Michael B. Austin

The chalcone synthase superfamily of type III polyketide synthases

This review covers the functionally diverse type III polyketide synthase (PKS) superfamily of plant and bacterial biosynthetic enzymes. from the discovery of chalcone synthase (CHS) in the 1970s through the end of 2001. A broader perspective is achieved by a comparison of these CHS-like enzymes to mechanistically and evolutionarily related families of enzymes, including the type I and type II PKSs, as well as the thiolases and beta-ketoacyl synthases of fatty acid metabolism. As CHS is both the most frequently occurring and best studied type III PKS, this enzymes structure and mechanism is examined in detail. The in vivo functions and biological activities of several classes of plant natural products derived from chalcones are also discussed. Evolutionary mechanisms of type III PKS divergence are considered, as are the biological functions and activities of each of the known and functionally divergent type III PKS enzymc families (currently twelve in plants and three in bacteria). A major focus of this review is the integration of information from genetic and biochemical studies with the unique insights gained from protein X-ray crystallography and homology modeling. This structural approach has generated a number of new predictions regarding both the importance and mechanistic role of various amino acid substitutions observed among functionally diverse type III PKS enzymes.

Structural control of polyketide formation in plant-specific polyketide synthases

BACKGROUND Polyketide synthases (PKSs) generate molecular diversity by utilizing different starter molecules and by controlling the final length of the polyketide. Although exploitation of this mechanistic variability has produced novel polyketides, the structural foundation of this versatility is unclear. Plant-specific PKSs are essential for the biosynthesis of anti-microbial phytoalexins, anthocyanin floral pigments, and inducers of Rhizobium nodulation genes. 2-Pyrone synthase (2-PS) and chalcone synthase (CHS) are plant-specific PKSs that share 74% amino acid sequence identity. 2-PS forms the triketide methylpyrone from an acetyl-CoA starter molecule and two malonyl-CoAs. CHS uses a p-coumaroyl-CoA starter molecule and three malonyl-CoAs to produce the tetraketide chalcone. Our goal was to elucidate the molecular basis of starter molecule selectivity and control of polyketide length in this class of PKS. RESULTS The 2.05 A resolution crystal structure of 2-PS complexed with the reaction intermediate acetoacetyl-CoA was determined by molecular replacement. 2-PS and CHS share a common three-dimensional fold, a set of conserved catalytic residues, and similar CoA binding sites. However, the active site cavity of 2-PS is smaller than the cavity in CHS. Of the 28 residues lining the 2-PS initiation/elongation cavity, four positions vary in CHS. Point mutations at three of these positions in CHS (T197L, G256L, and S338I) altered product formation. Combining these mutations in a CHS triple mutant (T197L/G256L/S338I) yielded an enzyme that was functionally identical to 2-PS. CONCLUSIONS Structural and functional characterization of 2-PS together with generation of a CHS mutant with an initiation/elongation cavity analogous to 2-PS demonstrates that cavity volume influences the choice of starter molecule and controls the final length of the polyketide. These results provide a structural basis for control of polyketide length in other PKSs, and suggest strategies for further increasing the scope of polyketide biosynthetic diversity.

Structural Elucidation of Chalcone Reductase and Implications for Deoxychalcone Biosynthesis

4,2′,4′,6′-tetrahydroxychalcone (chalcone) and 4,2′,4′-trihydroxychalcone (deoxychalcone) serve as precursors of ecologically important flavonoids and isoflavonoids. Deoxychalcone formation depends on chalcone synthase and chalcone reductase; however, the identity of the chalcone reductase substrate out of the possible substrates formed during the multistep reaction catalyzed by chalcone synthase remains experimentally elusive. We report here the three-dimensional structure of alfalfa chalcone reductase bound to the NADP+ cofactor and propose the identity and binding mode of its substrate, namely the non-aromatized coumaryl-trione intermediate of the chalcone synthase-catalyzed cyclization of the fully extended coumaryl-tetraketide thioester intermediate. In the absence of a ternary complex, the quality of the refined NADP+-bound chalcone reductase structure serves as a template for computer-assisted docking to evaluate the likelihood of possible substrates. Interestingly, chalcone reductase adopts the three-dimensional structure of the aldo/keto reductase superfamily. The aldo/keto reductase fold is structurally distinct from all known ketoreductases of fatty acid biosynthesis, which instead belong to the short-chain dehydrogenase/reductase superfamily. The results presented here provide structural support for convergent functional evolution of these two ketoreductases that share similar roles in the biosynthesis of fatty acids/polyketides. In addition, the chalcone reductase structure represents the first protein structure of a member of the aldo/ketoreductase 4 family. Therefore, the chalcone reductase structure serves as a template for the homology modeling of other aldo/keto-reductase 4 family members, including the reductase involved in morphine biosynthesis, namely codeinone reductase.

Evolving biosynthetic tangos negotiate mechanistic landscapes

The dependence of polyketide synthase and terpene cyclase mechanistic adaptation on the chemistry of their oligomeric substrates illuminates a convergent evolutionary strategy for shaping cyclization in these otherwise disparate reactions. Evolution of these enzyme families relies on rhythmic tangos, in which the enzymes and substrates together determine product outcome by negotiating decision networks governing intrinsic and induced chemical reactivities.

