A plant type III polyketide synthase that produces pentaketide chromone

A novel plant-specific type III polyketide synthase (PKS) that catalyzes formation of a pentaketide chromone, 5,7-dihydroxy-2-methylchromone, from five molecules of malonyl-CoA, was cloned and sequenced from aloe (Aloe arborescens). Site-directed mutagenesis revealed that Met207 (corresponding to Thr197 in CHS) determines the polyketide chain length and the product specificity of the enzyme; remarkably, replacement of a single amino acid residue, Met207, with Gly yielded a mutant enzyme that efficiently produces aromatic octaketides, SEK4 and SEK4b, the products of the minimal PKS for actinorhodin (act from Streptomyces coelicolor), from eight molecules of malonyl-CoA. This provided new insights into the catalytic functions and specificities of the CHS-superfamily type III PKS enzymes.     

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.     

Enzymatic formation of unnatural novel polyketide scaffolds by plant-specific type III polyketide synthase

The catalytic potential of octaketide synthase (OKS), a plant-specific type III polyketide synthase (PKS) from Aloe arborescens, was investigated by phenylacetyl-CoA and benzoyl-CoA as starter substrates. As a result, a novel C₁₆ pentaketide coumarin was produced from phenylacetyl-CoA, whereas benzoyl-CoA was not a good substrate of OKS. Remarkably, a structure-guided OKS N222G mutant dramatically extended the product chain length to yield four novel polyketides including C₂₂ aromatic octaketides from the C₆-C₂ phenylacetyl starter, as well as a novel C₁₉ heptaketide benzophenone from the C₆-C₁ benzoyl starter.     

Enzymatic formation of quinolone alkaloids by a plant type III polyketide synthase

[Structure: see text] Benzalacetone synthase from Rheum palmatum efficiently catalyzed condensation of N-methylanthraniloyl-CoA (or anthraniloyl-CoA) with malonyl-CoA (or methylmalonyl-CoA) to produce 4-hydroxy-2(1H)-quinolones, a novel alkaloidal scaffold produced by a type III polyketide synthase (PKS). Manipulation of the functionally divergent type III PKSs by a nonphysiological substrate thus provides an efficient method for production of pharmaceutically important quinolone alkaloids.     

Exploiting the reaction flexibility of a type III polyketide synthase through in vitro pathway manipulation

A synthetic metabolic pathway has been constructed in vitro consisting of the type III polyketide synthase from Streptomyces coelicolor and peroxidases from soybean and Caldariomyces fumago (chloroperoxidase). This has resulted in the synthesis of the pentaketide flaviolin and its dimeric derivative, and a wide range of pyrones and their coupled derivatives with flaviolin, as well as their halogenated derivatives. The addition of acyl-CoA oxidase to the pathway prior to the polyketide synthase resulted in unsaturated pyrone side chains, further broadening the product spectrum that can be achieved. The approach developed in this work, therefore, provides a new model to exploit biocatalysis in the synthesis of complex natural product derivatives.     

Structure-based engineering of a plant type III polyketide synthase: formation of an unnatural nonaketide naphthopyrone

Pentaketide chromone synthase (PCS) from Aloe arborescens is a novel plant-specific type III polyketide synthase (PKS) that produces 5,7-dihydroxy-2-methylchromone from five molecules of malonyl-CoA. On the basis of the crystal structures of wild-type and M207G mutant PCS, the F80A/Y82A/M207G triple mutant was constructed and shown to produce an unnatural novel nonaketide naphthopyrone by sequential condensations of nine molecules of malonyl-CoA. This is the first demonstration of the formation of a nonaketide by the structurally simple type III PKS. A homology model predicted that the active-site cavity volume of the triple mutant is increased to 4 times that of the wild-type PCS.     

Structure-based dissociation of a type I polyketide synthase module

Individual modules of modular polyketide synthases (PKSs) such as 6-deoxyerythronolide B synthase (DEBS) consist of conserved, covalently linked domains separated by unconserved intervening linker sequences. To better understand the protein-protein and enzyme-substrate interactions in modular catalysis, we have exploited recent structural insights to prepare stand-alone domains of selected DEBS modules. When combined in vitro, ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP) domains of DEBS module 3 catalyzed methylmalonyl transfer and diketide substrate elongation. When added to a minimal PKS, ketoreductase domains from DEBS modules 1, 2, and 6 showed specificity for the beta-ketoacylthioester substrate, but not for either the ACP domain carrying the polyketide substrate or the KS domain that synthesized the substrate. With insights into catalytic efficiency and specificity of PKS modules, our results provide guidelines for constructing optimal hybrid PKS systems.     

