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PubMed2001

Analysis of the enzymatic domains in the modular portion of the coronafacic acid polyketide synthase.

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant pathogen Pseudomonas syringae. The CFA polyketide synthase (PKS) consists of two open reading frames (ORFs) that encode type I multifunctional proteins and several ORFs that encode monofunctional proteins. Sequence comparisons of the modular portions of the CFA PKS with other prokaryotic, modular PKSs elucidated the boundaries of the domains that are involved in the individual stages of polyketide assembly. The two beta-ketoacyl:acyl carrier protein synthase (KS) domains in the modular portion of the CFA PKS exhibit a high degree of similarity to each other (53%), but are even more similar to the KS domains of DEBS, RAPS, and RIF. Cfa6 possesses two acyltransferases- AT0, which is associated with a loading domain, and AT1, which uses ethylmalonyl-CoA (eMCoA) as a substrate for chain extension. Cfa7 contains an AT that uses malonyl-CoA as a substrate for chain extension. The Cfa6 AT0 shows 35 and 32% similarity to the DEBS1 and NidA1 AT0s, respectively, and 32 and 36% similarity to the Cfa6 and Cfa7 AT1s. Sequence motifs have previously been identified that correlate with AT substrates. The motifs in Cfa6 AT1 were found to correlate reasonably well with those predicted for methylmalonyl-CoA (mMCoA) ATs. The motifs in the AT of Cfa7 correlated more poorly with those predicted for MCoA ATs. Three ACP domains occur in the modular proteins of the COR PKS. The loading domain-associated ACP0 showed 38% similarity to the loading domain ACP0s of DEBS1 and NidA1 and 32-36% similarity to the two module-associated ACPs of the COR PKS. It exhibited a higher degree of similarity to the module-associated ACPs of RAPS. The two module-associated ACPs show 39% similarity to each other, but appear more closely related to module-associated ACP domains in RAPS and RIFS. Furthermore, the DH and KR domains of Cfa6 and Cfa7 show greater similarity to DH and KR domains in RAPS and RIFS than to each other. The CFA PKS includes a thioesterase domain (TE I) that resides at the C-terminus of Cfa7 and a second thioesterase, which exists as a separate ORF (Cfa9, a TE II). Analysis of a Cfa7 thioesterase mutant demonstrated that the TE domain is required for the production of CFA. The co-existence of TE domains within modular PKSs along with physically separated, monofunctional TEs (TE IIs) has been reported for a number of modular polyketide and non-ribosomal peptide synthases (NRPS). An analysis of the two types of thioesterases using Clustal X yielded a dendrogram showing that TE IIs from PKSs and NRPSs are more closely related to each other than to domain TEs from either PKSs or NRPSs. Furthermore, the dendrogram indicates that both types of TE IIs are more closely related to TE domains associated with PKSs than to TE domains in NRPSs. Finally, the overall % G+C content and the % G+C content at the third codon for all of the PKS genes in the COR cluster suggest that these genes may have been recruited from a gram-positive bacterium.

Acyl Carrier Protein↗

PubMed2002

Cryptosporidium parvum: the first protist known to encode a putative polyketide synthase.

