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Natural Product Reports Oct 2018Covering: up to April 5, 2018 Metabolites from type II fatty acid synthase (FAS) and polyketide synthase (PKS) pathways differ broadly in their identities and functional... (Review)
Review
Covering: up to April 5, 2018 Metabolites from type II fatty acid synthase (FAS) and polyketide synthase (PKS) pathways differ broadly in their identities and functional roles. The former are considered primary metabolites that are linear hydrocarbon acids, while the latter are complex aromatic or polyunsaturated secondary metabolites. Though the study of bacterial FAS has benefitted from decades of biochemical and structural investigations, type II PKSs have remained less understood. Here we review the recent approaches to understanding the protein-protein and protein-substrate interactions in these pathways, with an emphasis on recent chemical biology and structural applications. New approaches to the study of FAS have highlighted the critical role of the acyl carrier protein (ACP) with regard to how it stabilizes intermediates through sequestration and selectively delivers cargo to successive enzymes within these iterative pathways, utilizing protein-protein interactions to guide and organize enzymatic timing and specificity. Recent tools that have shown promise in FAS elucidation should find new approaches to studying type II PKS systems in the coming years.
Topics: Crystallography, X-Ray; Cyclization; Fatty Acid Synthase, Type II; Helicobacter pylori; Mutagenesis; Nuclear Magnetic Resonance, Biomolecular; Polyketide Synthases; Protein Interaction Maps; Proteins; Substrate Specificity
PubMed: 30046786
DOI: 10.1039/c8np00040a -
Current Opinion in Structural Biology Apr 2015Modular polyketide synthases (PKS) produce a vast array of bioactive molecules that are the basis of many highly valued pharmaceuticals. The biosynthesis of these... (Review)
Review
Modular polyketide synthases (PKS) produce a vast array of bioactive molecules that are the basis of many highly valued pharmaceuticals. The biosynthesis of these compounds is based on ordered assembly lines of multi-domain modules, each extending and modifying a specific chain-elongation intermediate before transfer to the next module for further processing. The first 3D structures of a full polyketide synthase module in different functional states were obtained recently by electron cryo-microscopy. The unexpected module architecture revealed a striking evolutionary divergence of the polyketide synthase compared to its metazoan fatty acid synthase homolog, as well as remarkable conformational rearrangements dependent on its biochemical state during the full catalytic cycle. The design and dynamics of the module are highly optimized for both catalysis and fidelity in the construction of complex, biologically active natural products.
Topics: Acyl Carrier Protein; Animals; Cryoelectron Microscopy; Evolution, Molecular; Humans; Polyketide Synthases; Protein Structure, Tertiary
PubMed: 25791608
DOI: 10.1016/j.sbi.2015.02.014 -
Methods in Enzymology 2022Polyketides have demonstrated their significance as therapeutics, industrial products, pesticides, and biological probes following intense study over the past decades....
Polyketides have demonstrated their significance as therapeutics, industrial products, pesticides, and biological probes following intense study over the past decades. Tagging polyketides with a bioorthogonal functionality enables various applications such as diversification, quantification, visualization and mode-of-action elucidation. The terminal alkyne moiety, as a small, stable and highly selective clickable functionality, is widely adopted in tagging natural products. De novo biosynthesis of alkyne-tagged polyketides offers the unique advantage of reducing the background from feeding the biorthogonal moiety itself, leading to the accomplishment of in situ generation of a clickable functionality for bioorthogonal reactions. Here, we introduce several engineering strategies to apply terminal alkyne biosynthetic machinery, represented by JamABC, which produces a short terminal alkyne-bearing fatty acyl chain on a carrier protein, to functions with different downstream polyketide synthases (PKSs). Successful results in engineering type III and type I PKSs provide engineering guidelines and strategies that are applicable to additional PKSs to produce targeted alkyne-tagged metabolites for chemical and biological applications.
Topics: Alkynes; Biological Products; Polyketide Synthases; Polyketides
PubMed: 35379442
DOI: 10.1016/bs.mie.2021.11.013 -
Scientific Reports Jun 2024Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have...
Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.
Topics: Cell-Free System; Promoter Regions, Genetic; Streptomyces griseus; Escherichia coli; Multigene Family; Bacterial Proteins; Polyketide Synthases; Codon; Acyltransferases
PubMed: 38839808
DOI: 10.1038/s41598-024-61376-w -
Mechanisms of Development Jun 2019Deflecting biomineralized crystals attached to vestibular hair cells are necessary for maintaining balance. Zebrafish (Danio rerio) are useful organisms to study these...
