(Last modification:
26.01.2010)
Type III PKS: A Superfamily of Polyketide Synthases in Plants, Bacteria, and Fungi
Our work was supported by grants from the Deutsche Forschungsgemeinschaft to Gudrun and Joachim Schröder
Click here for a complete list of our publications in this field!
Link to Index of type III PKS pages
Also have a look at: ---->
Book on Polyketide Synthases <----
Note: the overview in
this page focusses on our own work. However, the updates of our website (beginning 2007) extended that to a more general description of these interesting enzymes: follow the links on top of this page or look at the Index!
Brief Introduction/Overview
Plants, bacteria, and fungi contain a class of polyketide synthases (PKS) that appear to be unique in several aspects; they are often called Type III PKS because they do not really fit the definitions of type I or type II PKS: Type III PKS are relatively small dimeric proteins (subunit sizes about 40-45 kDa) that usually carry out iterative condensation reactions with malonyl-CoA; the numbers can range from one to seven. The basic mechanisms of their condensing reactions, follow the general model developed for all condensing enzymes. An overview of the diversity of the reactions can be found here.
In plants, the most well-known members of this superfamily (and actually the ones first described) are the enzymes synthesizing chalcones (CHS) and stilbenes
(STS); with the well-known and much discussed resveratrol the most prominent
product: more...). CHS catalyzes the first committed step in the biosynthesis of a large number of biologically important substances, e.g. flavonoids (flower colour) and phytoalexins (defense against pathogens). STS forms the backbone of the stilbene phytoalexins; these enzymes are rare in higher plants. The first sequences for a STS were published in 1988 (Schröder et al., 1988). The enzyme was called resveratrol synthase (RS) because resveratrol is the product from the preferred substrate 4-coumaroyl-CoA.
Very recent data raising lots of attention suggest that the stilbene resveratrol may have interesting applications in extending the life span of animals and man/woman. See some keywords and citations or go to resveratrol and its effects.
CHS and STS are plant-specific polyketide synthases. They perform, with a starter CoA-ester from the phenylpropanoid pathway, three sequential condensation reactions with malonyl-CoA, followed by a ring closure of a tetraketide intermediate to new aromatic ring systems. The condensing reactions are identical in CHS and STS, but the ring closures are different, leading either to a chalcone or a stilbene. The sequences of the two enzymes are closely related, and most likely
many if not all present-day STS in higher plants evolved from CHS several times
independently in the course of evolution (Tropf et al., 1994).
This may be true for plant STS in the strict sense, but the type of
ring-folding to a resorcinol ring system is apparently much older: Several
type III PKS in bacteria are known to carry out this reaction (more...).
In particular the enzymes using long-chain fatty acids are of interest: they
might well be the evolutionary ancestors. In this context it should be noted
that there are also bacterial type III PKS that carry out a CHS-type
ring-folding (more...).
A fascinating topic is that CHS and STS are only the well-known members of a superfamily of related proteins that use the same type of condensing reaction, but with widely varying substrates (e.g. phenylpropanoid-CoA esters, benzoyl-CoA derivatives, linear CoA-esters) and serving quite different pathways in natural product biosynthesis. There are also CHS/STS-type enzymes programmed for only one or two condensation reactions. Taken together, the use of different starter substrates, modifications of intermediates, and programming for one, two, or three condensing reactions indicates a large variety of products that can be synthesized by proteins of the CHS/STS-type enzyme superfamily (Schröder 1997,
Schröder
2000). The active site and its environment are highly conserved in all members of the family; see the
PROSITE pattern.
The superfamily concept also allowed predictions for not yet identified enzymes involved in the formation of compounds already described as natural plant products. What is interesting about the
aroma of raspberries? It is synthesized by an enzyme that carries out only one condensation reaction to a diketide! Most important: such a diketide intermediate must also be postulated in the biosynthesis of many other interesting natural products: more...
As noted aboe, type III PKS proteins are also present in bacteria (more...) and fungi (more...), and their analysis might tell us ultimately a lot about the evolution of the superfamily.
An excellent review of the field at that time was published in 2003: Austin et al. (2003), but there are lots of new findings and reports after that.
