A family of plant-specific polyketide synthases: facts and predictions

reviews

A family of plant-specific polyketide synthases: facts and predictions Joachim SchrSder The enzymes involved in the synthesis of chalcones, stilbenes and acridones are closely related plant-specific polyketide synthases. Evidence suggests that they belong to a family of condensing enzymes that catalyse the initial key reactions in the biosynthesis of a group of biologically and pharmaceutically interesting substances. Recent analysis has revealed that related sequences occur in bacteria, suggesting that the protein family is much older than previously assumed. halcone synthase catalyses the synthesis of the backbone required for a large number of biologically important substances, including those required for flower colour, protection from ultraviolet light, defence against pathogens, interaction with microorganisms and fertility. The effects of chalcone derivatives (e.g. the bioflavonoids) on humans has been investigated for decades, and many studies suggest positive effects on health. The enzyme is a relatively small homodimeric protein (subunit size 40-45 kDa). It uses a phenylpropanoid CoA-ester as its substrate and catalyses three sequential condensing reactions with malonyl-CoA in the synthesis of a tetraketide intermediate that folds into a new aromatic ring system (Fig. 1). The condensing reactions correspond to those in the biosynthesis of fatty acid and other polyketides, but there are significant differences. For example, chalcone synthase uses the CoA-esters directly rather than using acyl carrier protein derivatives to deliver the substrates for the condensing reactions. A diagram showing the condensing reaction, based on earlier results 1~ and the finding that chalcone synthase contains a single cysteine (Cys169) (Ref. 5) that is essential for catalysis, is presented in Fig. 2. The diagram shows that the 4-coumaroyt residue is transferred to the HS-group of Cys169 (confirmed by binding studies; T. Simpson, pers. commun.). The compound formed then reacts with an enzyme-stabilized acetyl-CoA carbanion formed by decarboxylation of malonyl-CoA, and the condensation product is a ~-keto CoA-ester that is often called a 'diketide' for simplicity. The subsequent two condensations follow the same principle after retransfer of the diketide or triketide residues to the active site Cys169. The retransfer appears to be a critical step, because byproducts resulting from the release of intermediates after one or two condensation reactions have often been observed in vitro ~ . Recent evidence suggests that there are enzymes that are specific for the synthesis of these substances. In most plants, chalcone synthesis does not involve a modification of enzyme-bound intermediates by other proteins. However, chalcone derivatives reduced at a specific position in the de novo synthesized aromatic ring are widespread in certain plants 9. The reduction is catalysed by an NADPH-dependent, monomeric reductase (35 kDa) that interacts with chalcone synthase to reduce a specific carbonyl group of an enzyme-bound intermediate after the second or third condensing reaction, probably prior to

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closure of the aromatic ring (Fig. 1). The oxygen is presumably lost by the removal of water during ring formation. The enzyme was named polyketide reductase to avoid the misleading name chalcone reductase 1°. It has been cloned from several plants, but most of the detailed functional studies have been performed with the soybean enzyme, either after purification from plant cells or after expression in other organisms 1~-13. The polyketide reductase has no similarity with enzymes that catalyse the reduction of the intermediates involved in the biosynthesis of fatty acids or other polyketides, but is similar to various aldo/keto-reductases, mostly from carbohydrate metabolism (30-39% identity) 12. The enzyme was apparently acquired from other pathways during the evolution of flavonoid biosynthesis. Proteins related to chalcone synthase

