Role of the active site cysteine of DpgA, a bacterial type III polyketide synthase

DpgA is a bacterial type III polyketide synthase (PKS) that decarboxylates and condenses four malonyl-CoA molecules to produce 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) in the biosynthetic pathway to 3,5-dihydroxyphenylglycine, a key nonproteinogenic residue in the vancomycin family of antibiotics. DpgA has the conserved catalytic triad of Cys/His/Asn typical of type III PKS enzymes, and has been assumed to use Cys160 as the catalytic nucleophile to create a series of elongating acyl-S-enzyme intermediates prior to the C(8) to C(3) cyclization step. Incubation of purified DpgA with [(14)C]-malonyl-CoA followed by acid quench during turnover leads to accumulation of 10-15% of the DpgA molecules covalently acylated. Mutation of the active site Cys160 to Ala abrogated detectable covalent acylation, but the C160A mutant retained 50% of the V(max) for DPA-CoA formation, with a k(cat) still at 0.5 catalytic turnovers/min. For comparison, a C190A mutant retained wild-type activity, while the H296A mutant, in which the side chain of the presumed catalytic His is removed, had a 6-fold drop in k(cat). During turnover, purified DpgA produced 1.2 equivalents of acetyl-CoA for each DPA-CoA, indicating 23% uncoupled decarboxylation competing with condensative C-C coupling. The C160A mutant showed an increased partition ratio for malonyl-CoA decarboxylation to acetyl-CoA vs condensation to DPA-CoA, reflecting more uncoupling in the mutant enzyme. The Cys-to-Ala mutant thus shows the unexpected result that, when the normal acyl-S-enzyme mechanism for this type III PKS elongation/cyclization catalyst is removed, it can still carry out the regioselective construction of the eight-carbon DPA-CoA skeleton with surprising efficiency.     

Properties and substrate specificity of RppA, a chalcone synthase-related polyketide synthase in Streptomyces griseus.

RppA, a chalcone synthase-related polyketide synthase (type III polyketide synthase) in the bacterium Streptomyces griseus, catalyzes the formation of 1,3,6,8-tetrahydroxynaphthalene (THN) from five molecules of malonyl-CoA. The K(m) value for malonyl-CoA and the k(cat) value for THN synthesis were determined to be 0.93 +/- 0.1 microm and 0.77 +/- 0.04 min(-1), respectively. RppA accepted aliphatic acyl-CoAs with the carbon lengths from C(4) to C(8) as starter substrates and catalyzed sequential condensation of malonyl-CoA to yield alpha-pyrones and phloroglucinols. In addition, RppA yielded a hexaketide, 4-hydroxy-6-(2',4',6'-trioxotridecyl)-2-pyrone, from octanoyl-CoA and five molecules of malonyl-CoA, suggesting that the size of the active site cavity of RppA is larger than any other chalcone synthase-related enzymes found so far in plants and bacteria. RppA was also found to synthesize a C-methylated pyrone, 3,6-dimethyl-4-hydroxy-2-pyrone, by using acetoacetyl-CoA as the starter and methylmalonyl-CoA as an extender. Thus, the broad substrate specificity of RppA yields a wide variety of products.     

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.     

Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex

An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex.     

Functional analysis of putative beta-ketoacyl:acyl carrier protein synthase and acyltransferase active site motifs in a type II polyketide synthase of Streptomyces glaucescens.

The significance of potential active site motifs for acyltransferase and beta-ketoacyl:acyl carrier protein synthase regions within the TcmK protein was investigated by determining the effects of mutations in the proposed active sites on the production of tetracenomycins F2 and C. In a Streptomyces glaucescens tcmGHI JKLMNO null mutant, plasmids carrying the S351A mutation produced high amounts of tetracenomycin F2 but plasmids carrying the C173A or C173S mutation or the H350L-S351A double mutation produced no detectable amount of any known intermediate. In a tcmK mutant, plasmids with the S351A mutation restored high production of tetracenomycin C and plasmids carrying the other mutations were able to complement the chromosomal defect to some extent. None of the mutations affected the amount of TcmK produced.     