Structure and mechanism of chalcone synthase-like polyketide synthases

Polyketide synthases (PKS) produce an array of natural products with different biological activities and pharmacological properties by varying the starter and extender molecules that form the final polyketide. Recent studies of the simplest PKS, the chalcone synthase (CHS)-like enzymes involved in the biosynthesis of flavonoids, anthocyanin pigments, and antimicrobial phytoalexins, have yielded insight on the molecular basis of this biosynthetic versatility. Understanding the structure–function relationship in these PKS provides a foundation for manipulating polyketide formation and suggests strategies for further increasing the scope of polyketide biosynthetic diversity. Journal of Industrial Microbiology & Biotechnology (2001) 27, 393–398.

Multiple Biochemical and Morphological Factors Underlie the Production of Methylketones in Tomato Trichomes

Genetic analysis of interspecific populations derived from crosses between the wild tomato species Solanum habrochaites f. sp. glabratum, which synthesizes and accumulates insecticidal methylketones (MK), mostly 2-undecanone and 2-tridecanone, in glandular trichomes, and cultivated tomato (Solanum lycopersicum), which does not, demonstrated that several genetic loci contribute to MK metabolism in the wild species. A strong correlation was found between the shape of the glandular trichomes and their MK content, and significant associations were seen between allelic states of three genes and the amount of MK produced by the plant. Two genes belong to the fatty acid biosynthetic pathway, and the third is the previously identified Methylketone Synthase1 (MKS1) that mediates conversion to MK of β-ketoacyl intermediates. Comparative transcriptome analysis of the glandular trichomes of F2 progeny grouped into low- and high-MK-containing plants identified several additional genes whose transcripts were either more or less abundant in the high-MK bulk. In particular, a wild species-specific transcript for a gene that we named MKS2, encoding a protein with some similarity to a well-characterized bacterial thioesterase, was approximately 300-fold more highly expressed in F2 plants with high MK content than in those with low MK content. Genetic analysis in the segregating population showed that MKS2s significant contribution to MK accumulation is mediated by an epistatic relationship with MKS1. Furthermore, heterologous expression of MKS2 in Escherichia coli resulted in the production of methylketones in this host.

Metabolite Induction of Caenorhabditis elegans Dauer Larvae Arises via Transport in the Pharynx

Caenorhabditis elegans sense natural chemicals in their environment and use them as cues to regulate their development. This investigation probes the mechanism of sensory trafficking by evaluating the processing of fluorescent derivatives of natural products in C. elegans. Fluorescent analogs of daumone, an ascaroside, and apigenin were prepared by total synthesis and evaluated for their ability to induce entry into a nonaging dauer state. Fluorescent imaging detailed the uptake and localization of every labeled compound at each stage of the C. elegans life cycle. Comparative analyses against natural products that did not induce dauer indicated that dauer-triggering natural products accumulated in the cuticle of the pharnyx. Subsequent transport of these molecules to amphid neurons signaled entry into the dauer state. These studies provide cogent evidence supporting the roles of the glycosylated fatty acid daumone and related ascarosides and the ubiquitous plant flavone apigenin as chemical cues regulating C. elegans development.

Emergent Decarboxylase Activity and Attenuation of α/β-Hydrolase Activity during the Evolution of Methylketone Biosynthesis in Tomato

Methylketones are insecticidal compounds that accumulate in certain plants as defense against herbivorous pests. Methylketone biosynthesis is a two-step process within the plant, with the second step, decarboxylation of 3-keto acids, catalyzed by Methylketone Synthase1 (MKS1). This study focuses on understanding the enzymatic mechanism of MKS1, which differs from proteins of the same structural class. Specialized methylketone-containing metabolites accumulate in certain plants, in particular wild tomatoes in which they serve as toxic compounds against chewing insects. In Solanum habrochaites f. glabratum, methylketone biosynthesis occurs in the plastids of glandular trichomes and begins with intermediates of de novo fatty acid synthesis. These fatty-acyl intermediates are converted via sequential reactions catalyzed by Methylketone Synthase2 (MKS2) and MKS1 to produce the n-1 methylketone. We report crystal structures of S. habrochaites MKS1, an atypical member of the α/β-hydrolase superfamily. Sequence comparisons revealed the MKS1 catalytic triad, Ala-His-Asn, as divergent to the traditional α/β-hydrolase triad, Ser-His-Asp. Determination of the MKS1 structure points to a novel enzymatic mechanism dependent upon residues Thr-18 and His-243, confirmed by biochemical assays. Structural analysis further reveals a tunnel leading from the active site consisting mostly of hydrophobic residues, an environment well suited for fatty-acyl chain binding. We confirmed the importance of this substrate binding mode by substituting several amino acids leading to an alteration in the acyl-chain length preference of MKS1. Furthermore, we employ structure-guided mutagenesis and functional assays to demonstrate that MKS1, unlike enzymes from this hydrolase superfamily, is not an efficient hydrolase but instead catalyzes the decarboxylation of 3-keto acids.

Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incana R. Br. (Brassicaceae)

For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/anthocyanin biosynthesis pathway belonging to the group of type III polyketide synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in E. Scherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes.

Chapter Five – Tomato Glandular Trichomes As a Model System for Exploring Evolution of Specialized Metabolism in a Single Cell

The tomato glandular trichomes function as a site of biosynthesis as well as the site of storage of specialized metabolites. This chapter aims to further study the mechanisms that underlie the divergence of primary metabolism (fatty acid biosynthesis) to specialized metabolism (methylketones) in these cells. Molecular factors that regulate this divergence will be identified by combining quantitative trait loci (QTL) analysis of interspecific crosses, derived from crosses between wild species and the cultivated tomato, with transcriptosome and proteome analysis of the glandular trichomes. This integrated approach will develop an infrastructure that will be used to import natural insecticide compounds into the cultivated varieties, and will set a model on how to explore specialized metabolism in other plants.