Emergent decarboxylase activity and attenuation of α/ß-hydrolase activity during the evolution of methylketone biosynthesis in tomato.

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/ß-hydrolase superfamily. Sequence comparisons revealed the MKS1 catalytic triad, Ala-His-Asn, as divergent to the traditional α/ß-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.

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 beta-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 MKS2's 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.

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.

Type III polyketide synthase beta-ketoacyl-ACP starter unit and ethylmalonyl-CoA extender unit selectivity discovered by Streptomyces coelicolor genome mining.

Polyketide synthases (PKSs) are involved in the biosynthesis of many important natural products. In bacteria, type III PKSs typically catalyze iterative decarboxylation and condensation reactions of malonyl-CoA building blocks in the biosynthesis of polyhydroxyaromatic products. Here it is shown that Gcs, a type III PKS encoded by the sco7221 ORF of the bacterium Streptomyces coelicolor, is required for biosynthesis of the germicidin family of 3,6-dialkyl-4-hydroxypyran-2-one natural products. Evidence consistent with Gcs-catalyzed elongation of specific beta-ketoacyl-ACP products of the fatty acid synthase FabH with ethyl- or methylmalonyl-CoA in the biosynthesis of germicidins is presented. Selectivity for beta-ketoacyl-ACP starter units and ethylmalonyl-CoA as an extender unit is unprecedented for type III PKSs, suggesting these enzymes may be capable of utilizing a far wider range of starter and extender units for natural product assembly than believed until now.

Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase.

Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.

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.

Structure-function relationships in plant phenylpropanoid biosynthesis.

Plants, as sessile organisms, evolve and exploit metabolic systems to create a rich repertoire of complex natural products that hold adaptive significance for their survival in challenging ecological niches on earth. As an experimental tool set, structural biology provides a high-resolution means to uncover detailed information about the structure-function relationships of metabolic enzymes at the atomic level. Together with genomic and biochemical approaches and an appreciation of molecular evolution, structural enzymology holds great promise for addressing a number of questions relating to secondary or, more appropriately, specialized metabolism. Why is secondary metabolism so adaptable? How are reactivity, regio-chemistry and stereo-chemistry steered during the multi-step conversion of substrates into products? What are the vestigial structural and mechanistic traits that remain in biosynthetic enzymes during the diversification of substrate and product selectivity? What does the catalytic landscape look like as an enzyme family traverses all possible lineages en route to the acquisition of new substrate and/or product specificities? And how can one rationally engineer biosynthesis using the unique perspectives of evolution and structural biology to create novel chemicals for human use?

An aldol switch discovered in stilbene synthases mediates cyclization specificity of type III polyketide synthases.

Stilbene synthase (STS) and chalcone synthase (CHS) each catalyze the formation of a tetraketide intermediate from a CoA-tethered phenylpropanoid starter and three molecules of malonyl-CoA, but use different cyclization mechanisms to produce distinct chemical scaffolds for a variety of plant natural products. Here we present the first STS crystal structure and identify, by mutagenic conversion of alfalfa CHS into a functional stilbene synthase, the structural basis for the evolution of STS cyclization specificity in type III polyketide synthase (PKS) enzymes. Additional mutagenesis and enzymatic characterization confirms that electronic effects rather than steric factors balance competing cyclization specificities in CHS and STS. Finally, we discuss the problematic in vitro reconstitution of plant stilbenecarboxylate pathways, using insights from existing biomimetic polyketide cyclization studies to generate a novel mechanistic hypothesis to explain stilbenecarboxylate biosynthesis.

Crystal structure of a bacterial type III polyketide synthase and enzymatic control of reactive polyketide intermediates.

In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthlene synthase (THNS) catalyzes the iterative condensation of five CoA-linked malonyl units to form a pentaketide intermediate. THNS subsequently catalyzes dual intramolecular Claisen and aldol condensations of this linear intermediate to produce the fused ring tetrahydroxynaphthalene (THN) skeleton. The type III PKS-catalyzed polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic triad in an internal active site cavity, is fairly well understood. However, the mechanistic basis for the unusual production of THN and dual cyclization of its malonyl-primed pentaketide is obscure. Here we present the first bacterial type III PKS crystal structure, that of Streptomyces coelicolor THNS, and identify by mutagenesis, structural modeling, and chemical analysis the unexpected catalytic participation of an additional THNS-conserved cysteine residue in facilitating malonyl-primed polyketide extension beyond the triketide stage. The resulting new mechanistic model, involving the use of additional cysteines to alter and steer polyketide reactivity, may generally apply to other PKS reaction mechanisms, including those catalyzed by iterative type I and II PKS enzymes. Our crystal structure also reveals an unanticipated novel cavity extending into the "floor" of the traditional active site cavity, providing the first plausible structural and mechanistic explanation for yet another unusual THNS catalytic activity: its previously inexplicable extra polyketide extension step when primed with a long acyl starter. This tunnel allows for selective expansion of available active site cavity volume by sequestration of aliphatic starter-derived polyketide tails, and further suggests another distinct protection mechanism involving maintenance of a linear polyketide conformation.

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 enzyme's 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.

Plant-like biosynthetic pathways in bacteria: from benzoic acid to chalcone.

Although phenylpropanoids and flavonoids are common plant natural products, these major classes of biologically active secondary metabolites are largely absent from bacteria. The ubiquitous plant enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are key biosynthetic catalysts in phenylpropanoid and flavonoid assembly, respectively. Until recently, few bacterial counterparts were known, thus reflecting the dearth of these plant natural products in bacteria. This review highlights our progress on the biochemical and genetic characterization of recently identified streptomycete biosynthetic pathways to benzoic acid and type III polyketide synthase (PKS)-derived products. The sediment-derived bacterium "Streptomyces maritimus" produces benzoyl-CoA in a plant-like manner from phenylalanine involving a PAL-mediated reaction through cinnamic acid during the biosynthesis of the polyketide antibiotic enterocin. All but one of the genes encoding benzoyl-CoA biosynthesis in "S. maritimus" have been cloned, sequenced, and inactivated, providing a model for benzoate biosynthesis not only in this bacterium, but in plants where benzoic acid is an important constituent of many products. The recent discovery that bacteria harbor homodimeric PKSs belonging to the plant CHS superfamily of condensing enzymes has further linked the biosynthetic capabilities of plants and bacteria. A bioinformatics approach led to the prediction that the model actinomycete Streptomyces coelicolor A3(2) contains up to three type III PKSs. Biochemical analysis of one of the recombinant type III PKSs from S. coelicolor demonstrated activity as a 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). A homology model of THNS based upon the known three-dimensional structure of CHS was constructed to explore the structural and mechanistic details of this new subclass of bacterial PKSs.