Stilbene synthase Stilbenes are rare in higher plants and occur in distantly related families. They contribute to the resistance of woody tissues to degradation, and in other plant parts act as phytoalexins and stress indicators 14. The stilbene backbone is synthesized via catalysis by stilbene synthase, an enzyme that is closely related to chalcone synthase (about 70% amino acid identity). The enzyme has been used to engineer new phytoalexins in transgenic plants 15. Recent findings suggest that stilbenes (in particular resveratrol; Fig. 1) might have uses in human cancer therap# 6. The reactions of chalcone synthase and stilbene synthase are identical up to the tetraketide stage, and the active site cysteine of the condensing reaction is in the same position in both proteins 5. The difference is in the formation of the new aromatic ring system: the intermediate is folded differently to form the stilbene-specific aromatic ring, and the mechanisms are formally different (acylation as opposed to aldol condensation; Fig. 1). All stilbene synthases analysed in vitro catalyse the removal of the terminal carboxyl group of the tetraketide. However, this is not an essential part of stilbene synthase-type ring closure, because stilbenoids containing the carboxyl group are well known ~4. Enzymes possessing both high chalcone synthase and stilbene synthase activities have not been found. They were also not detected during the conversion of chalcone synthase into stilbene synthase by site-directed mutagenesis ~7, or in attempts to produce hybrids between chalcone synthase and stilbene synthase subunits ~3. This indicates that there is an either/or switch in the chalcone synthase- and stilbene PII $1360-1385(97)01104-7

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synthase-type ring folding, although the mechanisms N-methylanthraniloyl-CoA and three malonyl-CoA molinvolved are not understood. It is likely that the present-day ecules to catalyse the synthesis of a new aromatic ring sysstilbene synthase in higher plants evolved from chalcone tem that is formed by chalcone synthase-type ring folding. The synthesis in vitro includes a second ring closure to form synthase on several independent occasions17. In view of the close relationship between chalcone syn- 1,3-dihydroxy-N-methylacridone (Fig. 3), but it is unclear thase and stilbene synthase, an intriguing question is whether this is caused by intrinsic activity of the enzyme or whether a comparable polyketide reductase exists in stil- a nonenzymatic process. The cDNA sequence for acridone bene biosynthesis. Various plants contain stilbene-type sub- synthase shows that it is a member of the polyketide synstances that lack one or even both of the hydroxyl groups thase family that also includes chalcone synthase and stilthat originate from the aromatization of the ring system 14. bene synthase (overall amino acid similarity to these two Interestingly, these substances often contain the carboxyl enzymes is approximately 65%). The enzyme function was group that is removed in the standard stilbene synthase confirmed by expression in Escherichia coli 19. reaction, and both carboxylated (e.g. lunularic acid) and decarboxylated forms (e.g. lunularin) are known in liver- Chalcone synthase-typeenzyme activitieswith other worts and other plants. Although not demonstrated experi- substrates mentally, the loss of the hydroxyl groups could be explained Benzophenonesynthase Xanthones are a group of natural products with interestby the action of a stilbene synthase-specific polyketide reductase. Figure I shows a proposal for shared reduction at ing pharmaceutical properties, and the majority occur in the same carbonyl group, as in the chalcone synthase- two plant families (Gentianaceae and Hypericaceae). Cell polyketide reductase interaction, with or without retention cultures from Centaurium erythraea and C. littorale were recently used to investigate xanthone biosynthesis. The first of the carboxyl group. reaction of the pathway has been proposed to involve the condensation of a benzoyl-CoA derivative with three malAcridone synthase Certain genera of the Rutaceae are the only plants that onyl-CoA molecules. The ring of the benzophenone formed contain acridone alkaloids. The formation of the acridone in this reaction is closed in the manner typical for chalcone backbone has been investigated with acridone synthase synthase 2° (Fig. 3). This was recently confirmed in vitro with ~urified from cell cultures of Ruta graveolens is. It uses a partially purified protein preparation incubated with 3-hydroxybenzoyl-CoA and malonyl-CoA (Ref. 21). The second ring closure to form CoAS....[I.,.~V[~OH the xanthone apparently O requires an additional 3X Malonyl-CoA+ lx 4-Coumaroyl-CoA enzyme. The enzyme also accepted benzoyl-CoA (44% 1 ~, PKR efficiency), but was comX~ (After condensation 2 or 3) pletely inactive with 2hydroxybenzoyl-CoA and 3 3 "°" °" 4-hydroxybenzoyl-CoA, indicating specificity for the 0 0 OH 0 o o o position of the hydroxyl Reduced group. It remains to be CHS ~ STS STS ~ CHS shown whether the protein is related to chalcone synthase, but a similarity is o ~ - , , ~ OH OH suggested by the reaction type and the finding that 0 0 0 OH 0 OH chalcone synthase isolated from plants not synthesizing benzophenone derivaOH rives also accepts benzoylCoA as a substrate for the synthesis of benzophenone OH (J. SchrSder, unpublished). Resveratrol Naringenin 6'-Deoxychalcone Reduced stilbene (stilbene) chalcone (carboxylicacid) Phloroisovalerophenone

