Biosynthesis of biphenyls and benzophenones - Evolution of benzoic acid-specific polyketide synthases in plants

LUDGER BEERHUES
Institut für Pharmazeutische Biologie
Technische Universität Braunschweig
Mendelssohnstraße 1
D-38106 Braunschweig
l.beerhues@tu-bs.de
http://www.ipb-bs.de

References
Liu, B., Falkenstein-Paul, H., Schmidt, W., Beerhues, L. (2003) Benzophenone synthase and chalcone synthase from Hypericum androsaemum cell cultures: cDNA cloning, functional expression, and site-directed mutagenesis of two polyketide synthases. Plant J. 34: 847-855.
Benzophenone derivatives such as polyprenylated benzoylphloroglucinols and xanthones are biologically active secondary metabolites. The formation of their C₁₃ skeleton is catalyzed by benzophenone synthase (BPS, EC 2.3.1.151) that has been cloned for the first time from cell cultures of Hypericum androsaemum. BPS is a novel member of the superfamily of plant polyketide synthases (PKSs), also termed type III PKSs, with 53-63% amino acid sequence identity. Heterologously expressed BPS was a homodimer with a subunit molecular mass of 42.8 kDa. Its preferred starter substrate was benzoyl-CoA that was stepwise condensed with three malonyl-CoAs to give 2,4,6-trihydroxybenzophenone. BPS did not accept activated cinnamic acids as starter molecules. In contrast, recombinant chalcone synthase (CHS, EC 2.3.1.74) from the same cell cultures used preferentially 4-coumaroyl-CoA and also converted CoA esters of benzoic acids. The enzyme shared 60.1% amino acid sequence identity with BPS. In a phylogenetic tree, the two PKSs occurred in different clusters. One cluster was formed by CHSs including the one from H. androsaemum. BPS grouped together with the PKSs that functionally differ from CHS. Site-directed mutagenesis of amino acids shaping the initiation/elongation cavity of CHS yielded a triple mutant (L263M/F265Y/S338G) that preferred benzoyl-CoA over 4-coumaroyl-CoA.

Liu, B., Beuerle, T., Klundt, T., Beerhues, L. (2004) Biphenyl synthase from yeast-extract-treated cell cultures of Sorbus aucuparia. Planta 218: 492-496.
Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The biphenyl aucuparin accumulated in Sorbus aucuparia cell cultures in response to yeast extract treatment. Incubation of cell-free extracts from challenged cell cultures with benzoyl-CoA and malonyl-CoA led to the formation of 3,5-dihydroxybiphenyl. This reaction was catalysed by a novel polyketide synthase which will be named biphenyl synthase. The most efficient starter substrate for the enzyme was benzoyl-CoA. Relatively high activity was also observed with 2-hydroxybenzoyl-CoA but, instead of the corresponding biphenyl, the derailment product 2-hydroxybenzoyltriacetic acid lactone was formed.

Beerhues, L., Abd El-Mawla, A., Beuerle, T., Boubakir, Z., Klingauf, P., Liu, B. (2005) Biosynthesis of active plant polyketide derivatives. Proceedings of the 9th International Congress "Phytopharm 2005" and the PSE Young Scientists Meeting on "Plants and Health" (Makarov, V.G., ed.), pp 39-46.
A number of polyprenylated polyketide derivatives exhibit interesting pharmacological properties and are difficult to synthesize due to their complex caged structures. Thus, they are attractive compounds for biotechnological research. For example, hyperforin is a major antidepressant constituent of Hypericum perforatum which is widely used for the treatment of mild to moderate depressions. The hyperforin skeleton is formed by isobutyrophenone synthase, a type III polyketide synthase (PKS). The first prenylation step is catalyzed by a soluble and ion-dependent dimethylallyl-transferase. These reactions have recently been detected in adhyperforin-containing cell cultures of H. calycinum.
Further type III PKSs in Hypericum species are chalcone synthase, the key enzyme of flavonoid biosynthesis, and benzophenone synthase (BPS). Their preferred starter substrates are 4-coumaroyl-CoA and benzoyl-CoA, respectively. Both enzymes were cloned and functionally expressed. BPS catalyzes the formation of phlorbenzophenone which can undergo stepwise prenylation and cyclization reactions. Many of the resulting caged compounds, e.g. garcinol, exhibit cytotoxic activity. Simple benzophenones can alternatively undergo regioselective oxidative phenol couplings to give xanthones. This class of compounds also contains caged molecules with cytotoxic properties such as gambogic acid. The intramolecular cyclizations are catalyzed by cytochrome P450 enzymes.
Another benzoic acid-specific type III PKS is biphenyl synthase (BIS). BIS and BPS form the same tetraketide intermediate which is subjected to either intramolecular aldol condensation by BIS or intramolecular Claisen condensation by BPS. BIS has also been cloned. Biphenyls are the phytoalexins of Rosaceae which include many important fruit trees such as apple and pear.
Biosynthesis of benzoic acids as precursors for PKSs is also studied. While benzoic acid in Hypericum species is formed via cinnamic acid, 3-hydroxybenzoic acid in Gentianaceae, another xanthone-producing family, is derived directly from the shikimate pathway.

