ABSTRACT
Innovative discovery approaches such as genome-mining and metabolomics-inspired methods have reshaped the natural product research field, complementing traditional bioactivity-based screens and allowing hitherto unseen compounds to be uncovered from previously investigated producers. In line with these trends, we report here imidacins, a novel class of secondary metabolites specific to the myxobacterial genus Stigmatella. A combination of secondary metabolome analysis, genome-mining techniques, spectroscopic analysis, and finally total synthesis was used to allow structure elucidation. Imidacins are urocanate-derived aliphatic acids with an adjacent cyclopropane moiety, structural features unprecedented in natural products to date.
Subject(s)
Stigmatella aurantiaca , Molecular Structure , Stigmatella aurantiaca/chemistry , Alkaloids/chemistry , Alkaloids/chemical synthesis , Biological Products/chemistry , Biological Products/pharmacology , Biological Products/chemical synthesis , Myxococcales/chemistry , Cyclopropanes/chemistry , Cyclopropanes/pharmacology , Cyclopropanes/chemical synthesisABSTRACT
The Ebola virus (EBOV) is a lethal pathogen causing hemorrhagic fever syndrome which remains a global health challenge. In the EBOV, two multifunctional proteins, VP35 and VP40, have significant roles in replication, virion assembly, and budding from the cell and have been identified as druggable targets. In this study, we employed in silico methods comprising molecular docking, molecular dynamic simulations, and pharmacological properties to identify prospective drugs for inhibiting VP35 and VP40 proteins from the myxobacterial bioactive natural product repertoire. Cystobactamid 934-2, Cystobactamid 919-1, and Cittilin A bound firmly to VP35. Meanwhile, 2-Hydroxysorangiadenosine, Enhypyrazinone B, and Sorangiadenosine showed strong binding to the matrix protein VP40. Molecular dynamic simulations revealed that, among these compounds, Cystobactamid 919-1 and 2-Hydroxysorangiadenosine had stable interactions with their respective targets. Similarly, molecular mechanics Poisson-Boltzmann surface area (MMPBSA) calculations indicated close-fitting receptor binding with VP35 or VP40. These two compounds also exhibited good pharmacological properties. In conclusion, we identified Cystobactamid 919-1 and 2-Hydroxysorangiadenosine as potential ligands for EBOV that target VP35 and VP40 proteins. These findings signify an essential step in vitro and in vivo to validate their potential for EBOV inhibition.
Subject(s)
Antiviral Agents , Biological Products , Ebolavirus , Molecular Docking Simulation , Molecular Dynamics Simulation , Ebolavirus/drug effects , Biological Products/pharmacology , Biological Products/chemistry , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Myxococcales/chemistry , Humans , Viral Regulatory and Accessory Proteins/antagonists & inhibitors , Viral Regulatory and Accessory Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Matrix Proteins/antagonists & inhibitors , Viral Matrix Proteins/metabolism , Viral Matrix Proteins/chemistry , Nucleocapsid ProteinsABSTRACT
In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active 'slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning ß-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.
Subject(s)
Gasdermins , Myxococcales , Cryoelectron Microscopy , Gasdermins/chemistry , Gasdermins/metabolism , Gasdermins/ultrastructure , Hydrophobic and Hydrophilic Interactions , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Molecular Dynamics Simulation , Myxococcales/chemistry , Myxococcales/cytology , Myxococcales/ultrastructure , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/metabolism , Proteolysis , PyroptosisABSTRACT
Covering: 2017.01 to 2023.11Natural products biosynthesized by myxobacteria are appealing due to their sophisticated chemical skeletons, remarkable biological activities, and intriguing biosynthetic enzymology. This review aims to systematically summarize the advances in the discovery methods, new structures, and bioactivities of myxobacterial NPs reported in the period of 2017-2023. In addition, the peculiar biosynthetic pathways of several structural families are also highlighted.
Subject(s)
Biological Products , Myxococcales , Biological Products/metabolism , Biological Products/chemistry , Myxococcales/metabolism , Myxococcales/chemistry , Molecular Structure , Biosynthetic Pathways , Drug DiscoveryABSTRACT
Myxobacteria represent an underinvestigated source of chemically diverse and biologically active secondary metabolites. Here, we report the discovery, isolation, structure elucidation, and biological evaluation of two new bacterial sterols, termed nannosterols A and B (1, 2), from the terrestrial myxobacterium Nannocystis sp. (MNa10993). Nannosterols feature a cholestanol core with numerous modifications including a secondary alcohol at position C-15, a terminal vicinal diol side chain at C-24-C-25 (1, 2), and a hydroxy group at the angular methyl group at C-18 (2), which is unprecedented for bacterial sterols. Another rare chemical feature of bacterial triterpenoids is a ketone group at position C-7, which is also displayed by 1 and 2. The combined exploration based on myxobacterial high-resolution secondary metabolome data and genomic in silico investigations exposed the nannosterols as frequently produced sterols within the myxobacterial suborder of Nannocystineae. The discovery of the nannosterols provides insights into the biosynthesis of these new myxobacterial sterols, with implications in understanding the evolution of sterol production by prokaryotes.
Subject(s)
Myxococcales , Phytosterols , Sterols , Myxococcales/chemistryABSTRACT
Structure elucidation and total synthesis of five unprecedented terpenoid-alkaloids, the sandacrabins, are reported, alongside with the first description of their producing organism Sandaracinus defensii MSr10575, which expands the Sandaracineae family by only its second member. The genome sequence of S. defensii as presented in this study was utilized to identify enzymes responsible for sandacrabin formation, whereby dimethylbenzimidazol, deriving from cobalamin biosynthesis, was identified as key intermediate. Biological activity profiling revealed that all sandacrabins except congener A exhibit potent antiviral activity against the human pathogenic coronavirus HCoV229E in the three digit nanomolar range. Investigation of the underlying mode of action discloses that the sandacrabins inhibit the SARS-CoV-2 RNA-dependent RNA polymerase complex, highlighting them as structurally distinct non-nucleoside RNA synthesis inhibitors. The observed segregation between cell toxicity at higher concentrations and viral inhibition opens the possibility for their medicinal chemistry optimization towards selective inhibitors.
Subject(s)
Antiviral Agents , DNA-Directed RNA Polymerases/antagonists & inhibitors , Myxococcales/chemistry , SARS-CoV-2/drug effects , Antiviral Agents/chemistry , Antiviral Agents/pharmacologyABSTRACT
Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals.
Subject(s)
Bacteria/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacteriophages/physiology , Pyroptosis , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/metabolism , Bacteria/metabolism , Bacteria/virology , Bradyrhizobium/chemistry , Cell Membrane/metabolism , Crystallography, X-Ray , Cytophagaceae/chemistry , Models, Molecular , Myxococcales/chemistry , Peptide Fragments/metabolism , Peptide Hydrolases/metabolism , Protein Conformation , Protein Conformation, alpha-Helical , Protein DomainsABSTRACT
Maltogenic amylase CoMA from Corallococcus sp. strain EGB catalyzes the hydrolysis and transglycosylation of maltooligosaccharides and soluble starch into maltose, the sole hydrolysate. This process yields pure maltose with potentially wide applications. Here, we identified and evaluated the role of phenylalanine 314 (F314), a key amino acid located near the active center, in the catalytic activities of the CoMA. Site-directed mutagenesis analysis showed that the activity of a F314L mutant on potato starch substrate decreased to 26% of that of wild-type protein. Compared with the wild-type, F314L exhibited similar substrate specificity, hydrolysis pattern, pH, and temperature requirements. Circular dichroism spectrum data showed that the F314L mutation did not affect the structure of the folded protein. In addition, kinetic analysis demonstrated that F314L exhibited an increased Km value with lower substrate affinity. Homology modeling showed that the benzene ring structure of F314L was involved in π-π conjugation, which might potentially affect the affinity of CoMA toward starch. Taken together, these data demonstrated that F314 is essential for the hydrolytic activity of the CoMA from Corallococcus sp. strain EGB.
Subject(s)
Maltose , Myxococcales , Humans , Maltose/chemistry , Kinetics , Phenylalanine , Coma , Myxococcales/chemistry , Myxococcales/genetics , Myxococcales/metabolism , Hydrolysis , Starch/chemistry , Substrate SpecificityABSTRACT
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products. They feature a wide range of intriguing post-translational modifications, as exemplified by the biarylitides. These are a family of cyclic tripeptides found in Planomonospora, carrying a biaryl linkage between two aromatic amino acids. Recent genomic analyses revealed that the minimal biosynthetic prerequisite of biarylitide biosynthesis consists of only one ribosomally synthesized pentapeptide precursor as the substrate and a modifying cytochrome-P450-dependent enzyme. In silico analyses revealed that minimal biarylitide RiPP clusters are widespread among natural product producers across phylogenetic borders, including myxobacteria. We report here the genome-guided discovery of the first myxobacterial biarylitide MeYLH, termed Myxarylin, from Pyxidicoccus fallax An d48. Myxarylin was found to be an N-methylated tripeptide that surprisingly exhibits a C-N biaryl crosslink. In contrast to Myxarylin, previously isolated biarylitides are N-acetylated tripeptides that feature a C-C biaryl crosslink. Furthermore, the formation of Myxarylin was confirmed by the heterologous expression of the identified biosynthetic genes in Myxococcus xanthus DK1622. These findings expand the structural and biosynthetic scope of biarylitide-type RiPPs and emphasize the distinct biochemistry found in the myxobacterial realm.
Subject(s)
Cross-Linking Reagents/metabolism , Myxococcales/chemistry , Peptides/metabolism , Cross-Linking Reagents/chemistry , Molecular Conformation , Peptides/chemistry , Protein Processing, Post-TranslationalABSTRACT
Myxobacteria are a prolific source of structurally diverse natural products, and one of the best-studied myxobacterial products is the siderophore myxochelin. Herein, we report two new compounds, myxochelins N (1) and O (2), that are nicotinic paralogs of myxochelin A, from the terrestrial myxobacterium Archangium sp. SDU34; 2 is functionalized with a rare 2-oxazolidinone. A precursor-feeding experiment implied that the biosynthesis of 1 or 2 was due to altered substrate specificity of the loading module of MxcE, which likely accepts nicotinic acid and benzoic acid instead of more conventional 2,3-dihydroxybenzoic acid. We also employed a phylogenomic approach to map the evolutionary relationships of the myxochelin biosynthetic gene clusters (BGCs) in all the available myxobacterial genomes, to pave the way for the future discovery of potentially hidden myxochelin derivatives. Although the biological function of 1 and 2 is unclear yet, this work underpins that even extensively studied BGCs in myxobacteria can still produce new chemistry.
Subject(s)
Biological Products/chemistry , Lysine/analogs & derivatives , Myxococcales/chemistry , Lysine/biosynthesis , Molecular Structure , Multigene Family , Myxococcales/geneticsABSTRACT
Myxobacteria represent a viable source of chemically diverse and biologically active secondary metabolites. The myxochelins are a well-studied family of catecholate-type siderophores produced by various myxobacterial strains. Here, we report the discovery, isolation, and structure elucidation of three new myxochelins N1-N3 from the terrestrial myxobacterium Corallococcus sp. MCy9049, featuring an unusual nicotinic acid moiety. Precursor-directed biosynthesis (PDB) experiments and total synthesis were performed in order to confirm structures, improve access to pure compounds for bioactivity testing, and to devise a biosynthesis proposal. The combined evaluation of metabolome and genome data covering myxobacteria supports the notion that the new myxochelin congeners reported here are in fact frequent side products of the known myxochelin A biosynthetic pathway in myxobacteria.
Subject(s)
Biological Products/chemistry , Lysine/analogs & derivatives , Myxococcales/chemistry , Niacin/chemistry , Biosynthetic Pathways/genetics , Genome, Bacterial/genetics , Lysine/chemistry , Metabolome/genetics , Myxococcales/genetics , Myxococcales/isolation & purification , Niacin/isolation & purificationABSTRACT
Myxobacteria belong to a group of bacteria that are known for their well-developed communication system and synchronized or coordinated movement. This typical behavior of myxobacteria is mediated through secondary metabolites. They are capable of producing secondary metabolites belonging to several chemical classes with unique and wide spectrum of bioactivities. It is predominantly significant that myxobacteria specialize in mechanisms of action that are very rare with other producers. Most of the metabolites have been explored for their medical and pharmaceutical values while a lot of them are still unexplored. This review is an attempt to understand the role of potential metabolites produced by myxobacteria in different applications. Different myxobacterial metabolites have demonstrated antibacterial, antifungal, and antiviral properties along with cytotoxic activity against various cell lines. Beside their metabolites, these myxobacteria have also been discussed for better exploitation and implementation in different industrial sectors.
Subject(s)
Industrial Microbiology , Myxococcales , Anti-Bacterial Agents/biosynthesis , Industrial Microbiology/trends , Myxococcales/chemistry , Myxococcales/metabolismABSTRACT
Sorangipyranone was isolated as a novel natural product featuring a unique 2,3-dihydro-γ-4H-pyrone scaffold from cultures of the myxobacterial strain MSr12020. We report here the full structure elucidation of sorangipyranone by spectroscopic techniques including 2D NMR and high-resolution mass spectrometry together with the analysis of the biosynthetic pathway. Determination of the absolute configuration was performed by time-dependent density functional theory-electronic circular dichroism calculations and determination of the applicability of the Snatzke's helicity rule, to correlate the high-wavelength nâπ* electronic circular dichroism (ECD) transition and the absolute configuration of the 2,3-dihydro-4H-γ-pyrone, was done by the analysis of low-energy conformers and the Kohn-Sham orbitals. Sorangipyranone outlines a new class of a γ-dihydropyrone-containing natural product comprised of malonyl-CoA-derived building blocks and features a unique polyketide scaffold. In silico analysis of the genome sequence of the myxobacterial strain MSr12020 complemented with feeding experiments employing stable isotope-labeled precursors allowed the identification and annotation of a candidate biosynthetic gene cluster that encodes a modular polyketide synthase assembly line. A model for the biosynthetic pathway leading to the formation of the γ-dihydropyrone scaffold is presented in this study.
Subject(s)
Myxococcales/metabolism , Base Sequence , Biological Products/chemistry , Biological Products/metabolism , Biosynthetic Pathways/genetics , Multigene Family , Myxococcales/chemistry , Myxococcales/genetics , Polyketide Synthases/metabolism , Polyketides/chemistry , Polyketides/metabolismABSTRACT
Eukaryotic protein synthesis is the highly conserved, complex mechanism of translating genetic information into proteins. Although this process is essential for cellular homoeostasis, dysregulations are associated with cellular malfunctions and diseases including cancer and diabetes. In the challenging and ongoing search for adequate treatment possibilities, natural products represent excellent research tools and drug leads for new interactions with the translational machinery and for influencing mRNA translation. In this review, bacterial-, marine- and plant-derived natural compounds that interact with different steps of mRNA translation, comprising ribosomal assembly, translation initiation and elongation, are highlighted. Thereby, the exact binding and interacting partners are unveiled in order to accurately understand the mode of action of each natural product. The pharmacological relevance of these compounds is furthermore assessed by evaluating the observed biological activities in the light of translational inhibition and by enlightening potential obstacles and undesired side-effects, e.g. in clinical trials. As many of the natural products presented here possess the potential to serve as drug leads for synthetic derivatives, structural motifs, which are indispensable for both mode of action and biological activities, are discussed. Evaluating the natural products emphasises the strong diversity of their points of attack. Especially the fact that selected binding partners can be set in direct relation to different diseases emphasises the indispensability of natural products in the field of drug development. Discovery of new, unique and unusual interacting partners again renders them promising tools for future research in the field of eukaryotic mRNA translation.
Subject(s)
Aquatic Organisms , Bacteria , Biological Products/pharmacology , Plant Extracts/pharmacology , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/metabolism , Ribosomes/drug effects , Animals , Aquatic Organisms/chemistry , Bacteria/chemistry , Biological Products/isolation & purification , Drug Development , Humans , Myxococcales/chemistry , Plant Extracts/isolation & purification , Protein Synthesis Inhibitors/isolation & purification , RNA, Messenger/genetics , Ribosomes/genetics , Ribosomes/metabolismABSTRACT
Epithelial-mesenchymal transition (EMT), the epithelial cells transdifferentiation into the mesenchymal cells, has been involved in cancer metastasis. Nannocystin ax (NAN) is a cyclodepsipeptide initially isolated from Myxobacterial genus, Nannocystis sp. with anticancer activities. This study was designed to explore the effect of NAN on TGF-ß1-induced EMT in lung cancer cells. The morphological alteration was observed with a microscope. Western blotting and immunofluorescence assays were used to detect the protein expression and the localization. The adhesion and migration were evaluated by adhesion assay and wound healing assay. The mRNA expression of TGF-ß receptor type I (TßRI) was determined by real-time PCR. NAN significantly restrained TGF-ß1-induced EMT morphological changes, the protein expression of E-cadherin, N-cadherin, and Vimentin, etc. TGF-ß1 activated phosphorylation and nuclear translocation of Smad2/3 were inhibited by NAN. Furthermore, NAN suppressed adhesion and migration triggered by TGF-ß1. In addition, NAN significantly down-regulated TßRI on the transcriptional level directly. In summary, these results showed that NAN restrained TGF-ß1-induced epithelial-mesenchymal transition, migration, and adhesion in human lung cancer cells. The underlying mechanism involved the inhibition of Smad2/3 and the TßRI signaling pathway. This study reveals the new anticancer effect and mechanism of NAN.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Adhesion/drug effects , Cell Movement/drug effects , Depsipeptides/pharmacology , Epithelial-Mesenchymal Transition/drug effects , Lung Neoplasms/drug therapy , Macrocyclic Compounds/pharmacology , Myxococcales/chemistry , Peptide Elongation Factor 1/antagonists & inhibitors , A549 Cells , Antineoplastic Agents/isolation & purification , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Depsipeptides/isolation & purification , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Macrocyclic Compounds/isolation & purification , Peptide Elongation Factor 1/metabolism , Phosphorylation , Receptor, Transforming Growth Factor-beta Type I/genetics , Receptor, Transforming Growth Factor-beta Type I/metabolism , Signal Transduction , Smad2 Protein/metabolism , Smad3 Protein/metabolismABSTRACT
An unprecedented 19-membered allenic macrolide archangiumide (1) was discovered from the myxobacterium Archangium violaceum SDU8 by integrating NMR-based metabolic profiling and genome mining. Its biosynthesis pathway was proposed based on the architectural analysis of the encoding trans-AT PKS genes and validated by isotope labeling. The methodology of combing 2D NMR-based metabolic profiling and bioinformatics-aided structure prediction, as exemplified by this study, is anticipated to improve discovery efficiency of a broader range of microbial "dark matter".