~

Diketide

Tetraketide

;

;

Fig. 1. The principle of reactions catalysed by stilbene synthase (STS) and chalcone synthase (CHS) from the substrate 4-coumaroyl-CoA (products resveratrol and naringenin chalcone, respectively). Numbers 1-3 indicate the three sequential condensation reactions that occur in this process. The function of the polyketide reductase (PKR) that catalyses the reduction of a specific carbonyl group of an enzyme-bound intermediate, leading to 6'-deoxychalcone, is also shown. The analogous reaction in stilbene synthesis has not yet been demonstrated in vitro, but reduced products with and without the carboxyl group are known in nature.

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synthase and phloroisobutyrophenone synthase The ripe cones of hop (Humulus lupulus) contain up to 20% bitter acids, which are converted during the brewing process into the isoforms important for the

reviews flavour and taste of beer. Based on the detection of phloroisovalerophenone and phloroisobutyrophenone as intermediates, it has been suggested that the first reaction of the biosynthesis uses isovaleryl-CoA and isobutyryl-CoA to perform three condensations, followed by a chalcone synthase-type ring closure22 (Fig. 3). This was supported by the demonstration of the necessary enzyme activities in vitro ~3. Immunoblots with a chalcone synthase-specific antiserum revealed a band of approximately 45 kDa, the size expected for chalcone synthase and related proteins. Chalcone synthase activity was also present, but the activity with isovaleryl-CoA was always higher than with 4-coumaroyl-CoA. Also, the activity showed different kinetics during the development from flower buds to ripe cones. This indicates that chalcone synthase, phloroisovalerophenone synthase and phloroisobutyrophenone synthase are different proteins. Chalcone synthase from other plants also accepts linear CoA-esters as substrates 3, and therefore a clear separation of the activities is necessary. Enzymes that catalyse one or two condensation reactions

Styrylpyrone synthase Styrylpyrones are common in fungi, but also occur in pteridophytes and in angiosperm families. The pteridophyte Equisetum arvense is an interesting plant system because it shows a developmental switch24: gametophytes and rhizomes accumulate styrylpyrones as major phenolic constituents, but contain no flavonoids; and the green sprouts contain various flavonoid glycosides, but no styrylpyrones. Thus extracts from gametophytes are a suitable system in which to investigate styrylpyrone synthase without interference from chalcone synthases that use the same substrates. Feeding experiments in vivo suggest that styrylpyrones are synthesized from the same precursors as used by chalcone synthase, but with only two condensing reactions preceding ring closure (Fig. 3). Results with a partially purified protein preparation demonstrated that the enzyme used 4-coumaroyl-CoA or caffeoyl-CoA, and malonyl-CoA, to catalyse the synthesis of the styrylpyrones bisnoryangonin and hispidin ~5. The mechanism of pyrone ring formation from the postulated triketide intermediate is unknown. Interestingly, both substances have been described as dominant products of chalcone synthase reactions under certain assay conditions 3'~-8, suggesting that the pyrone formation may be nonenzymatic or an intrinsic property of chalcone synthase action if the reaction stops at the triketide level. An evolutionary relationship between styrylpyrone synthase and chalcone synthase has been proposed25, but it remains to be shown that the two proteins are actually related.