Engineered biosynthesis of plant polyketides: chain length control in an octaketide-producing plant type III polyketide synthase

The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) produces a variety of plant secondary metabolites with remarkable structural diversity and biological activities (e.g., chalcones, stilbenes, benzophenones, acrydones, phloroglucinols, resorcinols, pyrones, and chromones). Here we describe an octaketide-producing novel plant-specific type III PKS from aloe (Aloe arborescens) sharing 50-60% amino acid sequence identity with other plant CHS-superfamily enzymes. A recombinant enzyme expressed in Escherichia coli catalyzed seven successive decarboxylative condensations of malonyl-CoA to yield aromatic octaketides SEK4 and SEK4b, the longest polyketides known to be synthesized by the structurally simple type III PKS. Surprisingly, site-directed mutagenesis revealed that a single residue Gly207 (corresponding to the CHS's active site Thr197) determines the polyketide chain length and product specificity. Small-to-large substitutions (G207A, G207T, G207M, G207L, G207F, and G207W) resulted in loss of the octaketide-forming activity and concomitant formation of shorter chain length polyketides (from triketide to heptaketide) including a pentaketide chromone, 2,7-dihydroxy-5-methylchromone, and a hexaketide pyrone, 6-(2,4-dihydroxy-6-methylphenyl)-4-hydroxy-2-pyrone, depending on the size of the side chain. Notably, the functional diversity of the type III PKS was shown to evolve from simple steric modulation of the chemically inert single residue lining the active-site cavity accompanied by conservation of the Cys-His-Asn catalytic triad. This provided novel strategies for the engineered biosynthesis of pharmaceutically important plant polyketides.     

Macrotetrolide biosynthesis: a novel type II polyketide synthase

Polyketide biosynthesis is catalyzed by polyketide synthase (PKS) and three types of bacterial PKS are known to date. Feeding experiments with isotope-labeled precursors established the polyketide origin of the macrotetrolides, but the labeling pattern cannot be rationalized according to the established PKS paradigm. Genetic analysis of the macrotetrolide biosynthesis unveiled an unprecedented organization for a polyketide gene cluster that features five genes encoding discrete ketoacyl synthase (KS) and four genes encoding discrete ketoreductase (KR) but lacking an acyl carrier protein (ACP). Macrotetrolide biosynthesis is proposed to involve a novel type II PKS that acts directly on acyl CoA substrates, functions noniteratively, and catalyzes both C-C and C-O bond formation. These findings demonstrate once again Nature's versatility in making complex molecules and suggests new strategies for PKS engineering to further expand the scope and diversity of polyketide library. They also should serve as an inspiration in searching for PKS with novel chemistry for combinatorial biosynthesis.     

Biochemical and structural characterization of germicidin synthase: analysis of a type III polyketide synthase that employs acyl-ACP as a starter unit donor

Germicidin synthase (Gcs) from Streptomyces coelicolor is a type III polyketide synthase (PKS) with broad substrate flexibility for acyl groups linked through a thioester bond to either coenzyme A (CoA) or acyl carrier protein (ACP). Germicidin synthesis was reconstituted in vitro by coupling Gcs with fatty acid biosynthesis. Since Gcs has broad substrate flexibility, we directly compared the kinetic properties of Gcs with both acyl-ACP and acyl-CoA. The catalytic efficiency of Gcs for acyl-ACP was 10-fold higher than for acyl-CoA, suggesting a strong preference toward carrier protein starter unit transfer. The 2.9 ? germicidin synthase crystal structure revealed canonical type III PKS architecture along with an unusual helical bundle of unknown function that appears to extend the dimerization interface. A pair of arginine residues adjacent to the active site affect catalytic activity but not ACP binding. This investigation provides new and surprising information about the interactions between type III PKSs and ACPs that will facilitate the construction of engineered systems for production of novel polyketides.     