We are reporting a putative multifunctional Type I polyketide synthase (PKS) gene from the apicomplexan Cryptosporidium parvum (CpPKS1). The 40 kb intronless open reading frame (ORF) predicts a single polypeptide of 13,414 amino acids with a molecular mass of 1516.5 kDa. Sequence analysis identified at least 29 enzymatic domains within this protein. These domains are organized into an N-terminal loading unit, seven polyketide chain elongation modules, and a carboxy terminator unit. The loading domain consists of an acyl-CoA ligase (AL) and an acyl carrier protein (ACP). All seven elongation modules contain between two and five of the six domains required for the elongation of two-carbon (C2) acyl units, i.e. ketoacyl synthase, acyl transferase, dehydrase, enoyl reductase, ketoreductase and/or ACP. The carboxy terminator is homologous to various reductases, suggesting that the final elongated product is not hydrolytically released by thioesterases as observed in most Type I PKS and all fatty acid synthetase (FAS) systems, but by a reducing reaction, which has been demonstrated in some non-ribosomal peptide synthase systems. The protein sequence and domain organization of CpPKS1 protein resembles a previously reported C. parvum fatty acid synthase (CpFAS1), which is encoded by a 25 kb ORF. Maximum likelihood phylogenetic analysis of acyl transferases within PKS/FAS from C. parvum and other organisms clearly differentiates acetate-extending clades from those incorporating propionate. All acyl transferase domains from CpPKS1, and a previously reported CpFAS1, clustered within the acetate-extending group, suggesting the likelihood that only non-methylated C2 units are incorporated by C. parvum polyketide and fatty acid synthases. The expression of CpPKS1 was confirmed by reverse transcription-polymerase chain reaction and immunofluorescence microscopy. Many polyketides are medically significant antibiotics, anticancer agents, toxins, or signaling molecules. Therefore, it is interesting to speculate what role CpPKS1 might play in this apicomplexan and the disease caused by this opportunistic infection of AIDS patients.

Amino Acid Sequence↗

PubMed2000

Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system.

BACKGROUND: Blooms of toxic cyanobacteria (blue-green algae) have become increasingly common in the surface waters of the world. Of the known toxins produced by cyanobacteria, the microcystins are the most significant threat to human and animal health. These cyclic peptides are potent inhibitors of eukaryotic protein phosphatases type 1 and 2A. Synthesized nonribosomally, the microcystins contain a number of unusual amino acid residues including the beta-amino polyketide moiety Adda (3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyl-4,6-decadienoic acid). We have characterized the microcystin biosynthetic gene cluster from Microcystis aeruginosa PCC7806. RESULTS: A cluster spanning 55 kb, composed of 10 bidirectionally transcribed open reading frames arranged in two putative operons (mcyA-C and mcyD-J), has been correlated with microcystin formation by gene disruption and mutant analysis. Of the 48 sequential catalytic reactions involved in microcystin synthesis, 45 have been assigned to catalytic domains within six large multienzyme synthases/synthetases (McyA-E, G), which incorporate the precursors phenylacetate, malonyl-CoA, S-adenosyl-L-methionine, glutamate, serine, alanine, leucine, D-methyl-isoaspartate, and arginine. The additional four monofunctional proteins are putatively involved in O-methylation (McyJ), epimerization (McyF), dehydration (McyI), and localization (McyH). The unusual polyketide amino acid Adda is formed by transamination of a polyketide precursor as enzyme-bound intermediate, and not released during the process. CONCLUSIONS: This report is the first complete description of the biosynthesis pathway of a complex cyanobacterial metabolite. The enzymatic organization of the microcystin assembly represents an integrated polyketide-peptide biosynthetic pathway with a number of unusual structural and enzymatic features. These include the integrated synthesis of a beta-amino-pentaketide precursor and the formation of beta- and gamma-carboxyl-peptide bonds, respectively. Other features of this complex system also observed in diverse related biosynthetic clusters are integrated C- and N-methyltransferases, an integrated aminotransferase, and an associated O-methyltransferase and a racemase acting on acidic amino acids.

Amino Acid Motifs↗

PubMed2000

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.

Anti-Bacterial Agents↗

PubMed2000

Formation of functional heterologous complexes using subunits from the picromycin, erythromycin and oleandomycin polyketide synthases.