Benzalacetonesynthase The aroma of raspberries is caused by 4-hydroxyphenylbutan-2-one ('raspberry ketone'). The biosynthetic enzymes have been investigated using extracts from raspberry fruits and tissue cultures 26. The first enzyme in the pathway, benzalacetone synthase, uses a phenylpropanoid starter CoA-ester, catalyses one condensation with malonylCoA and releases the decarboxylated product benzalacetone27 (Fig. 3). The second enzyme catalyses the reduction of the double bond of the propenoyl moiety with NADPH to yield the aromatic component. A preparation enriched 172-fold for benzalacetone synthase and 14-fold for chalcone synthase was used for the

.o o

oH

MalonyI-CoA 4-CoumaroyI-CoA 002"~V CoASH~--~E-Cys-SH CoAS.,~e

E-Cys-

~ CoA~,,.,,~

----~ E-Cys-SH

OH

Diketide intermediate Fig. 2. Diagram showing the first of the three condensing reactions catalysed by stilbene synthase and chalcone synthase. The subsequent two condensations follow the same principle, after retransfer of the diketide or triketide intermediate to the active site cysteine. Abbreviation: E-Cys, active site of the condensing enzyme (Cys169) (Ref. 5).

characterization of benzalacetone synthase 27. Stained protein electrophoresis gels revealed a single band, and the native and denatured proteins had sizes of 83 kDa and 41.5 kDa, respectively; these are typical for chalcone synthasetype enzymes. The partial copurification of chalcone synthase does not yet allow a definite conclusion as to whether the chalcone synthase activity represents contamination or whether benzalacetone synthase can catalyse either one or three condensation reactions. The latter possibility raises interesting questions about the regulatory mechanism in vivo, but at least one line of evidence suggests two enzymes: benzalacetone synthase was strongly induced after treatment with yeast extract, but chalcone synthase was not increased at all. Benzalacetone has been identified as a byproduct of chalcone synthase reactions under certain conditions 7'8, suggesting that the decarboxylation of the released unstable diketide may be nonenzymatic. One attractive hypothesis is that benzalacetone synthase corresponds to a chalcone synthase with modifications that block the retransfer of the diketide to the active site of the condensing reaction. How large is this family of condensing enzymes? In vivo feeding studies with acetate and precursors from the phenylpropanoid pathway suggest that condensing reactions with phenylpropanoyl-CoA esters and malonyl-CoA may be the initial reactions in the biosynthesis of several other natural products. Of particular interest is the possibility that benzalacetone synthase-like reactions are involved, and that a phenylpropanoid-derived diketide is the initial product that is further modified. One interesting group of substances are the psilotins, which are arylpyrones found in the Psilotaceae (a group closely related to the ferns). It was proposed that their biosynthesis involves the condensation of a phenylpropanoid CoA-ester with one

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CoAS-~ R 0 Starter CoA-ester

CoAS-r%
O •

CoAS.~I.---~ R o o o

C02

0

Benzalacetone

SPS Styrylpyrone

C°AS-~~ql/R o o o o

.,ws

OH 0

PIBPS

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~ H3

ACS

BPS ~Hs CH2)n-CH 2 -CH 3

Acridone

H°@ OH

OH O

n=l: Phlorisovalerophenone n=0: Phlorisobutyrophenone

OH

0

Benzophenone

Fig. 3. Enzymes that catalyse one, two or three condensation reactions (numbers 1-3) with malonyl-CoA, each from a different starter substrate. R is the variable component of the starter substrate. The carbon atoms added by the condensing reactions are marked with dots. BAS (benzalacetone synthase) has 4-coumaroyl-CoAas the starter substrate, and the reaction involves one condensation before final production of benzalacetone. The rest of the enzymes are: SPS (styrylpyrone synthase), with 4-coumaroyl-CoAas the starter substrate and two condensations; ACS (acridone synthase), using N-methylanthraniloyl-CoA and three condensations; BPS (benzophenone synthase), using 3-hydroxybenzoyl-CoA and three condensations); and £IVPS and PIBPS (phloroisovalerophenone and phloroisobutyrophenone synthase, respectively), using isovaleryl-CoA and isobutyryl-CoA, respectively, and three condensations. acetyl unit from malonyl-CoA, followed by the reduction of a carbonyl group, formation of a double bond by water removal and ring closure~s (Fig. 4). Arylpyrones are also described as byproducts of chalcone synthase-catalysed reactions 3,~, and an attractive hypothesis is that a chalcone synthase-related enzyme catalyses the condensing reaction. The biosynthesis of other substances could involve a onestep condensation to produce a diketide and subsequent reactions that couple a benzalacetone synthase-type decarboxylation with the introduction of other residues. Precursor feeding studies with [6]-gingerol (the pungent component of ginger, Zingiber officinale) indicated that its biosynthesis uses ferulic acid, malonate and hexanoate residues, and that it includes two reduction steps ~9. The short-chain fatty acid is variable in the gingerol family, but the phenylpropanoid moiety is not, and therefore the scheme in Fig. 4 proposes a common phenylpropanoid diketide intermediate. Replacing the fatty acid with a 376