The malonyl transferase activity of type II polyketide synthase acyl carrier proteins

Acyl carrier proteins (ACPs) play a fundamental role in directing intermediates among the enzyme active sites of fatty acid and polyketide synthases (PKSs). In this paper, we demonstrate that the Streptomyces coelicolor (S. coelicolor) actinorhodin (act) PKS ACP can catalyze transfer of malonate to type II S. coelicolor fatty acid synthase (FAS) and other PKS ACPs in vitro. The reciprocal transfer from S. coelicolor FAS ACP to a PKS ACP was not observed. Several mutations in both act ACP and S. coelicolor FAS ACP could be classified by their participation in either donation or acceptance of this malonyl group. These mutations indicated that self-malonylation and malonyl transfer could be completely decoupled, implying that they were separate processes and that a FAS ACP could be converted from a non-malonyl-transferring protein to one with malonyl transferase activity.     

Enzymatic formation of an unnatural methylated triketide by plant type III polyketide synthases

Octaketide synthase, a novel plant-specific type III polyketide synthase from Aloe arborescens, efficiently accepted (2RS)-methylmalonyl-CoA as a sole substrate to produce 6-ethyl-4-hydroxy-3,5-dimethyl-2-pyrone. On the other hand, a tetraketide-producing chalcone synthase from Scutellaria baicalensis and a diketide-producing benzalacetone synthase from Rheum palmatum also yielded the unnatural methylated C₉ triketide pyrone as a single product by sequential decarboxylative condensations of three molecules of (2RS)-methylmalonyl-CoA.     

Enzymatic formation of an unnatural C(6)-C(5) aromatic polyketide by plant type III polyketide synthases

[reaction: see text] Substrate specificities of plant polyketide synthases (PKSs) were investigated using analogues of malonyl-CoA, the extension unit of the polyketide chain elongation reactions. When incubated with methylmalonyl-CoA and 4-coumaroyl-CoA, plant PKSs (chalcone synthase from Scutellaria baicalensis, stilbene synthase from Arachis hypogaea, and benzalacetone synthase from Rheum palmatum) afforded an unnatural C(6)-C(5) aromatic polyketide, 1-(4-hydroxyphenyl)pent-1-en-3-one, formed by one-step decarboxylative condensation of the two substrates. In contrast, succinyl-CoA was not accepted as a substrate by the enzymes.     

Redirecting the cyclization steps of fungal polyketide synthase

Regiospecific cyclizations of the nascent poly-beta-ketone backbones dictate the structures of polyketide natural products. The fungal iterative megasynthases use terminal thioesterase/claisen cyclase (TE/CLC) domains to direct the fate of the polyketide chains. In this work, we present two strategies toward redirecting the cyclization steps of fungal PKSs using the Gibberella fujikuroi PKS4. First, inactivation or removal of the TE/CLC domain resulted in the synthesis of the new polyketide SMA93 2. Complementation of the mutant PKS4 with a standalone TE/CLC domain restored the regioselective cyclization steps of PKS4 and led to the synthesis of SMA76 1, demonstrating that cyclization enzymes can interact with the megasynthase in trans. This led to the second approach in which various dissociated, bacterial tailoring enzymes were added to the megasynthase in trans. Addition of the act KR led to the synthesis of mutactin 3, while the addition of first ring and second ring cyclases yielded anthraquinone compounds DMAC 5 and SEK26 6. The cooperative activities of fungal and bacterial PKS components are especially important and enable synthesis of polyketides utilizing enzymes from two distinct families of PKSs.     

Biosynthesis of the vancomycin group of antibiotics: characterisation of a type III polyketide synthase in the pathway to (S)-3,5-dihydroxyphenylglycine

3,5-dihydroxyphenylacetate, a precursor for the non-proteinogenic amino acid 3,5-dihydroxyphenylglycine occurring in glycopeptide antibiotics, is determined to be catalysed by a type III polyketide synthase using malonyl-CoA as a starter unit.     

