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Nature Structural & Molecular Biology Mar 2023The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets...
The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug development. Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions that carries out the condensation of two different long-chain fatty acids to produce mycolic acids. We determined structures by cryogenic-electron microscopy of dimeric multi-enzyme Pks13 purified from mycobacteria under normal growth conditions, captured with native substrates. Structures define the ketosynthase (KS), linker and acyl transferase (AT) domains at 1.8 Å resolution and two alternative locations of the N-terminal acyl carrier protein. These structures suggest intermediate states on the pathway for substrate delivery to the KS domain. Other domains, visible at lower resolution, are flexible relative to the KS-AT core. The chemical structures of three bound endogenous long-chain fatty acid substrates were determined by electrospray ionization mass spectrometry.
Topics: Polyketide Synthases; Mycobacterium tuberculosis; Mycolic Acids; Fatty Acids
PubMed: 36782050
DOI: 10.1038/s41594-022-00918-0 -
Nature Chemistry Sep 2022Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-selective incorporation...
Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-selective incorporation of fluorine in complex molecules have improved in recent years, approaches for the biosynthetic incorporation of fluorine in natural compounds are still rare. Here, we report a strategy to introduce fluorine into complex polyketides during biosynthesis. We exchanged the native acyltransferase domain of a polyketide synthase, which acts as the gatekeeper for the selection of extender units, with an evolutionarily related but substrate tolerant domain from metazoan type I fatty acid synthase. The resulting polyketide-synthase/fatty-acid-synthase hybrid can utilize fluoromalonyl coenzyme A and fluoromethylmalonyl coenzyme A for polyketide chain extension, introducing fluorine or fluoro-methyl units in polyketide scaffolds. We demonstrate the feasibility of our approach in the chemoenzymatic synthesis of fluorinated 12- and 14-membered macrolactones and fluorinated derivatives of the macrolide antibiotics YC-17 and methymycin.
Topics: Acyltransferases; Animals; Coenzyme A; Fluorine; Polyketide Synthases; Polyketides
PubMed: 35879443
DOI: 10.1038/s41557-022-00996-z -
Natural Product Reports Oct 2018Covering: up to the end of 2018 Polyketides are a valuable source of bioactive and clinically important molecules. The biosynthesis of these chemically complex molecules... (Review)
Review
Covering: up to the end of 2018 Polyketides are a valuable source of bioactive and clinically important molecules. The biosynthesis of these chemically complex molecules has led to the discovery of equally complex polyketide synthase (PKS) pathways. Crystallography has yielded snapshots of individual catalytic domains, di-domains, and multi-domains from a variety of PKS megasynthases, and cryo-EM studies have provided initial views of a PKS module in a series of defined biochemical states. Here, we review the structural and biochemical results that shed light on the protein-protein interactions critical to catalysis by PKS systems with an embedded acyltransferase. Interactions include those that occur both within and between PKS modules, as well as with accessory enzymes.
Topics: Acyltransferases; Catalytic Domain; Polyketide Synthases; Protein Interaction Domains and Motifs; Protein Multimerization
PubMed: 30188553
DOI: 10.1039/c8np00058a -
ACS Synthetic Biology Nov 2023Polyketide retrobiosynthesis, where the biosynthetic pathway of a given polyketide can be reversibly engineered due to the colinearity of the polyketide synthase (PKS)... (Review)
Review
Polyketide retrobiosynthesis, where the biosynthetic pathway of a given polyketide can be reversibly engineered due to the colinearity of the polyketide synthase (PKS) structure and function, has the potential to produce millions of organic molecules. Mixing and matching modules from natural PKSs is one of the routes to produce many of these molecules. Evolutionary analysis of PKSs suggests that traditionally used module boundaries may not lead to the most productive hybrid PKSs and that new boundaries around and within the ketosynthase domain may be more active when constructing hybrid PKSs. As this is still a nascent area of research, the generality of these design principles based on existing engineering efforts remains inconclusive. Recent advances in structural modeling and synthetic biology present an opportunity to accelerate PKS engineering by re-evaluating insights gained from previous engineering efforts with cutting edge tools.
Topics: Polyketide Synthases; Polyketides
PubMed: 37871264
DOI: 10.1021/acssynbio.3c00282 -
Microbial Cell Factories Aug 2019Actinobacteria are characterized as the most prominent producer of natural products (NPs) with pharmaceutical importance. The production of NPs from these actinobacteria... (Review)
Review
Actinobacteria are characterized as the most prominent producer of natural products (NPs) with pharmaceutical importance. The production of NPs from these actinobacteria is associated with particular biosynthetic gene clusters (BGCs) in these microorganisms. The majority of these BGCs include polyketide synthase (PKS) or non-ribosomal peptide synthase (NRPS) or a combination of both PKS and NRPS. Macrolides compounds contain a core macro-lactone ring (aglycone) decorated with diverse functional groups in their chemical structures. The aglycon is generated by megaenzyme polyketide synthases (PKSs) from diverse acyl-CoA as precursor substrates. Further, post-PKS enzymes are responsible for allocating the structural diversity and functional characteristics for their biological activities. Macrolides are biologically important for their uses in therapeutics as antibiotics, anti-tumor agents, immunosuppressants, anti-parasites and many more. Thus, precise genetic/metabolic engineering of actinobacteria along with the application of various chemical/biological approaches have made it plausible for production of macrolides in industrial scale or generation of their novel derivatives with more effective biological properties. In this review, we have discussed versatile approaches for generating a wide range of macrolide structures by engineering the PKS and post-PKS cascades at either enzyme or cellular level in actinobacteria species, either the native or heterologous producer strains.
