Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A thioesterase from an iterative fungal polyketide synthase shows macrocyclization and cross coupling activity and may play a role in controlling iterative cycling through product offloading.

ABSTRACT: Zearalenone, a fungal macrocyclic polyketide, is a member of the resorcylic acid lactone family. Herein, we characterize in vitro the thioesterase from PKS13 in zearalenone biosynthesis (Zea TE). The excised Zea TE catalyzes macrocyclization of a linear thioester-activated model of zearalenone. Zea TE also catalyzes the cross coupling of a benzoyl thioester with alcohols and amines. Kinetic characterization of the cross coupling is consistent with a ping-pong bi-bi mechanism, confirming an acyl-enzyme intermediate. Finally, the substrate specificity of the Zea TE indicates the TE may help control iterative cycling on PKS13 by rapidly offloading the final resorcylate-containing product.

SUBMITTER: Wang M

PROVIDER: S-EPMC2722786 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A thioesterase from an iterative fungal polyketide synthase shows macrocyclization and cross coupling activity and may play a role in controlling iterative cycling through product offloading.

Wang Meng M Zhou Hui H Wirz Monica M Tang Yi Y Boddy Christopher N CN

Biochemistry 20090701 27

Zearalenone, a fungal macrocyclic polyketide, is a member of the resorcylic acid lactone family. Herein, we characterize in vitro the thioesterase from PKS13 in zearalenone biosynthesis (Zea TE). The excised Zea TE catalyzes macrocyclization of a linear thioester-activated model of zearalenone. Zea TE also catalyzes the cross coupling of a benzoyl thioester with alcohols and amines. Kinetic characterization of the cross coupling is consistent with a ping-pong bi-bi mechanism, confirming an acyl- ...[more]

PMID: 19530704

Similar Datasets

Offloading Role of a Discrete Thioesterase in Type II Polyketide Biosynthesis.

Project description:Type II polyketides are a group of secondary metabolites with various biological activities. In nature, biosynthesis of type II polyketides involves multiple enzymatic steps whereby key enzymes, including ketoacyl-synthase (KSα), chain length factor (KSβ), and acyl carrier protein (ACP), are utilized to elongate the polyketide chain through a repetitive condensation reaction. During each condensation, the biosynthesis intermediates are covalently attached to KSα or ACP via a thioester bond and are then cleaved to release an elongated polyketide chain for successive postmodification. Despite its critical role in type II polyketide biosynthesis, the enzyme and its corresponding mechanism for type II polyketide chain release through thioester bond breakage have yet to be determined. Here, kinamycin was used as a model compound to investigate the chain release step of type II polyketide biosynthesis. Using a genetic knockout strategy, we confirmed that AlpS is required for the complete biosynthesis of kinamycins. Further in vitro biochemical assays revealed high hydrolytic activity of AlpS toward a thioester bond in an aromatic polyketide-ACP analog, suggesting its distinct role in offloading the polyketide chain from ACP during the kinamycin biosynthesis. Finally, we successfully utilized AlpS to enhance the heterologous production of dehydrorabelomycin in Escherichia coli by nearly 25-fold, which resulted in 0.50 g/liter dehydrorabelomycin in a simple batch-mode shake flask culture. Taken together, our results provide critical knowledge to gain an insightful understanding of the chain-releasing process during type II polyketide synthesis, which, in turn, lays a solid foundation for future new applications in type II polyketide bioproduction.

| S-EPMC7492732 | biostudies-literature

Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis.