An iterative type I polyketide synthase PKSN catalyzes synthesis of the decaketide alternapyrone with regio-specific octa-methylation

A biosynthetic gene cluster containing five genes, alt1-5, was cloned from Alternaria solani, a causal fungus of early blight disease to tomato and potato. Homology searching indicated that the alt1, 2, and 3 genes code for cytochrome P450s and the alt4 gene for a FAD-dependent oxygenase/oxidase. The alt5 gene encodes a polyketide synthase (PKS), named PKSN, that was found to be an iterative type I complex reduced-type PKS with a C-methyltransferase domain. To identify the PKSN function, the alt5 gene was introduced into the fungal host Aspergillus oryzae under an alpha-amylase promoter. The transformant produced a polyketide compound, named alternapyrone, whose structure is shown to be 3,5-dimethyl-4-hydroxy-6-(1,3,5,7,11,13-hexamethyl-3,5,11-pentadecatrienyl)-pyran-2-one. Labeling experiments confirmed that alternapyrone is a decaketide with octa-methylation from methionine on every C(2) unit except the third unit.     

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.     

Mechanisms of molecular recognition in the pikromycin polyketide synthase

BACKGROUND: Modular polyketide synthases (PKSs) produce a wide range of medically significant compounds. In the case of the pikromycin PKS of Streptomyces venezuelae, four separate polypeptides (PikAI-PikAIV), comprising a total of one loading domain and six extension modules, generate the 14-membered ring macrolactone narbonolide. The polypeptide PikAIV contains a thioesterase (TE) domain and is responsible for catalyzing both the last elongation step with methylmalonyl CoA, and subsequent release of the final polyketide chain elongation intermediate from the PKS. Under certain growth conditions this polypeptide is synthesized from an alternative translational start site, giving rise to an N-terminal truncated form of PikAIV, containing only half of the ketosynthase (KS(6)) domain. The truncated form of PikAIV is unable to catalyze the final elongation step, but is able to cleave a polyketide chain from the preceding module on PikAIII (ACP(5)), giving rise to the 12-membered ring product 10-deoxymethynolide. RESULTS: S. venezuelae mutants expressing hybrid PikAIV polypeptides containing acyl carrier protein (ACP) and malonyl CoA specific acyltransferase (AT) domains from the rapamycin PKS were unable to catalyze production of 12- or 14-membered ring macrolactone products. Plasmid-based expression of a hybrid PikAIV containing the native KS(6) and TE domains, however, restored production of both narbonolide and 10-deoxymethynolide in the S. venezuelae AX912 mutant that generates a TE-deleted form of PikAIV. Use of alternative KS domains or deletion of the KS(6) domain within the hybrid PikAIV resulted in loss of both products. Plasmid-based expression of a TE domain of PikAIV as a separate polypeptide in the AX912 mutant resulted in greater than 50% restoration of 10-deoxymethynolide, but not in mutants expressing a hybrid PikAIV bearing an unnatural AT domain. Mutants expressing hybrid PikAIV polypeptides containing the natural AT(6) domains and different ACP domains efficiently produced polyketide products, but with a significantly higher 10-deoxymethynolide/narbonolide ratio than observed with native PikAIV. CONCLUSIONS: Dimerization of KS(6) modules allows in vivo formation of a PKS heterodimer using PikAIV polypeptides containing different AT and ACP domains. In such heterodimers, the TE domain and the AT(6) domain responsible for formation of the narbonolide product are located on different polypeptide chains. The AT(6) domain of PikAIV plays an important role in facilitating TE-catalyzed chain termination (10-deoxymethynolide formation) at the proceeding module in PikAIII. The pikromycin PKS can tolerate the presence of multiple forms (active and inactive) of PikAIV, and decreased efficiency of elongation by PikAIV can result in increased levels of 10-deoxymethynolide. These results provide new insight into functional molecular interactions and interdomain recognition in modular PKSs.     

Nongenetic reprogramming of a fungal highly reducing polyketide synthase

The biosynthesis of the fungal metabolite tenellin from Beauveria bassiana CBS110.25 was investigated in the presence of the epigenetic modifiers 5-azacytidine and suberoyl bis-hydroxamic acid and under conditions where individual genes from the tenellin biosynthetic gene cluster were silenced. Numerous new compounds were synthesized, indicating that the normal predominant biosynthesis of tenellin is just one outcome out of a diverse array of possible products. The structures of the products reveal key clues about the programming selectivities of the tenellin polyketide synthase.     

Heterologous expression, purification, reconstitution and kinetic analysis of an extended type II polyketide synthase.

BACKGROUND: Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC). RESULTS: The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate. CONCLUSIONS: Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems.