Pentaketide resorcylic acid synthesis by type III polyketide synthase from Neurospora crassa

Type III polyketide synthases (PKSs) are responsible for aromatic polyketide synthesis in plants and bacteria. Genome analysis of filamentous fungi has predicted the presence of fungal type III PKSs, although none have thus far been functionally characterized. In the genome of Neurospora crassa, a single open reading frame, NCU04801.1, annotated as a type III PKS was found. In this report, we demonstrate that NCU04801.1 is a novel type III PKS catalyzing the synthesis of pentaketide alkylresorcylic acids. NCU04801.1, hence named 2'-oxoalkylresorcylic acid synthase (ORAS), preferred stearoyl-CoA as a starter substrate and condensed four molecules of malonyl-CoA to give a pentaketide intermediate. For ORAS to yield pentaketide alkylresorcylic acids, aldol condensation and aromatization of the intermediate, which is still attached to the enzyme, are presumably followed by hydrolysis for release of the product as a resorcylic acid. ORAS is the first type III PKS that synthesizes pentaketide resorcylic acids.     

Pyrone polyketides synthesized by a type III polyketide synthase from Drosophyllum lusitanicum

To isolate cDNAs involved in the biosynthesis of acetate-derived naphthoquinones in Drosophyllum lusitanicum, an expressed sequence tag analysis was performed. RNA from callus cultures was used to create a cDNA library from which 2004 expressed sequence tags were generated. One cDNA with similarity to known type III polyketide synthases was isolated as full-length sequence and termed DluHKS. The translated polypeptide sequence of DluHKS showed 51-67% identity with other plant type III PKSs. Recombinant DluHKS expressed in Escherichia coli accepted acetyl-coenzyme A (CoA) as starter and carried out sequential decarboxylative condensations with malonyl-CoA yielding α-pyrones from three to six acetate units. However, naphthalenes, the expected products, were not isolated. Since the main compound produced by DluHKS is a hexaketide α-pyrone, and the naphthoquinones in D. lusitanicum are composed of six acetate units, we propose that the enzyme provides the backbone of these secondary metabolites. An involvement of accessory proteins in this biosynthetic pathway is discussed.     

Biphenyl synthase,a novel type III polyketide synthase

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.     

An acridone-producing novel multifunctional type III polyketide synthase from Huperzia serrata

A cDNA encoding a novel plant type III polyketide synthase was cloned and sequenced from the Chinese club moss Huperzia serrata (Huperziaceae). The deduced amino acid sequence of Hu. serrata polyketide synthase 1 showed 44-66% identity to those of other chalcone synthase superfamily enzymes of plant origin. Further, phylogenetic tree analysis revealed that Hu. serrata polyketide synthase 1 groups with other nonchalcone-producing type III polyketide synthases. Indeed, a recombinant enzyme expressed in Escherichia coli showed unusually versatile catalytic potential to produce various aromatic tetraketides, including chalcones, benzophenones, phloroglucinols, and acridones. In particular, it is remarkable that the enzyme accepted bulky starter substrates such as 4-methoxycinnamoyl-CoA and N-methylanthraniloyl-CoA, and carried out three condensations with malonyl-CoA to produce 4-methoxy-2',4',6'-trihydroxychalcone and 1,3-dihydroxy-N-methylacridone, respectively. In contrast, regular chalcone synthase does not accept these bulky substrates, suggesting that the enzyme has a larger starter substrate-binding pocket at the active site. Although acridone alkaloids have not been isolated from Hu. serrata, this is the first demonstration of the enzymatic production of acridone by a type III polyketide synthase from a non-Rutaceae plant. Interestingly, Hu. serrata polyketide synthase 1 lacks most of the consensus active site sequences with acridone synthase from Ruta graveolens (Rutaceae).     