Topics: Actinobacteria; Biological Products; Genetic Engineering; Macrolides; Multigene Family; Polyketide Synthases; Polyketides
PubMed: 31409353
DOI: 10.1186/s12934-019-1184-z -
Chemical Reviews Apr 2017The enzymology of 135 assembly lines containing primarily cis-acyltransferase modules is comprehensively analyzed, with greater attention paid to less common phenomena.... (Review)
Review
The enzymology of 135 assembly lines containing primarily cis-acyltransferase modules is comprehensively analyzed, with greater attention paid to less common phenomena. Diverse online transformations, in which the substrate and/or product of the reaction is an acyl chain bound to an acyl carrier protein, are classified so that unusual reactions can be compared and underlying assembly-line logic can emerge. As a complement to the chemistry surrounding the loading, extension, and offloading of assembly lines that construct primarily polyketide products, structural aspects of the assembly-line machinery itself are considered. This review of assembly-line phenomena, covering the literature up to 2017, should thus be informative to the modular polyketide synthase novice and expert alike.
Topics: Acyltransferases; Polyketide Synthases; Polyketides; Substrate Specificity
PubMed: 28394118
DOI: 10.1021/acs.chemrev.6b00683 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Feb 2018Fungal polyketides display complex structures and variously biological activities. Their biosynthetic pathways generally contain novel enzyme-catalyzed reactions. This... (Review)
Review
Fungal polyketides display complex structures and variously biological activities. Their biosynthetic pathways generally contain novel enzyme-catalyzed reactions. This review provides a summary of recent research advances in molecular mechanism of the biosynthesis of fungal polyketides including highly-reducing polyketide synthases (HR-PKSs), non-reducing polyketide synthases (NR-PKSs), as well as polyketide-nonribosomal peptide synthase (PKS-NRPSs) and reducingnon- reducing polyketide synthase (HR-NR PKSs) hybrids. The elucidation of biosynthetic mechanism of many fungal polyketides provides guidance on the discovery of new biosynthetic gene cluster of fungal polyketide natural products and compounds with novel structures as well as their analogue.
Topics: Fungal Proteins; Fungi; Industrial Microbiology; Multigene Family; Polyketide Synthases; Polyketides; Secondary Metabolism
PubMed: 29424130
DOI: 10.13345/j.cjb.170219 -
Structure (London, England : 1993) Sep 2022The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KS, that primes polyketide synthesis. Unlike downstream KSs...
The first domain of modular polyketide synthases (PKSs) is most commonly a ketosynthase (KS)-like enzyme, KS, that primes polyketide synthesis. Unlike downstream KSs that fuse α-carboxyacyl groups to growing polyketide chains, it performs an extension-decoupled decarboxylation of these groups to generate primer units. When Pik127, a model triketide synthase constructed from modules of the pikromycin synthase, was studied by cryoelectron microscopy (cryo-EM), the dimeric didomain comprised of KS and the neighboring methylmalonyl-selective acyltransferase (AT) dominated the class averages and yielded structures at 2.5- and 2.8-Å resolution, respectively. Comparisons with ketosynthases complexed with their substrates revealed the conformation of the (2S)-methylmalonyl-S-phosphopantetheinyl portion of KS and KS substrates prior to decarboxylation. Point mutants of Pik127 probed the roles of residues in the KS active site, while an AT-swapped version of Pik127 demonstrated that KS can also decarboxylate malonyl groups. Mechanisms for how KS and KS domains catalyze carbon-carbon chemistry are proposed.
Topics: Acyltransferases; Carbon; Cryoelectron Microscopy; Macrolides; Polyketide Synthases
PubMed: 35738283
DOI: 10.1016/j.str.2022.05.021 -
Journal of Industrial Microbiology &... Jun 2021Polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) multienzymes produce numerous high value metabolites. The protein subunits which constitute these... (Review)
Review
Polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) multienzymes produce numerous high value metabolites. The protein subunits which constitute these megasynth(et)ases must undergo ordered self-assembly to ensure correct organisation of catalytic domains for the biosynthesis of a given natural product. Short amino acid regions at the N- and C-termini of each subunit, termed docking domains (DDs), often occur in complementary pairs, which interact to facilitate substrate transfer and maintain pathway fidelity. This review details all structurally characterised examples of NRPS and PKS DDs to date and summarises efforts to utilise DDs for the engineering of biosynthetic pathways.
Topics: Biological Products; Humans; Peptide Synthases; Polyketide Synthases
PubMed: 33640957
DOI: 10.1093/jimb/kuab018 -
Angewandte Chemie (International Ed. in... Apr 2017Modular redefinition: A long-standing paradigm in modular polyketide synthase enzymology, namely the definition of a module, has been challenged by Abe and co-workers in...
Modular redefinition: A long-standing paradigm in modular polyketide synthase enzymology, namely the definition of a module, has been challenged by Abe and co-workers in their recent study. With this new understanding has emerged renewed hope for engineering these assembly lines to produce new materials and medicines.
Topics: Acyl Carrier Protein; Acyltransferases; Animals; Bacteria; Biocatalysis; Computational Biology; Evolution, Molecular; Fatty Acid Synthases; Humans; Polyketide Synthases; Polyketides; Protein Domains; Protein Engineering; Substrate Specificity
PubMed: 28322495
DOI: 10.1002/anie.201701281