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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 -
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 -
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 -
The Science of the Total Environment Nov 2021Prymnesium parvum is a bloom forming haptophyte that has been responsible for numerous fish kill events across the world. The toxicity of P. parvum has been attributed...
Prymnesium parvum is a bloom forming haptophyte that has been responsible for numerous fish kill events across the world. The toxicity of P. parvum has been attributed to the production of large polyketide compounds, collectively called prymnesins, which based on their structure can be divided into A-, B- and C-type. The polyketide chemical nature of prymnesins indicates the potential involvement of polyketide synthases (PKSs) in their biosynthesis. However, little is known about the presence of PKSs in P. parvum as well as the potential molecular trade-offs of toxin biosynthesis. In the current study, we generated and analyzed the transcriptomes of nine P. parvum strains that produce different toxin types and have various cellular toxin contents. Numerous type I PKSs, ranging from 37 to 109, were found among the strains. Larger modular type I PKSs were mainly retrieved from strains with high cellular toxin levels and eight consensus transcripts were present in all nine strains. Gene expression variance analysis revealed potential molecular trade-offs associated with cellular toxin quantity, showing that basic metabolic processes seem to correlate negatively with cellular toxin content. These findings point towards the presence of metabolic costs for maintaining high cellular toxin quantity. The detailed analysis of PKSs in P. parvum is the first step towards better understanding the molecular basis of the biosynthesis of prymnesins and contributes to the development of molecular tools for efficient monitoring of future blooms.
Topics: Animals; Fishes; Haptophyta; Polyketide Synthases
PubMed: 34252778
DOI: 10.1016/j.scitotenv.2021.148878 -
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 -
Molecules (Basel, Switzerland) Feb 2017Modular polyketide synthases (mPKSs) build functionalized polymeric chains, some of which have become blockbuster therapeutics. Organized into repeating clusters... (Review)
Review
Modular polyketide synthases (mPKSs) build functionalized polymeric chains, some of which have become blockbuster therapeutics. Organized into repeating clusters (modules) of independently-folding domains, these assembly-line-like megasynthases can be engineered by introducing non-native components. However, poor introduction points and incompatible domain combinations can cause both unintended products and dramatically reduced activity. This limits the engineering and combinatorial potential of mPKSs, precluding access to further potential therapeutics. Different regions on a given mPKS domain determine how it interacts both with its substrate and with other domains. Within the assembly line, these interactions are crucial to the proper ordering of reactions and efficient polyketide construction. Achieving control over these domain functions, through precision engineering at key regions, would greatly expand our catalogue of accessible polyketide products. Canonical mPKS domains, given that they are among the most well-characterized, are excellent candidates for such fine-tuning. The current minireview summarizes recent advances in the mechanistic understanding and subsequent precision engineering of canonical mPKS domains, focusing largely on developments in the past year.
Topics: Catalysis; Models, Molecular; Polyketide Synthases; Protein Domains; Protein Engineering; Structure-Activity Relationship; Substrate Specificity
PubMed: 28165430
DOI: 10.3390/molecules22020235 -
Nature Communications Feb 2023Modular polyketide synthase (PKS) is an ingenious core machine that catalyzes abundant polyketides in nature. Exploring interactions among modules in PKS is very...
Modular polyketide synthase (PKS) is an ingenious core machine that catalyzes abundant polyketides in nature. Exploring interactions among modules in PKS is very important for understanding the overall biosynthetic process and for engineering PKS assembly-lines. Here, we show that intermodular recognition between the enoylreductase domain ER inside module 1/2 and the ketosynthase domain KS inside module 3 is required for the cross-module enoylreduction in azalomycin F (AZL) biosynthesis. We also show that KS of module 4 acts as a gatekeeper facilitating cross-module enoylreduction. Additionally, evidence is provided that module 3 and module 6 in the AZL PKS are evolutionarily homologous, which makes evolution-oriented PKS engineering possible. These results reveal intermodular recognition, furthering understanding of the mechanism of the PKS assembly-line, thus providing different insights into PKS engineering. This also reveals that gene duplication/conversion and subsequent combinations may be a neofunctionalization process in modular PKS assembly-lines, hence providing a different case for supporting the investigation of modular PKS evolution.
Topics: Polyketide Synthases; Macrolides; Polyketides
PubMed: 36739290
DOI: 10.1038/s41467-023-36213-9 -
Methods in Molecular Biology (Clifton,... 2022The enzymes that comprise type II polyketide synthases (PKSs) are powerful biocatalysts that, once well-understood and strategically applied, could enable cost-effective...
The enzymes that comprise type II polyketide synthases (PKSs) are powerful biocatalysts that, once well-understood and strategically applied, could enable cost-effective and sustainable access to a range of pharmaceutically relevant molecules. Progress toward this goal hinges on gaining ample access to materials for in vitro characterizations and structural analysis of the components of these synthases. A central component of PKSs is the acyl carrier protein (ACP), which serves as a hub during the biosynthesis of type II polyketides. Herein, we share methods for accessing type II PKS ACPs via heterologous expression in E. coli . We also share how the installation of reactive and site-specific spectroscopic probes can be leveraged to study the conformational dynamics and interactions of type II PKS ACPs.
Topics: Acyl Carrier Protein; Escherichia coli; Polyketide Synthases
PubMed: 35524054
DOI: 10.1007/978-1-0716-2273-5_13