Stilbenecarboxylate biosynthesis: a new function in the family of chalcone synthase-related proteins.

Chalcone (CHS), stilbene (STS) synthases, and related proteins are key enzymes in the biosynthesis of many secondary plant products. Precursor feeding studies and mechanistic rationalization suggest that stilbenecarboxylates might also be synthesized by plant type III polyketide synthases; however, the enzyme activity leading to retention of the carboxyl moiety in a stilbene backbone has not yet been demonstrated. Hydrangea macrophylla L. (Garden Hortensia) contains stilbenecarboxylates (hydrangeic acid and lunularic acid) that are derived from 4-coumaroyl and dihydro-4-coumaroyl starter residues, respectively. We used homology-based techniques to clone CHS-related sequences, and the enzyme functions were investigated with recombinant proteins. Sequences for two proteins were obtained. One was identified as CHS. The other shared 65-70% identity with CHSs and other family members. The purified recombinant protein had stilbenecarboxylate synthase (STCS) activity with dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA or other substrates. We propose that the enzyme is involved in the biosynthesis of lunularic acid. It is the first example of a STS-type reaction that does not lose the terminal carboxyl group during the ring folding to the end product. Comparisons with CHS, STS, and a pyrone synthase showed that it is the only enzyme exerting a tight control over decarboxylation reactions. The protein contains unusual residues in positions highly conserved in other CHS-related proteins, and mutagenesis studies suggest that they are important for the structure or/and the catalytic activity. The formation of the natural products in vivo requires a reducing step, and we discuss the possibility that the absence of a reductase in the in vitro reactions may be responsible for the failure to obtain stilbenecarboxylates from substrates like 4-coumaroyl-CoA.

[1]  Joachim Schröder,et al.  Molecular and biochemical characterization of three aromatic polyketide synthase genes from Rubus idaeus , 2001, Plant Molecular Biology.

[2]  U. Sankawa,et al.  Evidence for catalytic cysteine-histidine dyad in chalcone synthase. , 2000, Biochemical and biophysical research communications.

[3]  Y. Yamazaki,et al.  Chalcone and stilbene synthases expressed in eucaryotes exhibit reduced cross-reactivity in vitro. , 2000, Chemical and pharmaceutical bulletin.

[4]  H. Noguchi,et al.  Deoxychalcone synthase from cell suspension cultures of Pueraria lobata , 1988 .

[5]  J. Schröder A family of plant-specific polyketide synthases: facts and predictions , 1997 .

[6]  S. Horinouchi,et al.  Properties and Substrate Specificity of RppA, a Chalcone Synthase-related Polyketide Synthase in Streptomyces griseus * , 2002, The Journal of Biological Chemistry.

[7]  F. Lynen,et al.  Biosynthese von 6‐Methylsalicylsäure , 1970 .

[8]  I. Abe,et al.  Enzymatic formation of unnatural aromatic polyketides by chalcone synthase. , 2000, Biochemical and biophysical research communications.

[9]  Richard A. Dixon,et al.  Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis , 1999, Nature Structural Biology.

[10]  G. Hrazdina,et al.  Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits) , 1996, Plant physiology.

[11]  W. Wohlleben,et al.  A Polyketide Synthase in Glycopeptide Biosynthesis , 2001, The Journal of Biological Chemistry.

[12]  A. Schmidt,et al.  Plant polyketide synthases: a chalcone synthase-type enzyme which performs a condensation reaction with methylmalonyl-CoA in the biosynthesis of C-methylated chalcones. , 1998, Biochemistry.

[13]  R. Welle,et al.  Isolation of a novel NADPH‐dependent reductase which coacts with chalcone synthase in the biosynthesis of 6′‐deoxychalcone , 1988 .

[14]  C. Walsh,et al.  Glycopeptide antibiotic biosynthesis: Enzymatic assembly of the dedicated amino acid monomer (S)-3,5-dihydroxyphenylglycine , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[15]  I. Abe,et al.  Benzalacetone synthase. A novel polyketide synthase that plays a crucial role in the biosynthesis of phenylbutanones in Rheum palmatum. , 2001, European journal of biochemistry.

[16]  N. Murakami,et al.  Thunberginols C, D, and E, new antiallergic and antimicrobial dihydroisocoumarins, and thunberginol G 3'-O-glucoside and (-)-hydrangenol 4'-O-glucoside, new dihydroisocoumarin glycosides, from Hydrangeae Dulcis Folium. , 1992, Chemical & pharmaceutical bulletin.

[17]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[18]  Takumi Akiyama,et al.  Cross‐reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli , 1999, FEBS letters.

[19]  J. Gorham The biochemistry of the stilbenoids , 1995 .

[20]  Y. Ebizuka,et al.  p-Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii. , 1999, European journal of biochemistry.

[21]  C. Khosla,et al.  Malonyl-CoA:ACP transacylase from Streptomyces coelicolor has two alternative catalytically active nucleophiles. , 2001, Biochemistry.

[22]  Isao Fujii,et al.  A new pathway for polyketide synthesis in microorganisms , 1999, Nature.

[23]  H. Kindl,et al.  Zur Biosynthese pflanzlicher Stilbene, 2. Mitt.: Die Bildung von Ring A des Hydrangenols , 1962 .

[24]  Joachim Schröder,et al.  Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution , 1994, Journal of Molecular Evolution.

[25]  A. Udagawa,et al.  NAD(P)H-dependent 6'-deoxychalcone synthase activity in Glycyrrhiza echinata cells induced by yeast extract. , 1988, Archives of biochemistry and biophysics.

[26]  Y. Yamazaki,et al.  Identification of amino acid residues important in the cyclization reactions of chalcone and stilbene synthases. , 2000, The Biochemical journal.

[27]  R. Dixon,et al.  Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. , 2000, Biochemistry.

[28]  H. Shimoda,et al.  Development of bioactive functions in hydrangeae dulcis folium. V. On the antiallergic and antimicrobial principles of hydrangeae dulcis folium. (2). Thunberginols C, D, and E, thunberginol G 3'-O-glucoside, (-)-hydrangenol 4'-o-glucoside, and (+)-hydrangenol 4'-O-glucoside. , 1996, Chemical & pharmaceutical bulletin.

[29]  R. Verpoorte,et al.  4-Hydroxy-2-pyrone formation by chalcone and stilbene synthase with nonphysiological substrates. , 1998, Phytochemistry.

[30]  I. Abe,et al.  Novel polyketides synthesized with a higher plant stilbene synthase. , 2001, European journal of biochemistry.

[31]  H. Shimoda,et al.  Development of bioactive functions in Hydrangeae dulcis folium. VI. Syntheses of thunberginols A and F and their 3'-deoxy-derivatives using regiospecific lactonization of stilbene carboxylic acid: structures and inhibitory activity on histamine release of hydramacrophyllols A and B. , 1996, Chemical & pharmaceutical bulletin.

[32]  D. Oesterhelt,et al.  Reaction of yeast fatty acid synthetase with iodoacetamide. 3. Malonyl-coenzyme A decarboxylase as product of the reaction of fatty acid synthetase with iodoacetamide. , 1977, European journal of biochemistry.

[33]  G. Schröder,et al.  Induced plant responses to pathogen attack , 1991 .

[34]  H. Shimoda,et al.  Dihydroisocoumarin Constituents from the Leaves of Hydrangea macrophylla var. thunbergii. (2).: Absolute Stereostructures of Hydrangenol, Thunberginol I, and Phyllodulcin Glycosides and Isomerization Reaction at the 3-Positions of Phyllodulcin and Its Glycosides. , 1999 .

[35]  H. Shimoda,et al.  Development of bioactive functions in hydrangeae dulcis folium. III. On the antiallergic and antimicrobial principles of hydrangeae dulcis folium. (1). Thunberginols A, B, and F. , 1994, Chemical & pharmaceutical bulletin.

[36]  Y. Ohta,et al.  The concentrations of lunularic acid and prelunularic acid in liverworts , 1984 .

[37]  R. J. Pryce Biosynthesis of lunularic acid—a dihydrostilbene endogenous growth inhibitor of liverworts , 1971 .

[38]  J. Schröder Chapter Three - The Family of Chalcone Synthase-Related Proteins: Functional Diversity and Evolution , 2000 .

[39]  P. Dimroth,et al.  6-Methylsalicylic acid synthetase from Penicillium patulum. Some catalytic properties of the enzyme and its relation to fatty acid synthetase. , 1976, European journal of biochemistry.

[40]  Y. Helariutta,et al.  New pathway to polyketides in plants , 1998, Nature.

[41]  三川 潮 Polyketides and other secondary metabolites including fatty acids and their derivatives , 1999 .

[42]  G. Towers,et al.  Studies of hydrangenol in Hydrangea macrophylla Ser. II. Biosynthesis of hydrangenol from C14-labelled compounds. , 1962, Canadian Journal of Biochemistry and Physiology.

[43]  J. Noel,et al.  Mechanism of Chalcone Synthase , 2000, The Journal of Biological Chemistry.

[44]  H. Komura,et al.  Prelunularic acid in liverworts , 1984 .

[45]  M. Austin,et al.  Structural control of polyketide formation in plant-specific polyketide synthases. , 2000, Chemistry & biology.

[46]  Ralph G. Nuzzo,et al.  ADSORPTION OF BIFUNCTIONAL ORGANIC DISULFIDES ON GOLD SURFACES , 1983 .