Subject(s)
Macrolides/chemistry , Myxococcales/chemistry , Anti-Bacterial Agents/chemistry , Macrolides/metabolism , Molecular StructureABSTRACT
Natural products provide important lead structures for development of pharmaceutical agents or present attractive tools for medicinal chemistry. However, structurally complex and thus less accessible metabolites defying conventional drug-like properties, as expressed by Pfizer's rule of five, have received less attention as medicinal leads. Traditionally, research focus has been on realizing total syntheses rather than developing more readily available analogs to resolve the critical supply issue. However, very recent studies with complex myxobacterial polyketides have demonstrated that considerable structural simplification may be realized with retention of biological potencies. The context, underlying rationale and importance of tailored synthetic strategies of three such case studies are presented, which may inspire further related activities and may eventually help exploiting the largely untapped biological potential of complex metabolites in general.
Subject(s)
Biological Products/pharmacology , Drug Design , Polyketides/pharmacology , Biological Products/chemical synthesis , Biological Products/chemistry , Humans , Molecular Structure , Myxococcales/chemistry , Polyketides/chemical synthesis , Polyketides/chemistryABSTRACT
Herein, we describe a new plasmid found in Sandaracinusâ sp. MSr10575 named pSa001 spanning 209.7â kbp that harbors a cryptic secondary metabolite biosynthesis gene cluster (BGC). Activation of this BGC by homologous-recombination-mediated exchange of the native promoter sequence against a vanillate inducible system led to the production and subsequent isolation and structure elucidation of novel secondary metabolites, the sandarazolsâ A-G. The sandarazols contain intriguing structural features and very reactive functional groups such as an α-chlorinated ketone, an epoxyketone, and a (2R)-2-amino-3-(N,N-dimethylamino)-propionic acid building block. In-depth investigation of the underlying biosynthetic machinery led to a concise biosynthetic model for the new compound family, including several uncommon biosynthetic steps. The chlorinated congener sandarazolâ C shows an IC50 â value of 0.5â µm against HCTâ 116 cells and a MIC of 14â µm against Mycobacterium smegmatis, which points at the sandarazols' potential function as defensive secondary metabolites or toxins.
Subject(s)
Myxococcales/chemistry , Toxins, Biological/chemistry , Molecular Structure , Multigene Family , Myxococcales/metabolism , Toxins, Biological/genetics , Toxins, Biological/metabolismABSTRACT
Recent advances in genome sequencing have unveiled a large discrepancy between the genome-encoded capacity of microorganisms to produce secondary metabolites and the number detected. In this work, a two-platform mass spectrometry analysis for the comprehensive secondary metabolomics characterization of nine myxobacterial strains, focusing on extending the range of detectable secondary metabolites by diversifying analytical methods and cultivation conditions, is presented. Direct infusion measurements of crude extracts on a Fourier transform ion cyclotron resonance mass spectrometer are compared to a time-of-flight device coupled to liquid chromatography measurements. Both methods are successful in detecting known metabolites, whereas statistical analysis of unknowns highlights their complementarity: Strikingly, 82-99% of molecular features detected with one setup were not detectable with the other. Metabolite profile differences from our set of strains grown in liquid culture versus their swarming colonies on agar plates were evaluated. The detection of up to 96% more molecular features when both liquid and plate cultures were analyzed translates into increased chances to identify new secondary metabolites. Discrimination between primary and secondary metabolism in combination with GNPS molecular networking revealed strain Mx3 as particularly promising for the isolation of novel secondary metabolites among the nine strains investigated in this study.
Subject(s)
Biological Products/analysis , Metabolomics , Myxococcales/chemistry , Biological Products/chemistry , Chromatography, Liquid , Mass Spectrometry , Metabolomics/methods , Secondary MetabolismABSTRACT
The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.