BACKGROUND: Recently developed tools for the genetic manipulation of modular polyketide synthases (PKSs) have advanced the development of combinatorial biosynthesis technologies for drug discovery. Although many of the current techniques involve engineering individual domains or modules of the PKS, few experiments have addressed the ability to combine entire protein subunits from different modular PKSs to create hybrid polyketide pathways. We investigated this possibility by in vivo assembly of heterologous PKS complexes using natural and altered subunits from related macrolide PKSs. RESULTS: The pikAI and pikAII genes encoding subunits 1 and 2 (modules 1-4) of the picromycin PKS (PikPKS) and the eryAIII gene encoding subunit 3 (modules 5-6) of the 6-deoxyerythronolide B synthase (DEBS) were cloned in two compatible Streptomyces expression vectors. A strain of Streptomyces lividans co-transformed with the two vectors produced the hybrid macrolactone 3-hydroxynarbonolide. Co-expression of the same pik genes with the gene for subunit 3 of the oleandomycin PKS (OlePKS) was also successful. A series of hybrid polyketide pathways was then constructed by combining PikPKS subunits 1 and 2 with modified DEBS3 subunits containing engineered domains in modules 5 or 6. We also report the effect of junction location in a set of DEBS-PikPKS fusions. CONCLUSIONS: We show that natural as well as engineered protein subunits from heterologous modular PKSs can be functionally assembled to create hybrid polyketide pathways. This work represents a new strategy that complements earlier domain engineering approaches for combinatorial biosynthesis in which complete modules or PKS protein subunits, in addition to individual enzymatic domains, are used as building blocks for PKS engineering.

Anti-Bacterial Agents↗

PubMed2001

Construction of desosamine containing polyketide libraries using a glycosyltransferase with broad substrate specificity.

BACKGROUND: Combinatorial biosynthesis techniques using polyketide synthases (PKSs) in heterologous host organisms have enabled the production of macrolide aglycone libraries in which many positions of the macrolactone ring have been manipulated. However, the deoxysugar moieties of macrolides, absent in previous libraries, play a critical role in contributing to the antimicrobial properties exhibited by compounds such as erythromycin. Since the glycosidic components of polyketides dramatically alter their molecular binding properties, it would be useful to develop general expression hosts and vectors for synthesis and attachment of deoxysugars to expand the nature and size of such polyketide libraries. RESULTS: A set of nine deoxysugar biosynthetic and auxiliary genes from the picromycin/methymycin (pik) cluster was integrated in the chromosome of Streptomyces lividans to create a host which synthesizes TDP-D-desosamine. The pik desosaminyl transferase was also included so that when the strain was transformed with a previously constructed library of expression plasmids encoding genetically modified PKSs that produce different macrolactones, the resulting strains produced desosaminylated derivatives. Although conversion of the macrolactones was generally low, bioassays revealed that, unlike their aglycone precursors, these novel macrolides possessed antibiotic activity. CONCLUSIONS: Based on the structural differences among the compounds that were glycosylated it appears that the desosaminyl transferase from the pik gene cluster is quite tolerant of changes in the macrolactone substrate. Since others have demonstrated tolerance towards modifications in the sugar substituent, one can imagine employing this approach to alter both polyketide and deoxysugar pathways to produce 'unnatural' natural product libraries.

Amino Sugars↗

PubMed1998

Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase.

BACKGROUND: Modular polyketide synthases (PKSs) catalyse the biosynthesis of complex polyketides using a different set of enzymes for each successive cycle of chain extension. Directed biosynthesis starting from synthetic diketides is a potentially valuable route to novel polyketides. We have used a purified bimodular derivative of the erythromycin-producing polyketide synthase (DEBS 1-TE) to study chain extension starting from a variety of diketide analogues and, in some cases, from the alternative acyl-CoA thioester substrates. RESULTS: Chain initiation in vitro by DEBS 1-TE module 2 using a synthetic diketide analogue as a substrate was tolerant of significant structural variation in the starter unit of the synthetic diketide, but other changes completely abolished activity. Interestingly, a racemic beta-keto diketide was found to be reduced in situ on the PKS and utilised in place of its more complex hydroxy analogue as a substrate for chain extension. The presence of a diketide analogue strongly inhibited chain initiation via the loading module. Significantly higher concentrations of diketide N-acetylcysteamine analogues than their corresponding acyl-CoA thioesters are required to achieve comparable yields of triketide lactones. CONCLUSIONS: Although a broad range of variation in the starter residue is acceptable, the substrate specificity of module 2 of a typical modular PKS in vitro is relatively intolerant of changes at C-2 and C-3. This will restrict the usefulness of approaches to synthesise novel erythromycins using synthetic diketides in vivo. The use of synthetic beta-keto diketides in vivo deserves to be explored.

Catalysis↗

PubMed1999

Polyketide synthase acyl carrier protein (ACP) as a substrate and a catalyst for malonyl ACP biosynthesis.