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Examples demonstrating the versatility of functions in the superfamily
-> Link to an overview of the type III PKS reactions in plants
Evidence that type III PKSs are involved in the biosynthesis of such complex compounds as diarylheptanoids and phenylphenalenones
What is so interesting about it? Precursor feeding experiments suggested years ago that in all of these cases the biosynthesis should involve an initial, single condensation reaction to a diketide intermediate, just like in the biosynthesis of the aroma component of raspberries (see above). However, in the biosynthesis of diarylheptanoids and phenylphenalenones the intermediate is then processed further to complex and widely divergent end products. The work described in that paper provides the first directe evidence that this is indeed correct! More...
Link to publication
The folding to different end products with stilbene synthases (STSs) and chalcone synthases (CHSs): what are the mechanisms?
The reactions of the two enzymes are identical up to the tetraketide stage (see the comparison). What then is really responsible for the different ring-folding to the end product? One would think the answer should not be that complicated, but it really was: It took the group of Noel several years, even with the availability of crystal structures, to figure out a model explaining the difference. As it turns out, electronic effects rather than steric factors balance the competing cyclization specificities in CHS and STS.
Click here for going to a few pages summarizing the results: the crystal structure of a STS is published !
Link to citation and abstract or go to
resveratrol and its effects.
Stilbenecarboxylate
biosynthesis: an interesting new function in the family of chalcone synthase related proteins
This is an interesting variation in the versatile functions in the protein family: Eckermann et al. (2003b). The STS reaction is of particular interest because a similar activity can be predicted for the biosynthesis of stilbenecarboxylic acids. These substances and their derivatives are abundant in liverworts, but they also occur in some higher plants. Some of them have interesting properties, e.g.
phyllodulcin is a substance 600 to 800-fold sweeter than sucrose.
More on Hydrangea macrophylla and the liverwort Marchantia polymorpha.
Certain herbicides bind covalently to the active site cysteines of CHS and STS
Quite an interesting, new development! Previous data suggested that chloroacetamide herbicides act by covalent binding to the active site cysteines in the complex enzyme system responsible for the elongation of very-long-chain-fatty-acids. However, these enzyme complexes are membrane-bound and not easily amenable to a more detailed analysis. Active site cysteines are well-known and identified from the family of CHS-related proteins, and thus we investigated this, in a wonderful collaboration with colleagues in Konstanz ("the Herbicide people") and Braunschweig ("the mass specialist"). The results (Eckermann et al., 2003a) are complex, but with CHS and STS they were quite clear-cut: the chloroacetamide herbicide metazachlor indeed couples covalently to the active site cysteine, as shown by MS/MS analysis of tryptic digests!
The protein family contains a pyrone synthase (2PS) which synthesizes the backbone of gerberin in Gerbera hybrida
The enzyme performs only two condensation reactions, with acetyl-CoA as the physiological substrate (Eckermann et al., 1998). If you don't remember how Gerbera looks like: take a look at a flower!
Take a look at the latest data, from a very pleasant collaboration with the Noel group in USA: the crystal structure of the pyrone synthase shows how substrate specificity and number of condensation reactions are controlled in these proteins, and the work also demonstrates how to convert a chalcone synthase into a pyrone synthase (Jez
et al., 2000): you need modifications in just three amino acids!!! If you don't have time to read the paper: The results are summarized in a)
sequence comparison: what are key residues determining the size of the active site pocket (i.e. possible substrate size and number of condensation reaction), b) the mutagenesis experiments (conversion of CHS to 2PS activity), and c) model: comparison of the size and shape of the active site pockets in CHS and 2PS.
Note: Essential for this work was the availability of the crystal structure of CHS: it was published in 1999 by the group of J. Noel (Nature Struct. Biol. 6: 775-784, 1999): Excellent work that facilitates attempts to understand the diversity of functions in the family of CHS-related proteins (see comment in
Schröder 1999), and it will facilitate the design of enzymes with new properties.
C-methylated chalcones are probably synthesized by CHS-type enzymes with special properties
(Schröder et al., 1998).
