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Journal of the American Chemical Society Mar 2016Phenalenones are polyketide natural products that display diverse structures and biological activities. The core of phenalenones is a peri-fused tricyclic ring system...
Phenalenones are polyketide natural products that display diverse structures and biological activities. The core of phenalenones is a peri-fused tricyclic ring system cyclized from a linear polyketide precursor via an unresolved mechanism. Toward understanding the unusual cyclization steps, the phn biosynthetic gene cluster responsible for herqueinone biosynthesis was identified from the genome of Penicillium herquei. A nonreducing polyketide synthase (NR-PKS) PhnA was shown to synthesize the heptaketide backbone and cyclize it into the angular, hemiketal-containing naphtho-γ-pyrone prephenalenone. The product template (PT) domain of PhnA catalyzes only the C4-C9 aldol condensation, which is unprecedented among known PT domains. The transformation of prephenalenone to phenalenone requires an FAD-dependent monooxygenase (FMO) PhnB, which catalyzes the C2 aromatic hydroxylation of prephenalenone and ring opening of the γ-pyrone ring simultaneously. Density functional theory calculations provide insights into why the hydroxylated intermediate undergoes an aldol-like phenoxide-ketone cyclization to yield the phenalenone core. This study therefore unveiled new routes and biocatalysts for polyketide cyclization.
Topics: Catalysis; Chromatography, Liquid; Cyclization; Flavins; Fungi; Molecular Structure; Multigene Family; Oxygenases; Phenalenes; Polyketide Synthases
PubMed: 26978228
DOI: 10.1021/jacs.6b01528 -
Structure (London, England : 1993) May 2023Bacterial modular polyketide synthases (PKSs) generate diverse, complex and bioactive natural products that are constructed mainly based on principles of fatty acid...
Bacterial modular polyketide synthases (PKSs) generate diverse, complex and bioactive natural products that are constructed mainly based on principles of fatty acid biosynthesis. The cytotoxic oocydin-type polyketides contain a vinyl chloride moiety introduced during polyketide chain elongation. Required for modular polyketide backbone halogenation are a non-heme iron and ɑ-ketoglutarate-dependent halogenase OocP and OocQ lacking characterized homologs. This work provides structural insights into these unusual PKS components and their interactions via a high-resolution X-ray crystallography structure of the heterocomplex. By mapping the protein-protein interactions and comparison with structures of similar halogenases, we illustrate the potential of this heterodimer complex as a replacement for the conserved homodimeric structure of homologous enzymes. The OocPQ protein pair has thus evolved as a means of stabilizing the halogenase and facilitating chemical transformations with great synthetic utility.
Topics: Polyketide Synthases; Halogenation; Polyketides; Bacteria
PubMed: 36917986
DOI: 10.1016/j.str.2023.02.010 -
Angewandte Chemie (International Ed. in... Nov 2023Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite,...
Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA-A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.
Topics: Mupirocin; Polyketide Synthases; Hydroxylation; Anti-Bacterial Agents
PubMed: 37768840
DOI: 10.1002/anie.202312514 -
Marine Drugs Jul 2023Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS)...
Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS) pathways. However, the exact mechanisms of toxin synthesis are unknown due to a lack of genomic segregation of fat, toxins, and other PKS-based pathways. To better understand the underlying mechanisms, the actions and expression of the PKS proteins were investigated using the toxic dinoflagellate as a model. Cerulenin, a known ketosynthase inhibitor, was shown to reduce acetate incorporation into all fat classes with the toxins amphidinol and sulpho-amphidinol. The mass spectrometry analysis of cerulenin-reacted synthetic peptides derived from ketosynthase domains of multimodular PKS transcripts demonstrated a strong covalent bond that could be localized using collision-induced dissociation. One multi-modular PKS sequence present in all dinoflagellates surveyed to date was found to lack an AT domain in toxin-producing species, indicating -acting domains, and was shown by Western blotting to be post-transcriptionally processed. These results demonstrate how toxin synthesis in dinoflagellates can be differentiated from fat synthesis despite common underlying pathway.