BACKGROUND: Using an acyl-acyl carrier protein (ACP) as a starter unit, type II polyketide synthases (PKSs) generate a wide range of polyketide products by successive decarboxylative condensations with the two-carbon donor malonyl (ACP). In vitro experiments have demonstrated that polyketide biosynthesis in reconstituted PKS systems requires the fatty acid synthase (FAS) enzyme malonyl CoA:ACP acyltransferase (FabD) from streptomycetes. It has also been shown that holo-ACPs from a type II PKS can catalyze self-malonylation in the presence of malonyl CoA and negate this FabD requirement. The relative roles of FabD and ACP self-malonylation in PKS biosynthesis in vivo are still not known. RESULTS: We have examined the ACP specificity of the Streptomyces glaucescens FabD and shown that it reacts specifically with monomeric forms of ACP, with comparable k(cat)/K(M) values for ACPs from both type II PKS and FAS systems. Incubations of tetracenomycin ACP (TcmM) with the Escherichia coli FAS ACP (AcpP) unexpectedly revealed that, in addition to the self-malonylation process, TcmM can catalyze the malonylation of AcpP. The k(cat)/K(M) value for the TcmM-catalyzed malonylation of S. glaucescens FAS ACP is two orders of magnitude smaller than that observed for the FabD-catalyzed process. CONCLUSIONS: The ability of a PKS ACP to catalyze malonylation of a FAS ACP is a surprising finding and demonstrates for the first time that PKS ACPs and FabD can catalyze the same reaction. The differences in the catalytic efficiency of these two proteins rationalizes in vitro observations that FabD-independent polyketide biosynthesis proceeds only at high concentrations of a PKS ACP.

Acyl-Carrier Protein S-Malonyltransferase↗

PubMed2002

The polyketide synthase gene pks4 from Gibberella fujikuroi encodes a key enzyme in the biosynthesis of the red pigment bikaverin.

The ascomycete Gibberella fujikuroi mating population C (MP-C) is well known for the production of gibberellins, but also produces many other secondary metabolites, including the red polyketide pigment bikaverin. Here, we used a differential display method to clone a polyketide synthase gene pks4 responsible for the first step of bikaverin biosynthesis. Sequence analysis indicated that pks4 encoded a 2009-amino acid polypeptide consisting of four functional domains: beta-ketoacyl synthase (KS), acyltransferase (AT), acyl carrier (ACP), and thioesterase (TE). Disruption of pks4 resulted in the loss of both pks4 transcripts and bikaverin biosynthesis in G. fujikuroi cultures. Expression of pks4 is strongly repressed by high amounts of ammonium and basic pH. Unexpectedly, pks4 was overexpressed in mutants of the regulatory gene, areA, which is responsible for the activation of nitrogen assimilation genes. Three additional polyketide synthase genes have been cloned from G. fujikuroi MP-C by heterologous hybridization. The presence of these four PKS genes demonstrates the diversity of polyketide biosynthetic pathways in this fungus.

Amino Acid Sequence↗

PubMed2000

Substrate specificity of the loading didomain of the erythromycin polyketide synthase.

The priming of many modular polyketide synthases is catalyzed by a loading acyltransferase-acyl carrier protein (AT(L)-ACP(L)) didomain which initiates polyketide biosynthesis by transferring a primer unit to the ketosynthase domain of the first module. Because the AT(L) domain influences the choice of the starter unit incorporated into the polyketide backbone, its specificity is of considerable interest. The AT(L)-ACP(L) didomain of the 6-deoxyerythronolide B synthase (DEBS) was functionally expressed in Escherichia coli. Coexpression of the Sfp phosphopantetheinyl transferase from Bacillus subtilis in E. coli leads to efficient posttranslational modification of the ACP(L) domain with a phosphopantetheine moiety. Competition experiments were performed with the holo-protein to determine the relative rates of incorporation of a variety of unnatural substrates in the presence of comparable concentrations of labeled acetyl-CoA. Our results showed that the loading didomain of DEBS can accept a surprisingly broad range of substrates, although it exhibits a preference for unbranched alkyl chain substrates over branched alkyl chain, polar, aromatic, and charged substrates. In particular, its tolerance toward acetyl- and butyryl-CoA is unexpectedly strong. The studies described here present an attractive prototype for the expression, analysis, and engineering of acyltransferase domains in modular polyketide synthases.

