RESUMEN
The antibiotic cerulenin is a fungal natural product identified as a covalent inhibitor of ketosynthases within fatty acid and polyketide biosynthesis. Due to its selective and potent inhibitory activity, cerulenin has found significant utility in multidisciplinary biochemical, biomedical, and clinical studies. Although its covalent inhibition profile has been confirmed, cerulenin's mechanism has not been fully determined at a molecular level, frustrating the drug development of related analogues. Herein, we describe the use of stable isotopic tracking with NMR and MS methods to unravel the covalent mechanism of cerulenin against type II fatty acid ketosynthases. We detail the discovery of a unique C2-C3 retro-aldol bond cleavage and a structural rearrangement upon covalent inhibition of cerulenin at the active cysteine residue in E. coli type II fatty acid ketosynthases FabB and FabF.
Asunto(s)
Cerulenina , Cerulenina/farmacología , Cerulenina/química , Escherichia coli/enzimología , Escherichia coli/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Antibacterianos/farmacología , Antibacterianos/química , Acido Graso Sintasa Tipo II/antagonistas & inhibidores , Acido Graso Sintasa Tipo II/metabolismo , Modelos Moleculares , Estructura MolecularRESUMEN
Many bioactive plant cyclic peptides form side-chain-derived macrocycles. Lyciumins, cyclic plant peptides with tryptophan macrocyclizations, are ribosomal peptides (RiPPs) originating from repetitive core peptide motifs in precursor peptides with plant-specific BURP (BNM2, USP, RD22 and PG1beta) domains, but the biosynthetic mechanism for their formation has remained unknown. Here, we characterize precursor-peptide BURP domains as copper-dependent autocatalytic peptide cyclases and use a combination of tandem mass spectrometry-based metabolomics and plant genomics to systematically discover five BURP-domain-derived plant RiPP classes, with mono- and bicyclic structures formed via tryptophans and tyrosines, from botanical collections. As BURP-domain cyclases are scaffold-generating enzymes in plant specialized metabolism that are physically connected to their substrates in the same polypeptide, we introduce a bioinformatic method to mine plant genomes for precursor-peptide-encoding genes by detection of repetitive substrate domains and known core peptide features. Our study sets the stage for chemical, biosynthetic and biological exploration of plant RiPP natural products from BURP-domain cyclases.
Asunto(s)
Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Proteínas de Plantas/química , Secuencia de Aminoácidos , Catálisis , Permeabilidad de la Membrana Celular , Ciclización , Genoma de Planta , Espectrometría de Masas en TándemRESUMEN
The COVID-19 pandemic has spread throughout the whole globe, so it is imperative that all available resources be used to treat this scourge. In reality, the development of new pharmaceuticals has mostly benefited from natural products. The widespread medicinal usage of species in the Asteraceae family is extensively researched. In this study, compounds isolated from methanolic extract of Artemisia monosperma Delile, a wild plant whose grows in Egypt's Sinai Peninsula. Three compounds, stigmasterol 3-O-ß-D-glucopyranoside 1, rhamnetin 3, and padmatin 6, were first isolated from this species. In addition, five previously reported compounds, arcapillin 2, jaceosidin 4, hispidulin 5, 7-O-methyleriodictyol 7, and eupatilin 8, were isolated. Applying molecular modelling simulations revealed two compounds, arcapillin 2 and rhamnetin 3 with the best docking interactions and energies within SARS-CoV-2 Mpro-binding site (-6.16, and -6.70 kcal mol-1, respectively). The top-docked compounds (2-3) were further evaluated for inhibitory concentrations (IC50), and half-maximal cytotoxicity (CC50) of both SARS-CoV-2 and MERS-CoV. Interestingly, arcapillin showed high antiviral activity towards SARS-CoV-2 and MERS-CoV, with IC50 values of 190.8 µg mL-1 and 16.58 µg mL-1, respectively. These findings may hold promise for further preclinical and clinical research, particularly on arcapillin itself or in collaboration with other drugs for COVID-19 treatment.
RESUMEN
Broccoli is commonly consumed as food and as medicine. However, comprehensive metabolic profiling of two broccoli varieties, Romanesco broccoli (RB) and purple broccoli (PB), in relation to their anticholinergic activity has not been fully disclosed. A total of 110 compounds were tentatively identified using UPLC-Q-TOF-MS metabolomics. Distinctively different metabolomic profiles of the two varieties were revealed by principal component analysis (PCA). Furthermore, by volcano diagram analysis, it was found that PB had a significantly higher content of phenolic acids, flavonoids, and glucosinolates, indicating the different beneficial health potentials of PB that demonstrated higher antioxidant and anticholinergic activities. Moreover, Pearson's correlation analysis revealed 18 metabolites, mainly phenolic and sulfur compounds, as the main bioactive. The binding affinity of these biomarkers to the active sites of acetyl- and butyryl-cholinesterase enzymes was further validated using molecular docking studies. Results emphasize the broccoli significance as a functional food and nutraceutical source and highlight its beneficial effects against Alzheimer's disease.