Topics: Cerulenin; Polyketide Synthases; Dinoflagellida; Harmful Algal Bloom; Blotting, Western
PubMed: 37623706
DOI: 10.3390/md21080425 -
Zebrafish Aug 2019Otoliths (ear stones) are biomineralized complexes essential for the balancing and hearing function of the inner ears in fish. Their formation is controlled by a...
Otoliths (ear stones) are biomineralized complexes essential for the balancing and hearing function of the inner ears in fish. Their formation is controlled by a genetically programmed biological process that is yet to be defined. We have isolated and characterized a spontaneous genetic mutant zebrafish with a complete absence of otoliths, named (). mutants are unable to develop otoliths during embryonic stages and fail to respond to acoustic stimuli, indicating an inner ear defect. We identified a deleterious mutation (G239R) that altered a highly conserved amino acid residue in the zebrafish ortholog of type I polyketide synthase () to underlie these phenotypes and showed that expression of the polyketide synthase gene of Japanese medaka fish could rescue the otolith deficiency in mutant zebrafish. Our finding highlights a critical and conserved role of type I polyketide synthase in the initiation of otolith formation. Given the functional homology between otoliths in teleost fish and otoconia in mammals and humans, mutants provide a new animal model for the study of human otoconia-related diseases.
Topics: Animals; Embryonic Development; Organogenesis; Oryzias; Otolithic Membrane; Polyketide Synthases; Zebrafish; Zebrafish Proteins
PubMed: 31188077
DOI: 10.1089/zeb.2019.1734 -
Cell Chemical Biology Oct 2016Thioesters play essential roles in many biosynthetic pathways to fatty acids, esters, polyketides, and non-ribosomal peptides. Coenzyme A (CoA) and related... (Review)
Review
Thioesters play essential roles in many biosynthetic pathways to fatty acids, esters, polyketides, and non-ribosomal peptides. Coenzyme A (CoA) and related phosphopantetheine thioesters are typically employed as activated acyl units for diverse C-C, C-O, and C-N coupling reactions. To study and control these enzymatic assembly lines in vitro and in vivo structurally simplified analogs such as N-acetylcysteamine (NAC) thioesters have been developed. This review gives an overview on experimental strategies enabled by synthetic NAC thioesters, such as the elucidation of complex biosynthetic pathways and enzyme mechanisms as well as precursor-directed biosynthesis and mutasynthesis. The review also summarizes synthetic protocols and protection group strategies to access these versatile synthetic tools, which are reactive and often unstable compounds. In addition, alternative phosphopantetheine thioester mimics are presented that can be used as protein tags or suicide inhibitors for protein crosslinking and off-loading probes to elucidate polyketide intermediates.
Topics: Acetylation; Bacteria; Biomimetics; Biosynthetic Pathways; Coenzyme A; Cysteamine; Fatty Acid Synthases; Fungi; Peptide Synthases; Polyketide Synthases
PubMed: 27693058
DOI: 10.1016/j.chembiol.2016.08.014 -
Journal of Microbiology and... Jul 2023Type III polyketide synthase (PKS) found in bacteria is known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Microbial type III PKSs synthesize various compounds...
Type III polyketide synthase (PKS) found in bacteria is known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Microbial type III PKSs synthesize various compounds that possess crucial biological functions and significant pharmaceutical activities. Based on our sequence analysis, we have identified a putative type III polyketide synthase from sp. CS682 was named as ThnA. The role of ThnA, in sp. CS682 during the biosynthesis of 1,3,6,8 tetrahydroxynaphthalene (THN), which is the key intermediate of 1-(α-L-(2--methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene (IBR-3) was characterized. ThnA utilized five molecules of malonyl-CoA as a starter substrate to generate the polyketide 1,3,6,8-tetrahydroxynaphthalene, which could spontaneously be oxidized to the red flaviolin compound 2,5,7-trihydroxy-1,4-naphthoquinone. The amino acid sequence alignment of ThnA revealed similarities with a previously identified type III PKS and identified Cys, Phe, His, and Asn as four highly conserved active site amino acid residues, as found in other known polyketide synthases. In this study, we report the heterologous expression of the type III polyketide synthase in TK24 and the identification of THN production in a mutant strain. We also compared the transcription level of in TK24 and pIBR25-thnA and found that was only transcribed in the mutant.