Bacillus subtilis↗

PubMedDate not supplied

Enhancing the atom economy of polyketide biosynthetic processes through metabolic engineering.

Polyketides, a large family of bioactive natural products, are synthesized from building blocks derived from alpha-carboxylated Coenzyme A thioesters such as malonyl-CoA and (2S)-methylmalonyl-CoA. The productivity of polyketide fermentation processes in natural and heterologous hosts is frequently limited by the availability of these precursors in vivo. We describe a metabolic engineering strategy to enhance both the yield and volumetric productivity of polyketide biosynthesis. The genes matB and matC from Rhizobium trifolii encode a malonyl-CoA synthetase and a putative dicarboxylate transport protein, respectively. These proteins can directly convert exogenous malonate and methylmalonate into their corresponding CoA thioesters with an ATP requirement of 2 mol per mol of acyl-CoA produced. Heterologous expression of matBC in a recombinant strain of Streptomyces coelicolor that produces the macrolactone 6-deoxyerythronolide B results in a 300% enhancement of macrolactone titers. The unusual efficiency of the bioconversion is illustrated by the fact that approximately one-third of the methylmalonate units added to the fermentation medium are converted into macrolactones. The direct conversion of inexpensive feedstocks such as malonate and methylmalonate into polyketides represents the most carbon- and energy-efficient route to these high value natural products and has implications for cost-effective fermentation of numerous commercial and development-stage small molecules.

Acyl Coenzyme A↗

PubMed2001

Heterologous expression of the naphthocyclinone hydroxylase gene from Streptomyces arenae for production of novel hybrid polyketides.

Streptomyces arenae produces at least four different isochromanequinone antibiotics, the naphthocyclinones, of which the beta- and gamma-form are active against Gram-positive bacteria. The naphthocyclinone biosynthesis gene cluster was isolated from Streptomyces arenae DSM 40737 and by sequence analysis the minimal polyketide synthase genes and several genes encoding tailoring enzymes were identified. Southern blot analysis of the naphthocyclinone gene cluster indicated that a 3.5 kb BamHI fragment located approximately 9 kb downstream of the minimal PKS genes hybridizes to the schC hydroxylase DNA probe isolated from S. halstedii. Two complete and one incomplete open reading frames were identified on this fragment. Sequence analysis revealed strong homology to the genes of the actVA region of S. coelicolor, to several (suggested) hydroxylases and a putative FMN-dependent monooxygenase. The proposed hydroxylase, encoded by ncnH, could hydroxylate aloesaponarin II, a molecule that is produced by the actinorhodin minimal polyketide synthase in combination with the actinorhodin ketoreductase, aromatase and cyclase. Furthermore, this enzyme is capable of accepting additional polyketide core structures that contain a 5-hydroxy-1,4-naphthoquinone moiety as substrates which makes it an interesting tailoring enzyme for the modification of polyketide structures.

Amino Acid Sequence↗

PubMedDate not supplied

Homology between Streptomyces genes coding for synthesis of different polyketides used to clone antibiotic biosynthetic genes.

Many important antibiotics such as tetracyclines, erythromycin, adriamycin, monensin, rifamycin and avermectins are polyketides. In their biosynthesis, multifunctional synthases catalyse iterated condensation of thio-esters derived from acetate, propionate or butyrate to yield aliphatic chains of varying length and carrying different alkyl substituents. Subsequent modifications, including aromatic or macrolide ring closure or specific methylations or glycosylations, generate further chemical diversity. It has been suggested that, if different polyketide synthases had a common evolutionary origin, cloned DNA coding for one synthase might be used as a hybridization probe for the isolation of others. We show here that this is indeed possible. Study of a range of such synthase genes and their products should help to elucidate what determines the choice and order of condensation of different residues in polyketide assembly, and might yield, by in vitro recombination or mutagenesis, synthase genes capable of producing novel antibiotics. Moreover, because genes for entire antibiotic pathways are usually clustered in Streptomyces, cloned polyketide synthase genes are valuable in giving access to groups of linked biosynthetic genes.

