2-Pyrone synthase

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... 

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. 

Fig. 1 Biosynthesis of plant polyphenols by CHS-superfamily type III PKSs. CHS chalcone synthase STS stilbene synthase ACS acridone synthase VPS valerophenone synthase SPS styrylpyrone synthase BPS benzophenone synthase OAS olivetolic acid synthase 2PS 2-pyrone synthase BAS benzalacetone synthase CUS curcumin synthase...

The 2-pyrone synthase (2-PS) found in several plant species is the simplest Type III PKS.ii i 2-PS condenses three malonyl-CoAs and yields a spontaneously cyclized triketide lactone (TKL). The first malonyl-CoA is decarboxylated and serves as the acetate primer, analogous to the decarboxylative mechanism observed in Type II PKSs. The 2-PS is unable to accept larger starter units due to a considerable smaller active site than that of CHS. Mutating the residues that surround the CHS-binding pocket to those found in 2-PS yielded a mutant CHS that is biochemically indistinguishable from 2-PS." ... 

Figure 6 HMGS containing biosynthetic pathways. Portions of the PKS and PKS/NRPS pathways where the HMGS and related enzymes are located. Abbreviations A - Adenylation, AGP - acyl carrier protein, AT - acyltransferase, Cy - cyciization, DH - dehydratase, ER - enoyl reductase, GNAT -CCN5-related N-acetyltransferase, KS - ketosynthase, KR - ketoreductase, MT - methyltransferase. Ox - Oxidase, Oxy - Oxygenase, PGP - peptide carrier protein, PhyH - phytanoyl-CoA dioxygenase, PS - pyrone synthase, TE - thioesterase, - unknown function, - inactive domain.

Flavonoids serve as flower pigments, UV protectants, phytoalexins and signal molecules (1,2). Unlike BPS, CHS exhibits a broad substrate specificity (Table I). H. androsaemum CHS and the enzymes from other sources (27) accepted CoA-linked benzoic acids as minor subshates and formed the respective benzophenones. An efficient but unphysiological starter unit is benzoyl-CoA for 2-pyrone synthase (2-PS) that performs only two decarboxylative condensations with malonyl-CoA to produce 6-phenyl-4-hydroxy-2-pyrone (28). In cell cultures of H. androsaemum, benzoic acid originates from cinnamic acid by side-chain degradation (29). The underlying mechanism is CoA-dependent and non-) -oxidative. The complete sequence of enzymes involved was detected. 

Further analysis of the raspberry PKS genes has shown that the proteins coded by the five genes have the seven conserved cysteines at positions 30,60,84,130,164,190 and 341. Cysteines at these conserved locations are likely to have structural functions, because in addition to the CHS from which the consensus sequence derives, they were found at identical or similar locations in Arachis hypogea STS, in the Gerbera hybrida 2-pyrone synthase (2PS), in the Ruta graveolens acridone synthase (ACS), the Rheum palmatum benzalacetone synthase (BAS) and in the aleosone synthase (ALS) (Abe et al., 2004). 

Type I and Type II PKSs catalyze multiple rounds of reactions by catalytic modules encoded either by a single polypeptide (PKS I) or on separate polypeptides (PKS II) by analogy to FAS-I and FAS-II. In contrast, PKS Ills are dimers of KASs that catalyze multiple condensation reactions in one active site and include chalcone synthase, stilbene synthase, and 2-pyrone synthase (see Chapters 1.05, 1.07, and 1.04). In the case of chalcone synthase, three consecutive condensation reactions each utilizing malonyl-CoA, followed by a cyclization reaction, lead to the formation of 4, 2, 4, 6 -tetrahydroxychalcone from 4-hydroxycinnamoyl-CoA (Figure 3). Recruitment of a reductase leads to the formation of a product lacking the 6 -hydroxy group, a reaction that requires an intermediate in the synthesis of chalcone to dissociate from the synthase active site. 

Fig. 2 Reaction types catalyzed by plant type III polyketide synthases. PS, pyrone synthase CHS, chalcone synthase STS, stilbene synthase VPS, valerophenone synthase ACS, acridone synthase BPS, benzophe-none synthase...

Scheme 2.29 Biosynthesis of 6-methyi-4-hydroxy-2-pyrone (2-PS, 2-pyrone synthase).

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