Project description:Polyketide natural products possess diverse architectures and biological functions and share a subset of biosynthetic steps with fatty acid synthesis. The final transformation catalyzed by both polyketide synthases (PKSs) and fatty acid synthases is most often carried out by a thioesterase (TE). The synthetic versatility of TE domains in fungal nonreducing, iterative PKSs (NR-PKSs) has been shown to extend to Claisen cyclase (CLC) chemistry by catalyzing C-C ring closure reactions as opposed to thioester hydrolysis or O-C/N-C macrocyclization observed in previously reported TE structures. Catalysis of C-C bond formation as a product release mechanism dramatically expands the synthetic potential of PKSs, but how this activity was acquired has remained a mystery. We report the biochemical and structural analyses of the TE/CLC domain in polyketide synthase A, the multidomain PKS central to the biosynthesis of aflatoxin B(1), a potent environmental carcinogen. Mutagenesis experiments confirm the predicted identity of the catalytic triad and its role in catalyzing the final Claisen-type cyclization to the aflatoxin precursor, norsolorinic acid anthrone. The 1.7 A crystal structure displays an alpha/beta-hydrolase fold in the catalytic closed form with a distinct hydrophobic substrate-binding chamber. We propose that a key rotation of the substrate side chain coupled to a protein conformational change from the open to closed form spatially governs substrate positioning and C-C cyclization. The biochemical studies, the 1.7 A crystal structure of the TE/CLC domain, and intermediate modeling afford the first mechanistic insights into this widely distributed C-C bond-forming class of TEs.

| S-EPMC2851968 | biostudies-literature

Iterative polyketide biosynthesis by modular polyketide synthases in bacteria.

Project description:Modular polyketide synthases (type I PKSs) in bacteria are responsible for synthesizing a significant percentage of bioactive natural products. This group of synthases has a characteristic modular organization, and each module within a PKS carries out one cycle of polyketide chain elongation; thus each module is non-iterative in function. It was possible to predict the basic structure of a polyketide product from the module organization of the PKSs, since there generally existed a co-linearity between the number of modules and the number of chain elongations. However, more and more bacterial modular PKSs fail to conform to the canonical rules, and a particularly noteworthy group of non-canonical PKSs is the bacterial iterative type I PKSs. This review covers recent examples of iteratively used modular PKSs in bacteria. These non-canonical PKSs give rise to a large array of natural products with impressive structural diversity. The molecular mechanism behind the iterations is often unclear, presenting a new challenge to the rational engineering of these PKSs with the goal of generating new natural products. Structural elucidation of these synthase complexes and better understanding of potential PKS-PKS interactions as well as PKS-substrate recognition may provide new prospects and inspirations for the discovery and engineering of new bioactive polyketides.

| S-EPMC4706475 | biostudies-literature

Intrinsic and Extrinsic Programming of Product Chain Length and Release Mode in Fungal Collaborating Iterative Polyketide Synthases.

Project description:Combinatorial biosynthesis with fungal polyketide synthases (PKSs) promises to produce unprecedented bioactive "unnatural" natural products (uNPs) for drug discovery. Genome mining of the dothideomycete Rhytidhysteron rufulum uncovered a collaborating highly reducing PKS (hrPKS)-nonreducing PKS (nrPKS) pair. These enzymes produce trace amounts of rare S-type benzenediol macrolactone congeners with a phenylacetate core in a heterologous host. However, subunit shuffling and domain swaps with voucher enzymes demonstrated that all PKS domains are highly productive. This contradiction led us to reveal novel programming layers exerted by the starter unit acyltransferase (SAT) and the thioesterase (TE) domains on the PKS system. First, macrocyclic vs linear product formation is dictated by the intrinsic biosynthetic program of the TE domain. Next, the chain length of the hrPKS product is strongly influenced in trans by the off-loading preferences of the nrPKS SAT domain. Last, TE domains are size-selective filters that facilitate or obstruct product formation from certain priming units. Thus, the intrinsic programs of the SAT and TE domains are both part of the extrinsic program of the hrPKS subunit and modulate the observable metaprogram of the whole PKS system. Reconstruction of SAT and TE phylogenies suggests that these domains travel different evolutionary trajectories, with the resulting divergence creating potential conflicts in the PKS metaprogram. Such conflicts often emerge in chimeric PKSs created by combinatorial biosynthesis, reducing biosynthetic efficiency or even incapacitating the system. Understanding the points of failure for such engineered biocatalysts is pivotal to advance the biosynthetic production of uNPs.

| S-EPMC7659983 | biostudies-literature

Discovery and heterologous biosynthesis of glycosylated polyketide luteodienoside A reveals unprecedented glucinol-mediated product offloading by a fungal carnitine <i>O</i>-acyltransferase domain.