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phenylpropanoid unit and omitting the reductions could lead to curcumin (Fig. 4), the main pigment of turmeric (Curcuma longa). Similar C~-C7-C6 structures (diarylheptanoids) and the biosynthetically related phenylphenalenones are known from several other plants, including the genus Anigozanthos. As examples, Fig. 4 shows anigorufone and hydroxyanigorufone, which are major pigments in Anigozanthos preissii cell cultures. Recent experiments revealed that these are synthesized from two phenylpropanoid precursors via a diarylheptanoid intermediate, and that the central one-carbon unit originates from an acetyl residue 3°. It is possible that in diarylheptanoids this carbon is the remainder of a condensing reaction between the first phenylpropanoid unit and malonyl-CoA.

Lessons from sequences At present, public electronic databases contain > 100 chalcone synthase sequences from >40 plants; stilbene synthase sequences from five plants; and one acridone synthase sequence. Phylogenetic trees show that the large group of chalcone synthase proteins does not form a cluster that is clearly separate from either stilbene synthase or acridone synthase, and it is impossible to distinguish the enzymes by overall comparisons of the proteins 17. Essentially the same result is obtained in searches for differences in specific motifs. One of the problems is that too many chalcone synthase entries are solely identified by sequence similarity, and the suspected diagnostic signatures for stilbene synthase or acridone synthase mostly disappear if all sequences labelled as chalcone synthase are included in the analysis. The remaining amino acids are mostly single residues scattered throughout the protein, the functional significance of which is difficult to evaluate. Even the gene structures are yew similar: all chalcone synthase and stilbene synthase genes contain a conserved intron that splits a strictly conserved cysteine (Cys65 in the proteins). Crystal structures have yet to be obtained for any of the enzymes. The identification of a cloned protein as chalcone synthase, stilbene synthase, acridone synthase or another related enzyme therefore requires either genetic evidence or functional expression in another organism. The presence or absence of certain secondary products may provide some clues, but only a few plants have been thoroughly analysed. Stilbenes, for example, were recently described in monocotyledons, where they were not previously suspected to be present 31. It is likely that some of the sequences putatively identified as chalcone synthase on the basis of their sequence similarity actually encode related enzymes, and thus functional classification will be very interesting. The clone CHS2 from Gerbera hybrida is an example where the enzymatic function is certainly not as a chalcone synthase 32, although the protein has about 74% identity with two 'true' chalcone synthases from the same plant. The CHS2 protein had no activity with pheny]propanoid CoA-esters, but accepted benzoyl-CoA for the synthesis of an unidentified product. The in vivo substrate and role of CHS2 remain to be elucidated. Another example is provided by two chalcone synthase-type cDNAs from Pinus strobus (J. SchrSder, unpublished). The proteins are 87.6% identical, but expression in E. coli showed that only the CHS1 protein had chalcone synthase activity. The CHS2 protein was inactive with any phenylpropanoid substrates or other CoA-esters, even after purification of the protein close to homogeneity.