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).     

Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2).

The multifunctional 6-methylsalicylic acid synthase gene from Penicillium patulum was engineered for regulated expression in Streptomyces coelicolor. Production of significant amounts of 6-methylsalicylic acid by the recombinant strain was proven by nuclear magnetic resonance spectroscopy. These results suggest that it is possible to harness the molecular diversity of eukaryotic polyketide pathways by heterologous expression of biosynthetic genes in an easily manipulated model bacterial host in which prokaryotic aromatic and modular polyketide synthase genes are already expressed and recombined.     

A cryptic type I polyketide synthase (cpk) gene cluster in Streptomyces coelicolor A3(2)

The chromosome of Streptomyces coelicolor A3(2), a model organism for the genus Streptomyces, contains a cryptic type I polyketide synthase (PKS) gene cluster which was revealed when the genome was sequenced. The ca. 54-kb cluster contains three large genes, cpkA, cpkB and cpkC, encoding the PKS subunits. In silico analysis showed that the synthase consists of a loading module, five extension modules and a unique reductase as a terminal domain instead of a typical thioesterase. All acyltransferase domains are specific for a malonyl extender, and have a B-type ketoreductase. Tailoring and regulatory genes were also identified within the gene cluster. Surprisingly, some genes show high similarity to primary metabolite genes not commonly identified in any antibiotic biosynthesis cluster. Using western blot analysis with a PKS subunit (CpkC) antibody, CpkC was shown to be expressed in S. coelicolor at transition phase. Disruption of cpkC gave no obvious phenotype.     

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.     

Streptomyces coelicolor A3(2) produces a new yellow pigment associated with the polyketide synthase Cpk

Streptomyces coelicolor A3(2) is an extensively studied model organism for the genetic studies of Streptomycetes - a genus known for the production of a vast number of bioactive compounds and complex regulatory networks controlling morphological differentiation and secondary metabolites production. We present the discovery of a presumptive product of the Cpk polyketide synthase. We have found that on the rich medium without glucose S. coelicolor A3(2) produces a yellow compound secreted into the medium. We have proved by complementation that production of the observed yellow pigment is dependent on cpk gene cluster previously described as cryptic type I polyketide synthase cluster. The pigment production depends on the medium composition, does not occur in the presence of glucose, and requires high density of spore suspension used for inoculation.     

Analysis of type II polyketide beta-ketoacyl synthase specificity in Streptomyces coelicolor A3(2) by trans complementation of actinorhodin synthase mutants.

Complementation of defined actinorhodin beta-ketoacyl synthase (KS) mutants by various other KS genes suggested that the ORF1-encoded KS may be relatively generalized in function, whereas the ORF2-encoded KS component may provide specificity in polyketide chain construction. Evidence for differential temporal-spatial expression of the actinorhodin and spore pigment KSs in Streptomyces coelicolor was obtained.     

Cloning and analysis of a type II polyketide synthase gene cluster from Streptomyces toxytricini NRRL 15,443

A standard type II polyketide synthase (PKS) gene cluster was isolated while attempting to clone the biosynthetic gene for lipstatin from Streptomyces toxytricini NRRL 15,443. This result was observed using a Southern blot of a PstI-digested S. toxytricini chromosomal DNA library with a 444 bp amplified probe of a ketosynthase (KS) gene fragment. Four open reading frames [thioesterase (TE), beta-ketoacyl systhase (KAS), chain length factor (CLF), and acyl carrier protein (ACP)], were identified through the nucleotide sequence determination and analysis of a 4.5 kb cloned DNA fragment. In order to confirm the involvement of a cloned gene in lipstatin biosynthesis, a gene disruption experiment for the KS gene was performed. However, the resulting gene disruptant did not show any significant difference in lipstatin production when compared to wild-type S. toxytricini. This result suggests that lipstatin may not be synthesized by a type II PKS.     