Deflecting biomineralized crystals attached to vestibular hair cells are necessary for maintaining balance. Zebrafish (Danio rerio) are useful organisms to study these biomineralized crystals called otoliths, as many required genes are homologous to human otoconial development. We sought to identify and characterize the causative gene in a trio of homozygous recessive mutants, no content (nco) and corkscrew (csr), and vanished (vns), which fail to develop otoliths during early ear development. We show that nco, csr, and vns have potentially deleterious mutations in polyketide synthase (pks1), a multi-modular protein that has been previously implicated in biomineralization events in chordates and echinoderms. We found that Otoconin-90 (Oc90) expression within the otocyst is diffuse in nco and csr; therefore, it is not sufficient for otolith biomineralization in zebrafish. Similarly, normal localization of Otogelin, a protein required for otolith tethering in the otolithic membrane, is not sufficient for Oc90 attachment. Furthermore, eNOS signaling and Endothelin-1 signaling were the most up- and down-regulated pathways during otolith agenesis in nco, respectively. Our results demonstrate distinct processes for otolith nucleation and biomineralization in vertebrates and will be a starting point for models that are independent of Oc90-mediated seeding. This study will serve as a basis for investigating the role of eNOS signaling and Endothelin-1 signaling during otolith formation.
Topics: Animals; Base Sequence; Biomineralization; DNA; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Ions; Mutation; Oryzias; Otolithic Membrane; Plasmids; Polyketide Synthases; RNA, Messenger; Zebrafish; Zebrafish Proteins
PubMed: 30974150
DOI: 10.1016/j.mod.2019.04.001 -
Proceedings of the National Academy of... Sep 2023Animal cytoplasmic fatty acid synthase (FAS) represents a unique family of enzymes that are classically thought to be most closely related to fungal polyketide synthase...
Animal cytoplasmic fatty acid synthase (FAS) represents a unique family of enzymes that are classically thought to be most closely related to fungal polyketide synthase (PKS). Recently, a widespread family of animal lipid metabolic enzymes has been described that bridges the gap between these two ubiquitous and important enzyme classes: the animal FAS-like PKSs (AFPKs). Although very similar in sequence to FAS enzymes that produce saturated lipids widely found in animals, AFPKs instead produce structurally diverse compounds that resemble bioactive polyketides. Little is known about the factors that bridge lipid and polyketide synthesis in the animals. Here, we describe the function of EcPKS2 from , which synthesizes a complex polypropionate natural product found in this mollusc. EcPKS2 starter unit promiscuity potentially explains the high diversity of polyketides found in and among molluscan species. Biochemical comparison of EcPKS2 with the previously described EcPKS1 reveals molecular principles governing substrate selectivity that should apply to related enzymes encoded within the genomes of photosynthetic gastropods. Hybridization experiments combining EcPKS1 and EcPKS2 demonstrate the interactions between the ketoreductase and ketosynthase domains in governing the product outcomes. Overall, these findings enable an understanding of the molecular principles of structural diversity underlying the many molluscan polyketides likely produced by the diverse AFPK enzyme family.
Topics: Animals; Polyketide Synthases; Fatty Acid Synthases; Biological Products; Gastropoda; Polyketides; Lipids
PubMed: 37695909
DOI: 10.1073/pnas.2305575120 -
The Plant Journal : For Cell and... Oct 2022Type III polyketide synthases (PKSs) are key enzymes involved in the biosynthesis of a variety of plant specialized metabolites, including flavonoids, stilbenes, and...
Type III polyketide synthases (PKSs) are key enzymes involved in the biosynthesis of a variety of plant specialized metabolites, including flavonoids, stilbenes, and sporopollenin, to name a few. These enzymes likely played vital roles in plant adaptation during their transition from aquatic to terrestrial habitats and their colonization of specific ecological environments. Members of this supergene family have diverse functions, but how type III PKSs and their functions have evolved remains poorly understood. Here, we conducted comprehensive phylogenomics analysis of the type III PKS supergene family in 60 species representing the major plant lineages and elucidated the classification, origin, and evolutionary history of each class. Molecular evolutionary analysis of the typical chalcone synthase and stilbene synthase types revealed evidence for strong positive natural selection in both the Pinaceae and Fabaceae lineages. The positively selected sites of these proteins include residues at the catalytic tunnel entrance and homodimer interface, which might have driven the functional divergence between the two types. Our results also suggest that convergent evolution of enzymes involved in plant flavonoid biosynthesis is quite common. The results of this study provide new insights into the origin, evolution, and functional diversity of plant type III PKSs. In addition, they serve as a guide for the enzymatic engineering of plant polyketides.