Topics: Nocardia; Amino Acid Sequence; Naphthols; Polyketide Synthases
PubMed: 37254303
DOI: 10.4014/jmb.2303.03008 -
Natural Product Reports Nov 2018Covering: up to early 2018 The Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) are families of modular enzymes that produce a tremendous... (Review)
Review
Covering: up to early 2018 The Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) are families of modular enzymes that produce a tremendous diversity of natural products, with antibacterial, antifungal, immunosuppressive, and anticancer activities. Both enzymes utilize a fascinating modular architecture in which the synthetic intermediates are covalently attached to a peptidyl- or acyl-carrier protein that is delivered to catalytic domains for natural product elongation, modification, and termination. An investigation of the structural mechanism therefore requires trapping the often transient interactions between the carrier and catalytic domains. Many novel chemical probes have been produced to enable the structural and functional investigation of multidomain NRPS and PKS structures. This review will describe the design and implementation of the chemical tools that have proven to be useful in biochemical and biophysical studies of these natural product biosynthetic enzymes.
Topics: Acetamides; Biochemistry; Biological Products; Carrier Proteins; Catalytic Domain; Crystallography, X-Ray; Enzymes; Molecular Probes; Peptide Synthases; Polyketide Synthases; Protein Domains; Protein Interaction Maps; Proteomics
PubMed: 30046790
DOI: 10.1039/c8np00044a -
Microbiology Spectrum Aug 2022Eremophilanes are a large group of "sesquiterpenes" produced by plants and fungi, with more than 180 compounds being known in fungi alone. Many of these compounds are...
Eremophilanes are a large group of "sesquiterpenes" produced by plants and fungi, with more than 180 compounds being known in fungi alone. Many of these compounds are phytotoxic, antimicrobial, anticancer and immunomodulators, and hence are of great economic values. Acremeremophilanes A to O have earlier been reported in a marine isolate of sp. We report here the presence of Acremeremophilane I, G, K, N, and O, in a plant beneficial fungus , in a strain-specific manner. We also describe a novel, P strain-specific polyketide synthase (PKS) gene cluster in . This gene cluster, designated cluster, is absent in the genome of a Q strain of , and in other spp.; instead, a near identical cluster is present in the genome of the toxic mold Stachybotrys chartarum. Using gene knockout, we provide evidence that acremeremophilanes are biosynthesized via a polyketide route, and not via the mevalonate/terpene synthesis route as believed. We propose here that the 10-carbon skeleton is a product of polyketide synthase, to which a five-carbon isoprene unit is added by a prenyl transferase (PT), a gene for which is present next to the PKS gene in the genome. Based on this evidence, we propose that at least some of the eremophilanes classified in literature as sesquiterpenes (catalyzed by terpene cyclase) are actually meroterpenes (catalyzed by PKSs and PTs), and that the core moiety is not a sesquiterpene, but a hybrid polyketide/isoprene unit. The article contradicts the established fact that acremeremophilane metabolites produced by fungi are sesquiterpenes; instead, our findings suggest that at least some of these well-studied metabolites are of polyketide origin. Acremeremophilane metabolites are of medicinal significance, and the present findings have implications for the metabolic engineering of these metabolites and also their overproduction in microbial cell factories.
Topics: Carbon; Polycyclic Sesquiterpenes; Polyketide Synthases; Polyketides; Terpenes; Trichoderma
PubMed: 35938791
DOI: 10.1128/spectrum.01793-22 -
BMC Biology May 2022During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term...
BACKGROUND
During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during "overwintering." Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear.
RESULTS
We analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type.
CONCLUSIONS
We show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.
Topics: Fungal Proteins; Gene Expression Regulation, Fungal; Melanins; Polyketide Synthases; Secondary Metabolism; Verticillium; Virulence
PubMed: 35637443
DOI: 10.1186/s12915-022-01330-2