Anthraquinones↗

PubMed1999

A chain initiation factor common to both modular and aromatic polyketide synthases.

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named 'chain-length factor', is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.

Acyl Carrier Protein↗

PubMed1996

Evidence for a double-helical structure for modular polyketide synthases.

Modular polyketide synthases are multienzymes responsible for the biosynthesis of a large number of clinically important natural products. They contain multiple sets, or modules, of enzymatic activities, distributed between a few giant multienzymes and there is one module for every successive cycle of polyketide chain extension. We show here that each multienzyme in a typical modular polyketide synthase forms a (possibly helical) parallel dimer, and that each pair of identical modules interacts closely across the dimer interface. Such an arrangement would allow identical modules to share active sites for chain extension, and thus to function independently of flanking modules, which would have important implications both for mechanisms of evolution of polyketide synthases and for their future genetic engineering.

Amino Acid Sequence↗

PubMed2001

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.

Acyltransferases↗

PubMed2000

Assembly of aryl-capped siderophores by modular peptide synthetases and polyketide synthases.

Bacterial siderophores assist pathogens in iron acquisition inside their hosts. They are often essential for achieving a successful infection, and their biosynthesis represents an attractive antibiotic target. Recently, several siderophore biosynthetic loci have been identified, and in vitro studies have advanced our knowledge of the biosynthesis of aryl-capped peptide and peptide-polyketide siderophores from Mycobacterium spp., Pseudomonas spp., Yersinia spp. and other bacteria. These studies also provided insights into the assembly of related siderophores and many secondary metabolites of medical relevance. Assembly of aryl-capped peptide and peptide-polyketide siderophores involves non-ribosomal peptide synthetase, polyketide synthase and non-ribosomal-peptide polyketide hybrid subunits. Analysis of these subunits suggests that their domains and modules are functionally and structurally independent. It appears that nature has selected a set of functional domains and modules that can be rearranged in different order and combinations to biosynthesize different products. Although much remains to be learned about modular synthetases and synthases, it is already possible to conceive strategies to engineer these enzymes to generate novel products.

Bacteria↗

PubMed2002

Biochemical evidence for an editing role of thioesterase II in the biosynthesis of the polyketide pikromycin.

The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an "editing" enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6-deoxyerythronolide B synthase; AT(L)-ACP(L)). The pikromycin TEII exhibited high K(m) values (>100 microm) with all substrates and no apparent ACP specificity, catalyzing cleavage of methylmalonyl-ACP from both AT(L)-ACP(L) (k(cat)/K(m) 3.3 +/- 1.1 m(-1) s(-1)) and PikAIII (k(cat)/K(m) 2.9 +/- 0.9 m(-1) s(-1)). The TEII exhibited some acyl-group specificity, catalyzing hydrolysis of propionyl (k(cat)/K(m) 15.8 +/- 1.8 m(-1) s(-1)) and butyryl (k(cat)/K(m) 17.5 +/- 2.1 m(-1) s(-1)) derivatives of AT(L)-ACP(L) faster than acetyl (k(cat)/K(m) 4.9 +/- 0.7 m(-1) s(-1)), malonyl (k(cat)/K(m) 3.9 +/- 0.5 m(-1) s(-1)), or methylmalonyl derivatives. PikAIV containing a TEI domain catalyzed cleavage of propionyl derivative of AT(L)-ACP(L) at a dramatically lower rate than TEII. These results provide the first unequivocal in vitro evidence that TEII can hydrolyze acyl-ACP thioesters and a model for the action of TEII in which the enzyme remains primarily dissociated from the polyketide synthase, preferentially removing aberrant acyl-ACP species with long half-lives. The lack of rigorous substrate specificity for TEII may explain the surprising observation that high level expression of the protein in Streptomyces venezuelae leads to significant (>50%) titer decreases.

Anti-Bacterial Agents↗