. The synthesis and
accumulation of pHPB correlates with that of anthocyanin and soluble solids
(degree Brix). pHPB is synthesized in cell-free extracts of fruits and
tissue cultures from p-coumaryl-CoA and malonyl-CoA in a manner similar to
the synthesis of chalcones and stilbenes. The specific biosynthetic pathway
for pHPB formation deviates from the general phenylpropanoid pathway at the
p-coumaryl-CoA stage and it is composed of two enzymes. The first enzyme is
the p-hydroxyphenylbut-3-ene-2-one synthase (pHPB-3-ene-2-one synthase) that
forms p-hydroxyphenylbut-3-ene-2-one by the condensation of malonyl-CoA with
p-coumaryl-CoA. The second enzyme, p- hydroxyphenylbut-3-ene-2-one reductase
(pHPB-3-ene-2-one reductase), reduces the p-hydroxyphenylbut-3-ene-2-one to
p-hydroxyphenylbutan-2-one, the raspberry ketone. We detected the activity
of both enzymes in crude extracts from raspberry fruits and their tissue
cultures, and identified their reaction products by HPLC, crystallization to
constant radioactivity and by GC-MS.
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Borejsza-Wysocki,
W., Hrazdina, G., 1996. Aromatic polyketide synthases. Purification,
characterization, and antibody development to benzalacetone synthase from
raspberry fruits. Plant Physiology 110, 791-799.
p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries
(Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and
malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by
benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and
G. Hrazdina 1994, Phytochemistry 35: 623-628). Benzalacetone synthase
condenses one malonate with p-coumarate to form the pathway intermediate p-
hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is
similar to those catalyzed by chalcone and stilbene synthases. We have
obtained an enzyme preparation from ripe raspberries that was preferentially
enriched in benzalacetone synthase (approximately 170-fold) over chalcone
synthase (approximately 14-fold) activity. This preparation was used to
characterize benzalacetone synthase and to develop polyclonal antibodies in
rabbits. Benzalacetone synthase showed similarity in its molecular
properties to chalcone synthase but differed distinctly in its substrate
specificity, response to 2-mercaptoethanol and ethylene glycol, and
induction in cell- suspension cultures. The product of the enzyme,
p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen
Phytophthora fragariae var rubi at 250 µM. We do not know whether the
dual activity in the benzalacetone synthase preparation is the result of a
bifunctional enzyme or is caused by contamination with chalcone synthase
that was also present. The rapid induction of the enzyme in cell-suspension
cultures upon addition of yeast extract and the toxicity of its product,
p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the
pathway may be part of a plant defense response.
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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:
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
(more...).
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Hrazdina, G., Zheng, D. S., 2006.
Expression and function of aromatic polyketide synthase genes in raspberries (Rubus
idaeus sp.). In: Rimando, A. M., Baerson, S. R. (Eds.), Polyketides:
Biosynthesis, Biological Activities and Genetic Engineering, American Chemical
Society, Washington, D.C., pp. 128-140.
The genus Rubus
contains a family of type III polyketide synthases which show differences in
structure and function. We have cloned and characterized five aromatic
polyketide synthase genes from raspberries. All five genes contain an intron
of varying length and have 1173 bp coding sequences, with the exception of
one gene that consists of 1149 bp. Four of the five genes encode proteins
with 391 amino acid residues with a calculated protein mass of 42 kDa, while
one gene coded for a shorter protein consisting of 383 amino acids. Sequence
comparison of the five polyketide synthase genes showed high similarity both
at the DNA and protein levels. Differences in the coding region were found
mainly in the flanking sequences. Analysis of the reaction products showed
that PKS1 and PKS5 were chalcone synthases, PKS2 that differs in six amino
acids from PKS1 is silent, PKS3 is a p-coumarate triacetic acid
lactone synthase (CTAS) and PKS4 is a benzalacetone synthase (BAS). The
structural variations and the architecture of these PKS genes and enzymes is
discussed.