Covalent binding of chloroacetamide herbicides to the active site cysteine of plant type III polyketide synthases

Chloroacetamide herbicides inhibit very-long-chain fatty acid elongase, and it has been suggested that covalent binding to the active site cysteine of the condensing enzyme is responsible [Pest Manage Sci 56 (2000), 497], but direct evidence was not available. The proposal implied that other condensing enzymes might also be targets, and therefore we have investigated four purified recombinant type III plant polyketide synthases. Chalcone synthase (CHS) revealed a high sensitivity to the chloroacetamide metazachlor, with 50% inhibition after a 10 min pre-incubation with 1-2 molecules per enzyme subunit, and the inactivation was irreversible. Stilbene synthase (STS) inactivation required 20-fold higher amounts, and 4-coumaroyltriacetic acid synthase and pyrone synthase revealed no response at the highest metazachlor concentrations tested. A similar spectrum of differential responses was detected with other herbicides that also inhibit fatty acid elongase (metolachlor and cafenstrole). The data indicate that type III polyketide synthases are potential targets of these herbicides, but each combination has to be investigated individually. The interaction of metazachlor with CHS was investigated by mass spectrometric peptide mapping, after incubation of the enzymes with the herbicides followed by tryptic digestion. A characteristic mass shift and MS/MS sequencing of the respective peptide showed that metazachlor was covalently bound to the cysteine of the active site, and the same was found with STS. This is the first direct evidence that the active site cysteine in condensing enzymes is the primary common target of these herbicides.     

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.     

Polyketide biosynthesis beyond the type I,II and III polyketide synthase paradigms

Recent literature on polyketide biosynthesis suggests that polyketide synthases have much greater diversity in both mechanism and structure than the current type I, II and III paradigms. These examples serve as an inspiration for searching novel polyketide synthases to give new insights into polyketide biosynthesis and to provide new opportunities for combinatorial biosynthesis.     

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.     

Cloning, analysis, and heterologous expression of a polyketide synthase gene cluster of Streptomyces curacoi.

Streptomyces curacoi produces curamycin, an antibiotic based on a modified orsellinic acid skeleton that is synthesized by the polyketide pathway. We have cloned, characterized, and partly sequenced a polyketide synthase gene cluster of S. curacoi. The sequence data reveal an organization of open reading frames that is similar to those of other polyketide synthetic clusters, although the biosynthetic products differ considerably in size and structure. We propose that one of the predicted open reading frames (curA) encodes polykeptide synthase, on the basis of its homology with other enzymes with similar functions. Expression of the cloned chromosomal fragment in the heterologous host S. lividans leads to the production of a brown pigment in large quantities. The analysis and expression of the cur genes for detailed molecular studies of the mechanism of polyketide biosynthesis is discussed.     

Functional replacement of genes for individual polyketide synthase components in Streptomyces coelicolor A3(2) by heterologous genes from a different polyketide pathway.

Streptomyces coelicolor A3(2) and Streptomyces violaceoruber Tü22 produce the antibiotics actinorhodin and granaticin, respectively. Both the aglycone of granaticin and the half-molecule of actinorhodin are derived from one acetyl coenzyme A starter unit and seven malonyl coenzyme A extender units via the polyketide pathway to produce benzoisochromane quinone moieties with identical structures (except for the stereochemistry at two chiral centers). In S. coelicolor and S. violaceoruber, the type II polyketide synthase (PKS) is encoded by clusters of five and six genes, respectively. We complemented a series of S. coelicolor mutants (act) defective in different components of the PKS (actI for carbon chain assembly, actIII for ketoreduction, and actVII for cyclization-dehydration) by the corresponding genes (gra) from S. violaceoruber introduced in trans on low-copy-number plasmids. This procedure showed that four of the act PKS components could be replaced by a heterologous gra protein to give a functional PKS. The analysis also served to identify which of three candidate open reading frames (ORFs) in the actI region had been altered in each of a set of 13 actI mutants. It also proved that actI-ORF2 (whose putative protein product shows overall similarity to the beta-ketoacyl synthase encoded by actI-ORF1 but whose function is unclear) is essential for PKS function. Mutations in each of the four complemented act genes (actI-ORF1, actI-ORF2, actIII, and actVII) were cloned and sequenced, revealing a nonsense or frameshift mutation in each mutant.