Topics: Polyketide Synthases; Plants; Polyketides; Stilbenes; Flavonoids
PubMed: 36004534
DOI: 10.1111/tpj.15953 -
Natural Product Reports Sep 2018Covering: up to 2018 Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide... (Review)
Review
Covering: up to 2018 Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide synthetases, polyketide synthases, and their hybrid biosynthetic complexes. This reductive off-loading of a natural product yields an aldehyde or alcohol, can initiate the formation of a macrocyclic imine, and contributes to important intermediates in a variety of biosyntheses, including those for polyketide alkaloids and pyrrolobenzodiazepines. Compounds that arise from reductase-terminated biosynthetic gene clusters are often reactive and exhibit biological activity. Biomedically important examples include the cancer therapeutic Yondelis (ecteinascidin 743), peptide aldehydes that inspired the first therapeutic proteasome inhibitor bortezomib, and numerous synthetic derivatives and antibody drug conjugates of the pyrrolobenzodiazepines. Recent advances in microbial genomics, metabolomics, bioinformatics, and reactivity-based labeling have facilitated the detection of these compounds for targeted isolation. Herein, we summarize known natural products arising from this important category, highlighting their occurrence in Nature, biosyntheses, biological activities, and the technologies used for their detection and identification. Additionally, we review publicly available genomic data to highlight the remaining potential for novel reductively tailored compounds and drug leads from microorganisms. This thorough retrospective highlights various molecular families with especially privileged bioactivity while illuminating challenges and prospects toward accelerating the discovery of new, high value natural products.
Topics: Alkaloids; Azabicyclo Compounds; Benzodiazepinones; Biological Products; Biosynthetic Pathways; Cyclization; Depsipeptides; Dipeptides; Indoles; Lactams; Leupeptins; Lysine; Multigene Family; Peptide Synthases; Polyketide Synthases; Protein Domains
PubMed: 29916519
DOI: 10.1039/c8np00013a -
International Journal of Molecular... May 2023Polyketides are a diverse set of natural products with versatile applications as pharmaceuticals, nutraceuticals, and cosmetics, to name a few. Of several types of... (Review)
Review
Polyketides are a diverse set of natural products with versatile applications as pharmaceuticals, nutraceuticals, and cosmetics, to name a few. Of several types of polyketides, aromatic polyketides comprising type II and III polyketides contain many chemicals important for human health such as antibiotics and anticancer agents. Most aromatic polyketides are produced from soil bacteria or plants, which are difficult to engineer and grow slowly in industrial settings. To this end, metabolic engineering and synthetic biology have been employed to efficiently engineer heterologous model microorganisms for enhanced production of important aromatic polyketides. In this review, we discuss the recent advancement in metabolic engineering and synthetic biology strategies for the production of type II and type III polyketides in model microorganisms. Future challenges and prospects of aromatic polyketide biosynthesis by synthetic biology and enzyme engineering approaches are also discussed.
Topics: Humans; Polyketides; Metabolic Engineering; Synthetic Biology; Polyketide Synthases; Biological Products
PubMed: 37240269
DOI: 10.3390/ijms24108923 -
Nature Communications Mar 2023Assembly-line polyketide synthases (PKSs) are molecular factories that produce diverse metabolites with wide-ranging biological activities. PKSs usually work by...
Assembly-line polyketide synthases (PKSs) are molecular factories that produce diverse metabolites with wide-ranging biological activities. PKSs usually work by constructing and modifying the polyketide backbone successively. Here, we present the cryo-EM structure of CalA3, a chain release PKS module without an ACP domain, and its structures with amidation or hydrolysis products. The domain organization reveals a unique "∞"-shaped dimeric architecture with five connected domains. The catalytic region tightly contacts the structural region, resulting in two stabilized chambers with nearly perfect symmetry while the N-terminal docking domain is flexible. The structures of the ketosynthase (KS) domain illustrate how the conserved key residues that canonically catalyze C-C bond formation can be tweaked to mediate C-N bond formation, revealing the engineering adaptability of assembly-line polyketide synthases for the production of novel pharmaceutical agents.
Topics: Polyketide Synthases; Catalytic Domain
PubMed: 36899013
DOI: 10.1038/s41467-023-36989-w