Chalcone-synthase

5 1 Chalcone Synthase.- Enzyme preparations from parsley (Petrosellnum hortense) giving the flavanone naringenin from p-coumaroyl CoA and malonyl CoA have been shown to be contaminated with chalcone isomerase, and further purification has 

Matthiola incana has been correlated with interruption of 

CHS carries out a series of sequential decarboxylation and condensation reactions, using 4-courmaroyl-CoA (in most species) and three molecules of malonyl-CoA, to produce a poly-ketide intermediate that then undergoes cyclization and aromatization reactions that form the A-ring and the resultant chalcone structure. The chalcone formed from 4-courmaroyl-CoA is naringenin chalcone. However, enzyme preparations and recombinant CHS proteins from some species have been shown to accept other HCA-CoA esters as substrates, such as cinnamoyl-CoA (see, e.g., Ref. 37). In particular, the Hordeum vulgare (barley) CHS2 cDNA encodes a CHS protein that converts feruloyl-CoA and caffeoyl-CoA at the highest rate, and cinnamoyl-CoA and 4-courmaroyl-CoA at lower rates. 

The key role of CHS in flavonoid biosynthesis has made it a focus of research for many years, and it is now very well characterized. The isolation of a cDNA for CHS represented the first gene cloned for a flavonoid enzyme. CHS sequences, and a series of C//S -like sequences, have now been characterized from many species, and Austin and Noel have identified close to 650 C//S -like sequences in public databases. 

Native CHS is a homodimer with subunits of 40 to 44kDa. The structure of the protein produced from the CHS2 cDNA of M. sativa has been determined and the residues of the active site defined. It belongs to the polyketide synthase (PKS) group of enzymes that occur in bacteria, fungi, and plants, and is a type III PKS. All the reactions are carried out at a single active site without the need for cofactors. 

PKSs are characterized by their ability to catalyze the formation of polyketide chains from the sequential condensation of acetate units from malonate thioesters. In plants they produce a range of natural products with varied in vivo and pharmacological properties. PKSs of particular note include acridone synthase, bibenzyl synthase, 2-pyrone synthase, and stilbene synthase (STS). STS forms resveratrol, a plant defense compound of much interest with regard to human health. STS shares high sequence identity with CHS, and is considered to have evolved from CHS more than once. ° Knowledge of the molecular structure of the CHS-like enzymes has allowed direct engineering of CHS and STS to alter their catalytic activities, including the number of condensations carried out (reviewed in Refs. 46, 51, 52). These reviews also present extensive, and superbly illustrated, discussions of CHS enzyme structure and reaction mechanism. 

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Lawton, M.A., Dixon, R.A., Rowell, P.M., Bailey, J.A. Lamb, C.J. (1983). Rapid induction of the synthesis of phenylalanine ammonia-lyase and of chalcone synthase in elicitor-treated plant cells. European Journal of Biochemistry, 129, 593-601. 

Fig. 5 Scheme of the flavonoid pathway leading to synthesis of proanthocyanidins. The enzymes involved in the pathway are shown as follows CHS = chalcone synthase CHI = chalcone isomerase F3H = flavanone-3B-hydroxylase DFR = dihydroflavonol-4-reductase LDOX = leucoanthocynidin dioxygenase LAR = leucoanthocyanidin reductase ANR = anthocyanidin reductase adapted from [27] and [28]... 

Flavonoids are the largest class of phenylpropanoids in plants. The basic flavonoid structure is two aromatic rings (one from phenylalanine and the other from the condensation of three malonic acids) linked by three carbons (Fig. 3.6). Chalcone is converted to naringenin by the enzyme chalcone isomerase, which is a key enzyme in flavonoid synthesis. This enzyme, like PAL and chalcone synthase (CHS), is under precise control and is inducible by both internal and external signals. Naringenin is the... 

Figure 5.4. Abbreviated scheme for biosynthesis of major flavonoid subclasses, showing the primary enzymes and substrates leading to different subclasses. Bold-faced, uppercase abbreviations refer to enzyme names, whereas substrate names are presented in lowercase letters. PAL, phenylalanine ammonia lyase C4H, cinnamate 4-hydroxylase 4CL, 4-coumarate CoA ligase CHS, chalcone synthase CHI, chalcone isomerase CHR, chalcone reductase IPS, isoflavone synthase F3H, flavonone 3-hydroxylase F3 H, flavonoid 3 -hydroxylase F3 5 H, flavonoid 3 5 -hydroxylase FNSI/II, flavone synthase DFR, dihydroflavonol 4-reductase FLS, flavonol synthase ANS, anthocyanidin synthase LAR, leucoanthocyanidin reductase ANR, anthocyanidin reductase UFGT, UDP-glucose flavonoid 3-O-glucosyltransferase. R3 = H or OH. R5 = H or OH. Glc = glucose. Please refer to text for more information.

Austin MB and Noel IP. 2003. The chalcone synthase superfamily of type III polyketide synthases. Nat Prod Rep 20 79-110. 

Figure 6.1 Major branch pathways of flavonoid biosynthesis in Arabidopsis. Branch pathways, enzymes, and end products present in other plants but not Arabidopsis are shown in light gray. Abbreviations cinnamate-4-hydroxylase (C4H), chalcone isomerase (CHI), chalcone synthase (CHS), 4-coumarate CoA-ligase (4CL), dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), flavonoid 3 or 3 5 hydroxylase (F3 H, F3 5 H), leucoanthocyanidin dioxygenase (LDOX), leucoanthocyanidin reductase (LCR), O-methyltransferase (OMT), phenylalanine ammonia-lyase (PAL), rhamnosyl transferase (RT), and UDP flavonoid glucosyl transferase (UFGT).

Table 6.1 Abbreviations BAN, BANYULS bHLH, basic helix-loop-helix CHS, chalcone synthase CHI, chalcone isomerase DFR, dihydroflavonol reductase F3H, flavonol 3-hydroxylase F3 H, flavonoid 3 -hydroxylase FLS, flavonol synthase icx, increased chalcone synthase expression LDOX, leucoanthocyanidin dioxygenase LCR, leucoanthocyanidin reductase MATE, multidrug and toxic compound extrusion NR, not yet reported tt, transparent testa ttg, transparent testa glabrous the WD40 and WRKY transcription factors are named for conserved amino acid sequences within these proteins. PC = personal communication.

FEINBAUM, R.L., AUSUBEL, F.M., Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene, Mol. Cell. Biol., 1988, 8, 1985-1992. 

WADE, H.K., BIBIKOVA, T.N., VALENTINE, W.J., JENKINS, G.I., Interactions within a network of phytochrome, cryptochrome and UV-B phototransduction pathways regulate chalcone synthase gene expression in Arabidopsis leaf tissue, Plant J., 2001, 25, 675-685. 

SASLOWSKY, D.E., DANA, C.D., WINKEL-SHIRLEY, B., An allelic series for the chalcone synthase locus in Arabidopsis, Gene, 2000, 255,127-138. 

PELLETIER, M.K., SHIRLEY, B.W., Analysis of flavanone 3-hydroxylase in Arabidopsis seedlings Coordinate regulation with chalcone synthase and chalcone isomerase, Plant Physiol., 1996, 111, 339-345. 

CAIN, C.C., SASLOWSKY, D.E., WALKER, R.A., SHIRLEY, B.W., Expression of chalcone synthase and chalcone isomerase proteins in Arabidopsis seedlings, Plant Mol. Biol., 1997, 35,377-381. 

CL, 4-coumarate CoA ligase CHS, chalcone synthase CHI, chalcone isomerase F3H, flavanone 3-hydroxylase DFR, dihydroflavonol 4-reductase ANS, anthocyanidin synthase FGT, flavonoid 3-O-glucosyltransferase. 

Chalcone Synthase / Stilbene Synthase Family of Plant Polyketide Synthases. 199... 

Chalcone Synthase, a Model Plant Polyketide Synthase. 199... 

Recently, a new polyketide biosynthetic pathway in bacteria that parallels the well studied plant PKSs has been discovered that can assemble small aromatic metabolites.8,9 These type III PKSs10 are members of the chalcone synthase (CHS) and stilbene synthase (STS) family of PKSs previously thought to be restricted to plants.11 The best studied type III PKS is CHS. Physiologically, CHS catalyzes the biosynthesis of 4,2, 4, 6 -tetrahydroxychalcone (chalcone). Moreover, in some organisms CHS works in concert with chalcone reductase (CHR) to produce 4,2 ,4 -trihydroxychalcone (deoxychalcone) (Fig. 12.1). Both natural products constitute plant secondary metabolites that are used as precursors for the biosynthesis of anthocyanin pigments, anti-microbial phytoalexins, and chemical inducers of Rhizobium nodulation genes.12... 

In this chapter, we describe the atomic resolution structural elucidation of several plant type III polyketide synthases, including chalcone synthase, 2-pyrone synthase, and stilbene synthase. Manipulation of the catalytic activity and specificity of these biosynthetic enzymes by using a structurally guided approach offers a novel... 

CHALCONE SYNTHASE / STILBENE SYNTHASE FAMILY OF PLANT POLYKETIDE SYNTHASES... 

TROPF, S LANZ, T., RENSING, S.A., SCHRODER, J., SCHRODER, G., Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution, J. Mol. Evol., 1994,38, 610-618. 

FERRER, J.-L., JEZ, J.M., BOWMAN, M.E., DIXON, R.A., NOEL, J.P., Structure of chalcone synthase and the molecular basis of plant polyketide synthesis, Nature Struct. Biol., 1999,6, 775-784. 

AKIYAMA, T SHIBUYA, M., LIU, H.M., EBIZUKA, Y. p-Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii, Eur. J. Biochem., 1999, 263, 834-839. 

JEZ, J.M., NOEL, J.P., Mechanism of chalcone synthase pKa of the catalytic cysteine and the role of the conserved histidine in a plant polyketide synthase, J. Biol. Chem., 2000, 275, 39640-39646. 

LANZ T., TROPF, S., MARNER, F.J, SCHRODER, J., SCHRODER, G., 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., 1991, 266, 9971-9976. 

SUH, D.Y., KAGAMI, J., FUKUMA, K., SANKAWA, U., Evidence for catalytic cysteine-histidine dyad in chalcone synthase, Biochem. Biophy. Res. Comm., 2000, 275, 725-730. 

SCHUZ, R., HELLER, W., HAHLBROCK, K., Substrate specificity of chalcone synthase from Petroselinum hortense formation of phloroglucinol derivatives from aliphatic substrates, J. Biol. Chern., 1983,258, 6730-6734. 

ABE, I., MORITA, H NOMURA, A., NOGUCHI, H., Substrate specificity of chalcone synthase enzymatic formation of unnatural polyketides from synthetic cinnamoyl-CoA analogues, J. Am. Chem. Soc., 2000,122,11242-11243. 

MORITA, H., TAKAHASHI, Y., NOGUCHI, H., ABE, I., Enzymatic formation of unnatural aromatic polyketides by chalcone synthase, Biochem. Biophys. Res. Comm., 2000,279,190-195. 

CHRISTENSEN, A.B., GREGERSEN, P.L., SCHRODER, J., COLLINGE, D.B., A chalcone synthase with an unusual substrate preference is expressed in barley leaves in response to UV light and pathogen attack, Plant Mol. Biol., 1998,37, 849-857. 

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