This was discovered after the identification of two CHS-type cDNAs in Pinus strobus: the two proteins were 87.6% identical, but only one was a true CHS (Pinus strobus CHS1). The second was inactive with any of the typical substrates. Interestingly, P. strobus contains unusual C-methylated flavonoids. Their biosynthesis can be explained by a reaction sequence in which methylmalonyl-CoA instead of malonyl-CoA is used in the second condensation reaction as chain extender. The results suggest that CHS2 indeed carries out this condensation reaction with methylmalonyl-CoA. More...
Biosynthesis of isoflavonoids: the key reaction is the formation of 6'-deoxychalcone
This has been a long-standing puzzle which was resolved several years ago. It is the only reaction known sofar in which CHS coacts with another protein: with a NADPH-dependent reductase which reduces a CHS reaction intermediate prior to formation of the chalcone ring system (Welle et al. 1991). One of these proteins has now been crystallized, resulting in interesting proposals: More...
Why is a CHS-type reaction important for
beer?
The valerophenone synthase (VPS) in the cones from hop (Humulus lupulus) uses linear CoA-esters (e.g. isovaleryl-CoA) to form with a CHS-type reaction the backbone of the bitter acids in hop, and these are important factors for the taste and flavor of beer. The substrates are aliphatic CoA-esters: will typical CHS accept them also? The data with purified CHSs (e.g. from Pinus sylvestris) show that they do, suggesting that minor changes are sufficient to convert a CHS into a VPS (Zuurbier et al., 1998). Valerophenone synthases have been cloned, and the sequences confirmed that they belong to the CHS-superfamily: More...
Can a single amino acid difference change important properties of a stilbene synthase?
Yes, it can: see Raiber et al., 1995: the description of two enzymes from Pinus strobus, where a single Arg-His exchange makes a drastic difference in the enzyme properties. Interestingly, the crystal structure of CHS (published 1999) showed that we identified a residue that is involved in the binding of the CoA-moieties of the substrates! More...
Why are CHS and STS dimers?
Experiments with heterodimers show that a single active site in a dimer is sufficient to catalyze the formation of the end product (chalcone or stilbene). It seems likely that the subunits interact in the final formation of the end products. See Tropf et al. (1995) for the summary of the data on some mutants. The crystal structures of CHS and STS are clearly consistent with the dimer model.
Can one make new and interesting substances with these enzymes?
Take a look at the Abstract of a paper resulting from a very pleasant collaboration with a group in Japan. The work investigates the activities of a stilbene synthase with unusual substrates:
Morita
et al. (2001).
CHS-related proteins in bacteria
This is a fascinating topic that will be of interest for some time to come. More...
Or go to a specific publication where I was involved: Biosynthesis of
dihydroxyphenylglycine.
Note: this enzyme carries out an STS-type ring-folding, but it is
pretty unusual: more...!
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Review
Austin,M.B.; Noel,J.P.: The chalcone synthase superfamily of type III polyketide synthases. Natural Product Reports 20, 79-110 (2003)
Covering 1970–2001. This review covers the functionally diverse type III polyketide synthase (PKS) superfamily of plant and bacterial biosynthetic enzymes, from the discovery of chalcone synthase (CHS) in the 1970s through the end of 2001. A broader perspective is achieved by a comparison of these CHS-like enzymes to mechanistically and evolutionarily related families of enzymes, including the type I and type II PKSs, as well as the thiolases and -ketoacyl synthases of fatty acid metabolism. As CHS is both the most frequently occurring and best studied type III PKS, this enzyme's structure and mechanism is examined in detail. The in vivo functions and biological activities of several classes of plant natural products derived from chalcones are also discussed. Evolutionary mechanisms of type III PKS divergence are considered, as are the biological functions and activities of each of the known and functionally divergent type III PKS enzyme families (currently twelve in plants and three in bacteria). A major focus of this review is the integration of information from genetic and biochemical studies with the unique insights gained from protein X-ray crystallography and homology modeling. This structural approach has generated a number of new predictions regarding both the importance and mechanistic role of various amino acid substitutions observed among functionally diverse type III PKS enzymes.
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