Project description:Luteodienoside A is a novel glycosylated polyketide produced by the Australian fungus Aspergillus luteorubrus MST-FP2246, consisting of an unusual 1-O-β-d-glucopyranosyl-myo-inositol (glucinol) ester of 3-hydroxy-2,2,4-trimethylocta-4,6-dienoic acid. Mining the genome of A. luteorubrus identified a putative gene cluster for luteodienoside A biosynthesis (ltb), harbouring a highly reducing polyketide synthase (HR-PKS, LtbA) fused at its C-terminus to a carnitine O-acyltransferase (cAT) domain. Heterologous pathway reconstitution in Aspergillus nidulans, substrate feeding assays and gene truncation confirmed the identity of the ltb cluster and demonstrated that the cAT domain is essential for offloading luteodienoside A from the upstream HR-PKS. Unlike previously characterised cAT domains, the LtbA cAT domain uses glucinol as an offloading substrate to release the product from the HR-PKS. Furthermore, the PKS methyltransferase (MT) domain is capable of catalysing gem-dimethylation of the 3-hydroxy-2,2,4-trimethylocta-4,6-dienoic acid intermediate, without requiring reversible product release and recapture by the cAT domain. This study expands the repertoire of polyketide modifications known to be catalysed by cAT domains and highlights the potential of mining fungal genomes for this subclass of fungal PKSs to discover new structurally diverse secondary metabolites.

| S-EPMC10901484 | biostudies-literature

Deconstruction of iterative multidomain polyketide synthase function.

Project description:PksA, which initiates biosynthesis of the environmental carcinogen aflatoxin B1, is one of the multidomain iterative polyketide synthases (IPKSs), a large, poorly understood family of biosynthetic enzymes. We found that dissection of PksA and its reconstitution from selected sets of domains allows the accumulation and characterization of advanced octaketide intermediates bound to the enzyme, permitting the reactions controlled by individual catalytic domains to be identified. A product template (PT) domain unites with the ketosynthase and thioesterase in this IPKS system to assemble precisely seven malonyl-derived building blocks to a hexanoyl starter unit and mediate a specific cyclization cascade. Because the PT domain is common among nonreducing IPKSs, these mechanistic features should prove to be general for IPKS-catalyzed production of aromatic polyketides.

| S-EPMC2480491 | biostudies-literature

The iterative gramicidin s thioesterase catalyzes peptide ligation and cyclization.

Project description:Here, we present a comprehensive in vitro characterization of the excised iterative, bimodular PCP-TE of the gramicidin S synthetase GrsB, which is able to act both as a ligation and a cyclization catalyst. Using the native pentapeptidyl-thioester substrates, GrsB PCP-TE catalyzes the dimerization and subsequent formation of the decapeptide lactam gramicidin S. Interestingly, the detection of linear decapeptidyl-SNAC as an enzyme-dependent intermediate supports the iterative mechanism in vivo, in which two pentapeptides, one bound as an ester to the active site serine of the TE domain and the second bound as a thioester to the adjacent pan-PCP, are ligated to a decapeptidyl-pan-PCP that subsequently transferred to the adjacent TE domain and cyclized. Moreover, GrsB PCP-TE can handle different substrates length, leading not only to dimerization, but also to trimerization and the formation of different ring sizes.

| S-EPMC7129787 | biostudies-literature

Macrodiolide formation by the thioesterase of a modular polyketide synthase.

Project description:Elaiophylin is an unusual C2 -symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in?vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C2 -symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in?vitro construction of novel diolides.

| S-EPMC4471547 | biostudies-literature

Macrodiolide Formation by the Thioesterase of a Modular Polyketide Synthase.

Project description:Elaiophylin is an unusual C2-symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C2-symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides.

| S-EPMC4535664 | biostudies-literature

A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst.

Project description:Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and synthetic chemistry. In the preceding paper , we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural substrates in the pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular dynamics simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrinsic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the observed hydrolysis. Accordingly, we engineered the stereoselective Pik TE to yield a variant (TES148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TES148C and TEWT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition-elimination (TEWT) to a lower energy concerted acyl substitution (TES148C), accounting for the gain-of-function and improved reaction kinetics. Finally, we introduced the S148C mutation into a polyketide synthase module (PikAIII-TE) to impart increased substrate flexibility, enabling the production of diastereomeric macrolactones.

| S-EPMC5617804 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data