Cloning and characterization of a type III polyketide synthase from Aspergillus niger

Type III polyketide synthases (PKSs) are the condensing enzymes that catalyze the formation of a myriad of aromatic polyketides in plant, bacteria, and fungi. Here we report the cloning and characterization of a putative type III PKS from Aspergillusniger, AnPKS. This enzyme catalyzes the synthesis of alkyl pyrones from C2 to C18 starter CoA thioesters with malonyl-CoA as an extender CoA through decaboxylative condensation and cyclization. It displays broad substrate specificity toward fatty acyl-CoA starters to yield triketide and tetraketide pyrones, with benzoyl-CoA as the most preferred starter. The optimal temperature and pH of AnPKS are 50°C and 8, respectively. Under optimal conditions, the enzyme shows the highest catalytic efficiency (k(cat)/K(m)) of 7.4×10(5)s(-1)M(-1) toward benzoyl-CoA. Homology modeling and site-directed mutagenesis were used to probe the molecular basis of its substrate specificity. This study should open doors for further engineering of AnPKS as a biocatalyst for synthesis of value-added polyketides.     

Alkylresorcylic acid synthesis by type III polyketide synthases from rice Oryza sativa

Alkylresorcinols, produced by various plants, bacteria, and fungi, are bioactive compounds possessing beneficial activities for human health, such as anti-cancer activity. In rice, they accumulate in seedlings, contributing to protection against fungi. Alkylresorcylic acids, which are carboxylated forms of alkylresorcinols, are unstable compounds and decarboxylate readily to yield alkylresorcinols. Genome mining of the rice Oryza sativa identified two type III polyketide synthases, named ARAS1 (alkylresorcylic acid synthase) and ARAS2, that catalyze the formation of alkylresorcylic acids. Both enzymes condensed fatty acyl-CoAs with three C₂ units from malonyl-CoA and cyclized the resulting tetraketide intermediates via intramolecular C-2 to C-7 aldol condensation. The alkylresorcylic acids thus produced were released from the enzyme and decarboxylated non-enzymatically to yield alkylresorcinols. This is the first report on a plant type III polyketide synthase that produces tetraketide alkylresorcylic acids as major products.     

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.     

Identification of a cryptic type III polyketide synthase (1,3,6,8-tetrahydroxynaphthalene synthase) from Streptomyces peucetius ATCC 27952

We identified a 1,134-bp putative type III polyketide synthase from the sequence analysis of Streptomyces peucetius ATCC 27952, named Sp-RppA, which is characterized as 1,3,6,8-tetrahydroxynaphthalene synthase and shares 33% identity with SCO1206 from S. coelicolor A3(2) and 32% identity with RppA from S. griseus. The 1,3,6,8-tetrahydroxynaphthalene synthase is known to catalyze the sequential decarboxylative condensation, intramolecular cyclization, and aromatization of an oligoketide derived from five units of malonyl-CoA to give 1,3,6,8-tetrahydroxynaphthalene, which spontaneously oxidizes to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). In this study, we report the in vivo expression and in vitro synthesis of flaviolin from purified gene product (Sp-RppA).     

Phenolic lipids synthesized by type III polyketide synthase confer penicillin resistance on Streptomyces griseus

Type III polyketide synthases (PKSs) found in plants, fungi, and bacteria synthesize a variety of aromatic polyketides. A Gram-positive, filamentous bacterium Streptomyces griseus contained an srs operon, in which srsA encoded a type III PKS, srsB encoded a methyltransferase, and srsC encoded a flavoprotein hydroxylase. Consistent with this annotation, overexpression of the srs genes in a heterologous host, Streptomyces lividans, showed that SrsA was a type III PKS responsible for synthesis of phenolic lipids, alkylresorcinols, and alkylpyrones, SrsB was a methyltransferase acting on the phenolic lipids to yield alkylresorcinol methyl ethers, and SrsC was a hydroxylase acting on the alkylresorcinol methyl ethers. In vitro SrsA reaction showed that SrsA synthesized alkylresorcinols from acyl-CoAs of various chain lengths as a starter substrate, one molecule of methylmalonyl-CoA, and two molecules of malonyl-CoA. SrsA was thus unique in that it incorporated the extender substrates in a strictly controlled order of malonyl-CoA, malonyl-CoA, and methylmalonyl-CoA to produce alkylresorcinols. An srsA mutant, which produced no phenolic lipids, was highly sensitive to beta-lactam antibiotics, such as penicillin G and cephalexin. Together with the fact that the alkylresorcinols were fractionated mainly in the cell wall fraction, this observation suggests that the phenolic lipids, perhaps associated with the cytoplasmic membrane because of their amphiphilic property, affect the characteristic and rigidity of the cytoplasmic membrane/peptidoglycan of a variety of bacteria. An srs-like operon is found widely among Gram-positive and -negative bacteria, indicating wide distribution of the phenolic lipids.     