reviews How old is this enzyme family? There is no significant similarity between, on the one hand, chalcone synthase, stilbene synthase and acridone synthase and, on the other hand, condensing enzymes involved in the biosynthesis of other polyketides - even the active sites have only superficial similarity 5. They are also distinguished from most other polyketide synthases by the use of CoA-esters rather than acyl carrier protein (ACP) derivatives. This suggests a long interval of separate evolution, although they probably developed from ancestral condensing enzymes. An attractive evolutionary scenario for the pathway includes changes in the starter specificity (forming benzalacetone); changes in the capacity for chain elongation with two acetyl units (forming styrylpyrones) and three acetyl units (forming chalcones, stilbenes and others); and the concomitant development of protein structures for the stabilization of intermediates. Benzalacetone synthase and styrylpyrone synthase may be considered to formally represent these evolutionary stages, but the actual relationship has yet to be shown, and the present-day enzymes could also be more recent developments from proteins that catalyse three condensation reactions. It has been proposed that the present-day stilbene synthase evolved from chalcone synthase on several independent occasions17, and this must have involved complex changes in the protein to allow the formation of an entirely different ring system. Benzalacetone and styrylpyrones are byproducts of chalcone synthase reactions in vitro. At least in theory it could be argued that a loss of one or two of the three condensing reactions would require less complex changes. Interestingly, in this scenario the parent enzyme could be either chalcone synthase or stilbene synthase, because differences between them are only apparent after the third condensation reaction. It is thought that chalcone synthase (and thus chalcone formation) first appeared in Charophyceae or in simple bryophytes33, although there is no direct information on these enzymes. It is therefore interesting that related sequences are present in bacteria (e.g. Pseudomonas fluorescens 34, Bacillus subtilis ~5, Streptomyces griseus ~ and Mycobacterium tuberculosis (GenBank database accession numbers Z81011 and Z85982). The proteins are a rather diverse group (about 30% identity), but all of them share 25-30% overall identity with chalcone synthase-type proteins. Several motifs characteristic for these enzymes can be readily identified, and all contain a cysteine in the position corresponding to the cysteine of the active site in chalcone synthase and stilbene synthase 5. The reactions catalysed are not yet known, but the chalcone synthase-like gene phlD, which is located in the 6.5 kbp gene cluster responsible for the biosynthesis of 2,4-diacetylphloroglucino134, may provide the first indication. It seems a reasonable possibility that the encoded protein catalyses the synthesis of monoacetylphloroglucinol from acetyl-CoA and three malonyl-CoA molecules with a chalcone synthase-type ringfolding of the tetraketide intermediate. The successful demonstration of this activity in vitro would indicate that chalcone synthase-type reactions are probably much older than previously suspected. Conclusions The enzymes chalcone synthase, stilbene synthase and acridone synthase are the best-characterized examples of a family of related proteins that share the same type of condensation reaction. Benzophenone synthase, phloroiso-

R2

R2

R2 H3C~[CH2]n

OH O Curcumin RI=OH; R2=OCH3 Secondp h e n y l - ~ propanoidunit / ' ~

[6]-Gingerol

n=4; RI=OH;R2=OCH3

" cO2 ~

I

CoASH~

Sht~yrtch(~innit, 2x reduction

R2 C o A ~ ~

R1

SreCOnoien:'-

Diketide

R2

H20

~SC~A R1

• 002

OH

R2

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R1

o ~

CoASH

o ~ R 1

Diarylheptanoid

OH

R2

o

RI=OH, R2=H:Psilotinin RI=R2=OH:Hydroxypsilotinin

RI=R2=H:Anigorufone R1=OH:Hydroxyanigorufone

Fig. 4. DiagTam showing the possible involvement of a diketide intermediate in the biosynthesis of natural compounds. The carbon atoms added by the condensing reactions are marked with dots. valerophenone synthase, phloroisobutyrophenone synthase, styrylpyrone synthase and benzalacetone synthase are prominent candidates likely to be included in the family, but sequence analysis will be necessary to confirm this prediction. Other possibilities, such as in the biosynthesis of psilotinins, gingerols and diarylheptanoids, remain speculative at present. However, the results of precursor feeding studies are consistent with a proposal that the initial reaction may be a condensation of phenylpropanoid CoA-esters with malonyl-CoA. The concept of a protein family should now allow the direct application of molecular techniques to obtain related sequences and to identify unknown enzyme activities.