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Jiang, C., Kim, S. Y., Suh, D.-Y., 2008. Divergent
evolution of the thiolase superfamily and chalcone synthase family.
Molecular Phylogenetics and Evolution 49,691-701.
Enzymes of the
thiolase superfamily catalyze the formation of carbon-carbon bond via the
Claisen condensation reaction. Thiolases catalyze the reversible
non-decarboxylative condensation of acetoacetyl-CoA from two molecules of
acetyl-CoA, and possess a conserved Cys-His catalytic diad. Elongation
enzymes (beta-ketoacyl-acyl carrier protein synthase (KAS) I and KAS II and
the condensing domain of polyketide synthase) have invariant Cys and two His
residues (CHH triad), while a Cys-His-Asn (CHN) triad is found in initiation
enzymes (KAS III, 3-ketoacyl-CoA synthase (KCS) and the chalcone synthase (CHS)
family). These enzymes all catalyze decarboxylative condensation reactions.
3-Hydroxyl-3-methylglutaryl-CoA synthase (HMGS) also contains the CHN triad,
although it catalyzes a non-decarboxylative condensation. That the enzymes
of the thiolase superfamily share overall similarity in protein structure
and function suggested a common evolutionary origin. All thiolases were
found to have, in addition to the Cys-His diad, either Asn or His (thus C(N/H)H)
at a position corresponding to the His in the CHH and CHN triads. In our
phylogenetic analyses, the thiolase superfamily was divided into four main
clusters according to active site architecture. During the functional
divergence of the superfamily, the active architecture was suggested to
evolve from the CHH in archaeal thiolases to the C(N/H)H in non-archaeal
thiolases, and subsequently to the CHH in the elongation enzymes and the CHN
in the initiation enzymes. Based on these observations and available
biochemical and structural evidences, a plausible evolutionary history for
the thiolase superfamily is proposed that includes the emergence of
decarboxylative condensing enzymes accompanied by a recruitment of the His
in the CHH and CHN triads for a catalytic role during decarboxylative
condensation. In addition, phylogenetic analysis of the plant CHS family
showed separate clustering of CHS and non-CHS members of the family with a
few exceptions, suggesting repeated gene birth-and-death and re-invention of
non-CHS functions throughout the evolution of angiosperms. Based on these
observations, predictions on the enzymatic functions are made for several
members of the CHS family whose functions are yet to be characterized.
Further, a moss CHS-like enzyme that is functionally similar to a
cyanobacterial enzyme was identified as the most recent common ancestor to
the plant CHS family.
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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.
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:
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
(more...).
Return
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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 coafactors 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 degrees 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 muM did not affect the enzyme activity. Apparent molecular weights of
approx. 120 000, 50 000, and 70 000, 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- methylsalicylic 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:
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
(more...).
Return
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Morita, H.,
Noguchi, H., Schröder, J., Abe, I., 2001. Novel polyketides
synthesized with a higher plant stilbene synthase. European Journal of
Biochemistry 268, 3759-3766.
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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Morita, H., Takahashi, Y., Noguchi, H., Abe, I., 2000. Enzymatic formation
of unnatural aromatic polyketides by chalcone synthase. Biochemical and
Biophysical Research Communications 279, 190-195.
Substrate specificity of recombinant chalcone synthase (CHS) from
Scutellaria baicalensis (Labiatae) was investigated using chemically
synthesized aromatic and aliphatic CoA esters. It was demonstrated for the
first time that CHS converted benzoyl-CoA to phlorobenzophenone
(2,4,6-trihydroxybenzophenone) along with pyrone by-products. On the other
hand, phenylacetyl-CoA was enzymatically converted to an unnatural aromatic
polyketide, phlorobenzylketone (2,4,6-trihydroxyphenylbenzylketone), whose
structure was finally confirmed by chemical synthesis. Furthermore, in
agreement with earlier reports, S. baicalensis CHS also accepted
aliphatic CoA esters, isovaleryl-CoA and isobutyryl-CoA, to produce
phloroacylphenones. In contrast, hexanoyl-CoA only afforded pyrone
derivatives without formation of a new aromatic ring. It was noteworthy that
both aromatic and aliphatic CoA esters were accepted in the active site of
the enzyme as a starter substrate for the complex condensation reaction. The
low substrate specificity of CHS thus provided further insight into the
structure and function of the enzyme.
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Saleh,
N. A. M., Fritsch, H., Kreuzaler, F., Grisebach, H., 1978. Flavanone synthase
from cell suspension cultures of Haplopappus gracilis and
comparison with the synthase from parsley. Phytochemistry 17, 183-186.
Flavanone synthase was isolated and purified
ca 62-fold from cell suspension
cultures of Haplopappus gracilis.
The enzyme preparation catalysed the formation of naringenin from
4-coumaryl-CoA and malonyl-CoA with a pH optimum of
ca 8. The same enzyme was also
capable of synthesizing eriodictyol from caffeyl-CoA and malonyl-CoA; in
this case the pH optimum lay between 6.5 and 7. The homogeneous flavanone
synthase from cell suspension cultures of parsley showed the same dependence
of the pH optimum on the nature of the cinnamyl-CoA. It can be concluded
that both naringenin and eriodictyol are natural products of the synthase
reaction.
Note:
The enzyme was first labelled as 'flavanone synthase', because the chalcone was so quickly converted in a non-enzymatic reaction to the flavanone that it was not detectable as the initial product. That was corrected a few years later with improved techniques
(see reference below), but the wrong name was used in the publications up to
that time: more...
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Schröder, J., Raiber, S., Berger, T., Schmidt, A.,
Schmidt, J., Soares-Sello, A. M., Bardshiri, E., Strack, D., Simpson, T. J.,
Veit, M., Schröder, G., 1998. 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.
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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C-Methylated Flavonoids
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Schröder,
J., 1999. The chalcone/stilbene synthase-type family of condensing enzymes.
In: Sankawa, U. (Ed.), Polyketides and Other Secondary Metabolites Including
Fatty Acids and Their Derivatives, Vol. 1.
Elsevier, Amsterdam, pp. 749-771.
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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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Suh,
D.-Y., Kagami, J., Fukuma, K., Iwanami, N., Yamazaki, Y., Yurimoto, H.,
Sakai, Y., Kato, N., Shibuya, M., Ebizuka, Y., Sankawa, U., 2000. Chalcone and
stilbene synthases expressed in eucaryotes exhibit reduced cross-reactivity in vitro. Chemical & Pharmaceutical Bulletin 48, 1051-1054.
Chalcone synthase (CHS) and stilbene synthase (STS) catalyze different
cyclization reactions of the common tetraketide to give different products,
naringenin chalcone and resveratrol, respectively. We have previously
observed in vitro cross-reaction of CHS and STS overexpressed in
Escherichia coli, resveratrol production by CHS and chalcone production
by STS. When expressed in eucaryotic cells, or in E. coli as
thioredoxin-fusion proteins, CHS and STS exhibited reduced cross-reaction.
STS refolded from inclusion bodies also showed reduced cross-reaction. While
addition of bovine serum albumin and pH in the reaction were without
noticeable effect, addition of glycerol decreased the cross-reaction of CHS
likely due to its stabilizing effect on enzyme conformation. These results
were interpreted to provide supporting evidence to our earlier proposition (Yamaguchi
T. et al., FEBS Lett., 460, 457-461 (1999)) that the in vitro cross-
reaction of CHS and STS is due to intrinsic capability of these enzymes to
catalyze different types of cyclization, which, in turn, is endowed by
conformational flexibility of their active sites.
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Yamaguchi, T., Kurosaki, F., Suh, D.-Y., Sankawa, U., Nishioka, M.,
Akiyama, T., Shibuya, M., Ebizuka, Y., 1999. Cross-reaction of chalcone
synthase and stilbene synthase overexpressed in Escherichia coli.
FEBS Letters 460, 457-461.
Chalcone synthase (CHS) and
stilbene synthase (STS) are related plant polyketide synthases belonging to
the CHS superfamily. CHS and STS catalyze common condensation reactions of
p-coumaroyl-CoA and three C-2-units from malonyl-CoA but different
cyclization reactions to produce naringenin chalcone and resveratrol,
respectively. Using purified Pueraria lobata CHS and Arachis
hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY,
the derailed lactone after two condensations) and p-coumaroyltriacetic acid
lactone (the derailed lactone after three condensations) were detected from
the reaction products. More importantly, we found a crossreaction between
CHS and STS, i.e. resveratrol production by CHS (2.7-4.2% of naringenin) and
naringenin production by STS (1.4-2.3% of resveratrol), possibly due to the
conformational flexibility of their active sites.
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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.
A chalcone
synthase (CHS)-like gene, SbCHS8, with high expressed sequence tag
abundance in a pathogen-induced cDNA library, was identified previously in
sorghum (Sorghum bicolor). Genomic Southern analysis revealed that
SbCHS8 represents a single-copy gene. SbCHS8 expression was
induced in sorghum mesocotyls following inoculation with Cochliobolus
heterotrophus and Colletotrichum sublineolum, corresponding to
nonhost and host defense responses, respectively. However, the induction was
delayed by approximately 24 h when compared to the expression of at least
one of the other SbCHS genes. In addition, SbCHS8 expression
was not induced by light and did not occur in a tissue-specific manner.
SbCHS8, together with SbCHS2, was overexpressed in transgenic
Arabidopsis (Arabidopsis thaliana) tt4 (transparent testa)
mutants defective in CHS activities. SbCHS2 rescued the ability of
these mutants to accumulate flavonoids in seed coats and seedlings. In
contrast, SbCHS8 failed to complement the mutation, suggesting that
the encoded enzyme does not function as a CHS. To elucidate their
biochemical functions, recombinant proteins were assayed with different
phenylpropanoid-Coenzyme A esters. Flavanones and stilbenes were detected in
the reaction products of SbCHS2 and SbCHS8, respectively. Taken together,
our data demonstrated that SbCHS2 encodes a typical CHS that
synthesizes naringenin chalcone, which is necessary for the formation of
different flavonoid metabolites. On the other hand, SbCHS8, now
retermed SbSTS1, encodes an enzyme with stilbene synthase activity,
suggesting that sorghum accumulates stilbene-derived defense metabolites in
addition to the well-characterized 3-deoxyanthocyanidin phytoalexins.
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Zheng, D., Hrazdina, G., 2008.
Molecular and
biochemical characterization of benzalacetone synthase and chalcone synthase
genes and their proteins from raspberry (Rubus idaeus L). Archives of
Biochemistry and Biophysics 470,
139-145.
Two new members of the polyketide synthase (PKS) gene family (RiPKS4
and RiPKS5) were cloned from raspberry fruits (Rubus idaeus L.,
cv Royalty) and expressed in E. coli. Characterization of the
recombinant enzyme products indicated that RiPKS4 is a
bifunctional polyketide synthase producing both 4-hydroxybenzalacetone and
naringenin chalcone. The recombinant RiPKS4 protein, like the native
protein from raspberry fruits accepted p-coumaryl-CoA and
ferulyl-CoA as starter substrates and catalyzed the formation of both
naringenin chalcone, 4-hydroxy-benzalacetone and
3-methoxy-4-hydroxy-benzalacetone. Although activity of RiPKS4 was
higher with ferulyl-CoA than with p-coumaryl-CoA, the corresponding
product, 3-methoxy-4-hydroxy phenylbutanone could not be detected in
raspberries to date. Sequence analysis of the genes and proteins suggested
that this feature of RiPKS4 was created by variation in the C-terminus
due to DNA recombination at the 3'region of its coding sequence. RiPKS5
is a typical chalcone synthase (CHS) that uses p-coumaryl-CoA only as
starter substrate and produces naringenin chalcone exclusively as the reaction
product.
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