Characterization of the substrate specificity of PhlD, a type III polyketide synthase from Pseudomonas fluorescens

PhlD, a type III polyketide synthase from Pseudomonas fluorescens, catalyzes the synthesis of phloroglucinol from three molecules of malonyl-CoA. Kinetic analysis by direct measurement of the appearance of the CoASH product (k(cat) = 24 +/- 4 min(-1) and Km = 13 +/- 1 microM) gave a k(cat) value more than an order of magnitude higher than that of any other known type III polyketide synthase. PhlD exhibits broad substrate specificity, accepting C4-C12 aliphatic acyl-CoAs and phenylacetyl-CoA as the starters to form C6-polyoxoalkylated alpha-pyrones from sequential condensation with malonyl-CoA. Interestingly, when primed with long chain acyl-CoAs, PhlD catalyzed extra polyketide elongation to form up to heptaketide products. A homology structural model of PhlD showed the presence of a buried tunnel extending out from the active site to assist the binding of long chain acyl-CoAs. To probe the structural basis for the unusual ability of PhlD to accept long chain acyl-CoAs, both site-directed mutagenesis and saturation mutagenesis were carried out on key residues lining the tunnel. Three mutations, M21I, H24V, and L59M, were found to significantly reduce the reactivity of PhlD with lauroyl-CoA while still retaining its physiological activity to synthesize phloroglucinol. Our homology modeling and mutational studies indicated that even subtle changes in the tunnel volume could affect the ability of PhlD to accept long chain acyl-CoAs. This suggested novel strategies for combinatorial biosynthesis of unnatural pharmaceutically important polyketides.     

Enzymatic formation of long-chain polyketide pyrones by plant type III polyketide synthases

Recombinant chalcone synthase (CHS) from Scutellaria baicalensis and stilbene synthase (STS) from Arachis hypogaea accepted CoA esters of long-chain fatty acid (CHS up to the C12 ester, while STS up to the C14 ester) as a starter substrate, and carried out sequential condensations with malonyl-CoA, leading to formation of triketide and tetraketide alpha-pyrones. Interestingly, the C6, C8, and C10 esters were kinetically favored by the enzymes over the physiological starter substrate; the kcat/KM values were 1.2- to 1.9-fold higher than that of p-coumaroyl-CoA. The catalytic diversities of the enzymes provided further mechanistic insights into the type III PKS reactions, and suggested involvement of the CHS-superfamily enzymes in the biosynthesis of long-chain alkyl polyphenols such as urushiol and ginkgolic acid in plants.     

Kingdom-wide analysis of the evolution of the plant type III polyketide synthase superfamily

The emergence of type III polyketide synthases (PKSs) was a prerequisite for the conquest of land by the green lineage. Within the PKS superfamily, chalcone synthases (CHSs) provide the entry point reaction to the flavonoid pathway, while LESS ADHESIVE POLLEN 5 and 6 (LAP5/6) provide constituents of the outer exine pollen wall. To study the deep evolutionary history of this key family, we conducted phylogenomic synteny network and phylogenetic analyses of whole-genome data from 126 species spanning the green lineage including Arabidopsis thaliana, tomato (Solanum lycopersicum), and maize (Zea mays). This study thereby combined study of genomic location and context with changes in gene sequences. We found that the two major clades, CHS and LAP5/6 homologs, evolved early by a segmental duplication event prior to the divergence of Bryophytes and Tracheophytes. We propose that the macroevolution of the type III PKS superfamily is governed by whole-genome duplications and triplications. The combined phylogenetic and synteny analyses in this study provide insights into changes in the genomic location and context that are retained for a longer time scale with more recent functional divergence captured by gene sequence alterations.