Acknowledgements Work in the author's lab is supported by the Deutsche Forschungsgemeinschaft and Fends der Chemischen Industrie. References 1 Kreuzaler, F. and Hahlbreck, K. (1975) Enzymic synthesis of an aromatic ring from acetate units. Partial purification and properties of flavanone synthase from cell-suspension cultures of Petroselinum hortense, Eur. J. Biochem. 56, 205-213

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2 Kreuzaler, F., Light, R.J, and Hahlbrock, K. (1978) Flavanone synthase catalyzes CQ exchange and decarboxytation of malonyl-CoA, FEBS Lett. 94, 175-178 3 Schtiz, R., Heller, W. and Hahlbrock, K. (1983) Substrate specificity of chalcone synthase from Petroselinum hortense. Formation of phloroglucinol derivatives from aliphatic substrates, J. Biol. Chem. 258, 6730-6734 4 Kreuzaler, F. et al. (1979) Flavanone synthase from Petroselinum hortense. Molecular weight, subunit composition, size of messenger RNA, and absence of pantetheinyl residue, Eur. J. Biochem. 99, 89-96 5 Lanz, T. et al. (1991) The role of cysteines in polyketide synthases: site-directed mutagenesis of resveratrol and chalcone synthases, two key enzymes in different plant-specific pathways, J. Biol. Chem. 266, 9971-9976 6 Kreuzaler, F. and Hahlbrock, K. (1975) 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, Arch. Biochem. Biophys. 169, 84-90 7 Hrazdina, G. et al. (1976) Substrate specificity of flavanone synthase from cell suspension cultures of parsley and structure of release products in vitro, Arch. Biochem. Biophys. 175, 392-399 8 Saleh, N.A.M. et al. (1978) Flavanone synthase from cell suspension cultures ofHaplopappus gracilis and comparison with the synthase from parsley, Phytochemistry 17, 183-186 9 Dewick, P.M. (1994) Isoflavonoids, in The Flavonoids: Advances in Research since 1986 (Harborne, J.B., ed.), pp. 117-238, Chapman & Hall 10 Heller, W. and Forkmann, G. (1994) Biosynthesis of flavonoids, in The Flavonoids: Advances in Research since 1986 (Harborne, J.B., ed.), pp. 499-535, Chapman & Hall ll Welle, R. and Grisebach, H. (1988) Isolation of a novel NADPHdependent reductase which coacts with chalcone synthase in the biosynthesis of 6'-deoxychalcone, FEBS Lett. 236, 221-225 12 Welle, R. et al. (1991) Induced plant responses to pathogen attack: analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean (Glycine max L. Merr. cv. Harosoy 63), Eur. J. Biochem. 196, 423-430 13 Tropf, S. et al. (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, J. Biol. Chem. 270, 7922-7928 14 Gorham, J. (1995) The Biochemistry of the Stilbenoids, Chapman & Halt 15 Hain, R. et al. (1993) Disease resistance results from foreign phytoalexin expression in a novel plant, Nature 361, 153-156 16 Jang, M. et al. (1997) Cancer chemopreventive activity of resveratrol, a natural product derived from grapes, Science 275, 218-220 17 Tropf, S. et al. (1994) Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution, J. Mol. Evol. 38, 610-618 18 Baumert, A. et al. (1994) Purification and properties of acridone synthase from cell suspension cultures of Ruta graveolens, Z. Naturforsch. 49c, 26-32 19 Junghanns, K.T. et al. (1995) Molecular cloning and recombinant expression of acridone synthase from elicited Ruta graveolens L. cell suspension cultures, Plant Mol. Biol. 27, 681-692 20 Sultanbawa, M.U.S. (1980) Xanthonoids of tropical plants, Tetrat~vdron 36, 1465-1506 21 Beerhues, L. (1996) Benzophenone synthase from cultured cells of Centaurium erythraea, FEBS Lett. 383, 264-266 22 Fung, S-Y. et al. (1994) Analysis of proposed aromatic precursors of hop bitter acids, J. Nat. Prod. 57, 452-459

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Joachim SchrSder is at the Institut fur Biologie II, Universit&t Freiburg, D-79104 Freiburg, Germany (tel ÷49 761 203 2691; fax +49 761 203 2601; e-mail [email protected]).

Plants on the Net The Botanical Society of America http://www.botany.org/ USDA National PLANTS Database and Projects http://plants.usda.gov/ Canadian Botanical Conservation Network http://www.science.mcmaster.ca/Biology/CBCN/ homepage.htmt