Beerhues, L., Liu, B., Raeth, T., Klundt, T., Beuerle, T., Bocola, M. (2006) Benzoic acid-specific type III polyketide synthases. American Chemical Society Symposium Series 955 (Rimando, A.M., Baerson, S.R., eds.), pp 97-108.
Benzoic acid is a rare starter substrate for polyketide synthases. Benzophenone synthase (BPS) and biphenyl synthase (BIS) are plant type III PKSs that prefer benzoyl-CoA as starter substrate. They catalyze the iterative condensation of benzoyl-CoA with three molecules of malonyl-CoA to give identical linear tetraketides. BPS cyclizes this intermediate via an intramolecular Claisen condensation, whereas BIS catalyzes an intramolecular aldol condensation and decarboxylative elimination of the terminal carboxyl group. The enzymes share 54% amino acid sequence identity. They are inactive with 4-coumaroyl-CoA, the preferred starter molecule for the ubiquitous plant PKS, chalcone synthase (CHS). CHS-derived BPS and BIS homology models guided the generation of site-directed mutants. CHSs and functionally diverse PKSs were used for phylogenetic analysis. Benzophenones undergo either polyprenylation to give polycyclic caged molecules or regioselective intramolecular cyclizations to yield xanthones. Biphenyls are the phytoalexins of many economically important fruit trees, such as apple and pear.

Liu, B., Raeth, T., Beuerle, T., Beerhues, L. (2007) Biphenyl synthase, a novel type III polyketide synthase. Planta 225: 1495-1503.
Biphenyls and dibenzofurans are the phytoalexins of the Maloideae, a subfamily of the economically important Rosaceae. The carbon skeleton of the two classes of antimicrobial secondary metabolites is formed by biphenyl synthase (BIS). A cDNA encoding this key enzyme was cloned from yeast-extract-treated cell cultures of Sorbus aucuparia. BIS is a novel type III polyketide synthase (PKS) that shares about 60 % amino acid sequence identity with other members of the enzyme superfamily. Its preferred starter substrate is benzoyl-CoA that undergoes iterative condensation with three molecules of malonyl-CoA to give 3,5-dihydroxybiphenyl via intramolecular aldol condensation. BIS did not accept CoA-linked cinnamic acids such as 4-coumaroyl-CoA. This substrate, however, was the preferential starter molecule for chalcone synthase (CHS) that was also cloned from S. aucuparia cell cultures. While BIS expression was rapidly, strongly and transiently induced by yeast extract treatment, CHS expression was not. In a phylogenetic tree, BIS grouped together closely with benzophenone synthase (BPS) that also uses benzoyl-CoA as starter molecule but cyclizes the common intermediate via intramolecular Claisen condensation. The molecular characterization of BIS thus contributes to the understanding of the functional diversity and evolution of type III PKSs.

Charchoglyan, A., Abrahamyan, A., Fujii, I., Boubakir, Z., Gulder, T.A.M., Kutchan, T.M., Vardapetyan, H., Bringmann, G., Ebizuka, Y., Beerhues, L. (2007) Differential accumulation of hyperforin and secohyperforin in Hypericum perforatum tissue cultures. Phytochemistry, in press.
Hyperforin is a pharmacologically active constituent of Hypericum perforatum (St. John's wort). In vitro cultures of this medicinal plant were found to contain hyperforin and three related polyprenylated acylphloroglucinol derivatives. The accumulation of these compounds was coupled to shoot regeneration, with secohyperforin being the major constituent in morphogenic cultures. The structure of secohyperforin was elucidated online by LC-DAD, -MS, and -NMR. In multiple shoot cultures, the ratio of hyperforin to secohyperforin was strongly influenced by the phytohormones N⁶-benzylaminopurine (BAP) and naphthalene-1-acetic acid (NAA). While increasing concentrations of BAP stimulated the formation of hyperforin, increasing concentrations of NAA elevated the level of secohyperforin. No differential stimulation was observed after elicitor treatment. Hyperforin and secohyperforin are proposed to arise from a branch point in the biosynthetic pathway.

Beerhues, L., Liu, B. (2007) Polyketide metabolism in Hypericum perforatum and related species. Proceedings of the 11^th IAPTC&B congress (Xu, Z., Li, J., Vasil, I.K., Xue, Y., Yang, W., eds.), in press.
Extracts from St. John's wort (Hypericum perforatum L., Clusiaceae) are widely used for the treatment of mild to moderate depression. The major antidepressant constituent is hyperforin, a prenylated polyketide derivative and a novel broad-band neurotransmitter reuptake inhibitor. Clusiaceae are rich in polyprenylated phloroglucinol derivatives. Bridged polycyclic compounds with caged skeletons result from cyclizations of prenyl side chains. The nuclei of acyl- and benzoylphloroglucinols are formed by type III polyketide synthases (PKSs) which use isobutyryl-CoA and benzoyl-CoA, respectively, as starter substrates. In cell cultures of H. calycinum, three PKSs, isobutyrophenone synthase (BUS), benzophenone synthase (BPS), and chalcone synthase (CHS), were detected. cDNAs were cloned and the recombinant proteins functionally expressed. In a phylogenetic tree, CHSs and the functionally diverse PKSs form individual clusters. CHS-derived homology models are guiding the generation of site-directed mutants. The first prenylation step in hyperforin biosynthesis is catalyzed by a soluble and Fe²⁺-dependent dimethylallyl transferase. Besides polyprenylation, simple benzophenones can undergo intramolecular cyclizations to give xanthones. These regioselective oxidative phenol couplings are catalyzed by cytochrome P450 enzymes. P450s are also responsible for subsequent hydroxylations. Another novel PKS is biphenyl synthase which forms the same linear intermediate like BPS but catalyzes a different cyclization mechanism. Biphenyls are phytoalexins of Maloideae including economically important fruit trees such as apple and pear. One precursor of biphenyls and benzophenones is benzoic acid. In cell cultures of H. androsaemum, it is formed from cinnamic acid via a CoA-dependent and non-ß-oxidative route. Three CoA ligases with different substrate specificities were detected. Transformation and regeneration protocols are being established for H. perforatum.

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