Phylogenetic and syntenic analyses of whole genome data reveal that macroevolution of the type III polyketide synthase superfamily is mainly governed by whole-genome duplications and triplications.     

The first plant type III polyketide synthase that catalyzes formation of aromatic heptaketide

A cDNA encoding a novel plant type III polyketide synthase (PKS) was cloned from rhubarb (Rheum palmatum). A recombinant enzyme expressed in Escherichia coli accepted acetyl-CoA as a starter, carried out six successive condensations with malonyl-CoA and subsequent cyclization to yield an aromatic heptaketide, aloesone. The enzyme shares 60% amino acid sequence identity with chalcone synthases (CHSs), and maintains almost identical CoA binding site and catalytic residues conserved in the CHS superfamily enzymes. Further, homology modeling predicted that the 43-kDa protein has the same overall fold as CHS. This provides new insights into the catalytic functions of type III PKSs, and suggests further involvement in the biosynthesis of plant polyketides.     

Structural insights into biosynthesis of resorcinolic lipids by a type III polyketide synthase in Neurospora crassa

Microbial type III polyketide synthases (PKSs) have revealed remarkable mechanistic as well as functional versatility. Recently, a type III PKS homolog from Azotobacter has been implicated in the biosynthesis of resorcinolic lipids, thus adding a new functional significance to this class of proteins. Here, we report the structural and mutational investigations of a novel type III PKS protein from Neurospora crassa involved in the biosynthesis of resorcinolic metabolites by utilizing long chain fatty acyl-CoAs. The structure revealed a long hydrophobic tunnel responsible for its fatty acyl chain length specificity resembling that of PKS18, a mycobacterial type III PKS. Structure-based mutational studies to block the tunnel not only altered the fatty acyl chain specificity but also resulted in change of cyclization pattern affecting the product profile. This first structural characterization of a resorcinolic lipid synthase provides insights into the coordinated functioning of cyclization and a substrate-binding pocket, which shows mechanistic intricacy underlying type III PKS catalysis.     

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.     

A type III polyketide synthase from Wachendorfia thyrsiflora and its role in diarylheptanoid and phenylphenalenone biosynthesis

Chalcone synthase (CHS) related type III plant polyketide synthases (PKSs) are likely to be involved in the biosynthesis of diarylheptanoids (e.g. curcumin and polycyclic phenylphenalenones), but no such activity has been reported. Root cultures from Wachendorfia thyrsiflora (Haemodoraceae) are a suitable source to search for such enzymes because they synthesize large amounts of phenylphenalenones, but no other products that are known to require CHSs or related enzymes (e.g. flavonoids or stilbenes). A homology-based RT-PCR strategy led to the identification of cDNAs for a type III PKS sharing only approximately 60% identity with typical CHSs. It was named WtPKS1 (W. thyrsiflora polyketide synthase 1). The purified recombinant protein accepted a large variety of aromatic and aliphatic starter CoA esters, including phenylpropionyl- and side-chain unsaturated phenylpropanoid-CoAs. The simplest model for the initial reaction in diarylheptanoid biosynthesis predicts a phenylpropanoid-CoA as starter and a single condensation reaction to a diketide. Benzalacetones, the expected release products, were observed only with unsaturated phenylpropanoid-CoAs, and the best results were obtained with 4-coumaroyl-CoA (80% of the products). With all other substrates, WtPKS1 performed two condensation reactions and released pyrones. We propose that WtPKS1 catalyses the first step in diarylheptanoid biosynthesis and that the observed pyrones are derailment products in the absence of downstream processing proteins.