Asunto(s)
Acetilcolinesterasa , Brassica , Inhibidores de la Colinesterasa , Metabolómica , Simulación del Acoplamiento Molecular , Brassica/química , Inhibidores de la Colinesterasa/farmacología , Acetilcolinesterasa/metabolismo , Glucosinolatos/metabolismo , Glucosinolatos/análisis , Flavonoides/análisis , Flavonoides/farmacología , Antioxidantes/farmacología , Antioxidantes/análisis , Cromatografía Liquida , Fenoles/análisis , Fenoles/farmacología , Análisis de Componente Principal , Hidroxibenzoatos/análisis , Hidroxibenzoatos/farmacología , Butirilcolinesterasa/metabolismo , Simulación por Computador , Extractos Vegetales/farmacología , Extractos Vegetales/química , Espectrometría de MasasRESUMEN
Microcystis spp. produce diverse secondary metabolites within freshwater cyanobacterial harmful algal blooms (cyanoHABs) around the world. In addition to the biosynthetic gene clusters (BGCs) encoding known compounds, Microcystis genomes harbor numerous BGCs of unknown function, indicating a poorly understood chemical repertoire. While recent studies show that Microcystis produces several metabolites in the lab and field, little work has focused on analyzing the abundance and expression of its broader suite of BGCs during cyanoHAB events. Here, we use metagenomic and metatranscriptomic approaches to track the relative abundance of Microcystis BGCs and their transcripts throughout the 2014 western Lake Erie cyanoHAB. The results indicate the presence of several transcriptionally active BGCs that are predicted to synthesize both known and novel secondary metabolites. The abundance and expression of these BGCs shifted throughout the bloom, with transcript abundance levels correlating with temperature, nitrate, and phosphorus concentrations and the abundance of co-occurring predatory and competitive eukaryotic microorganisms, suggesting the importance of both abiotic and biotic controls in regulating expression. This work highlights the need for understanding the chemical ecology and potential risks to human and environmental health posed by secondary metabolites that are produced but often unmonitored. It also indicates the prospects for identifying pharmaceutical-like molecules from cyanoHAB-derived BGCs. IMPORTANCE Microcystis spp. dominate cyanobacterial harmful algal blooms (cyanoHABs) worldwide and pose significant threats to water quality through the production of secondary metabolites, many of which are toxic. While the toxicity and biochemistry of microcystins and several other compounds have been studied, the broader suite of secondary metabolites produced by Microcystis remains poorly understood, leaving gaps in our understanding of their impacts on human and ecosystem health. We used community DNA and RNA sequences to track the diversity of genes encoding synthesis of secondary metabolites in natural Microcystis populations and assess patterns of transcription in western Lake Erie cyanoHABs. Our results reveal the presence of both known gene clusters that encode toxic secondary metabolites as well as novel ones that may encode cryptic compounds. This research highlights the need for targeted studies of the secondary metabolite diversity in western Lake Erie, a vital freshwater source to the United States and Canada.
Asunto(s)
Cianobacterias , Microcystis , Humanos , Microcystis/genética , Lagos/microbiología , Ecosistema , Cianobacterias/genética , Floraciones de Algas Nocivas , Familia de MultigenesRESUMEN
INTRODUCTION: Compared to synthetic herbicides, natural products with allelochemical properties can inhibit weed germination, aiding agricultural output with less phytotoxic residue in water and soil. OBJECTIVES: To identify natural product extracts of three Cassia species; C. javanica, C. roxburghii, and C. fistula and to investigate the possible phytotoxic and allelopathic potential. METHODS: Allelopathic activity of three Cassia species extracts was evaluated. To further investigate the active constituents, untergated metabolomics using UPLC-qTOF-MS/MS and ion-identity molecular networking (IIMN) approach was performed to identify and determine the distribution of metabolites in different Cassia species and plant parts. RESULTS: We observed in our study that the plant extracts showed consistent allelopathic activity against seed germination (P < 0.05) and the inhibition of shoot and root development of Chenopodium murale in a dose-dependent manner. Our comprehensive study identified at least 127 compounds comprising flavonoids, coumarins, anthraquinones, phenolic acids, lipids, and fatty acid derivatives. We also report the inhibition of seed germination, shoot growth, and root growth when treated with enriched leaf and flower extracts of C. fistula, and C. javanica, and the leaf extract of C. roxburghii. CONCLUSION: The present study recommends further evaluation of Cassia extracts as a potential source of allelopathic compounds in agricultural systems.
Asunto(s)
Cassia , Espectrometría de Masas en Tándem , Metabolómica , Germinación , Extractos Vegetales/farmacología , Extractos Vegetales/químicaRESUMEN
Discovery and structure elucidation of natural products available in infinitesimally small quantities are recognized challenge. This challenge is epitomized by the diphenazine class of molecules that contain three bridged stereocenters, several conformations, ring fusions, and multiple spatially isolated phenols. Because empirical NMR and spatial analyses using ROESY/NOESY were unsuccessful in tackling these challenges, we developed a computational pipeline to determine the relative and absolute configurations and phenol positions of diphenazines as inhibitors of eukaryotic translation initiation factor 4E (eIF4E) protein-protein interactions. In this pipeline, we incorporated ECD and GIAO NMR calculations coupled with a DP4+ probability measure, enabling the structure revision of phenazinolin D (4), izumiphenazine A (5), and baraphenazine G (7) and the structure characterization of two new diphenazines, baraphenazine H (3) and izumiphenazine E (6). Importantly, through these efforts, we demonstrate the feasibility of NMR/DP4+ analysis for the determination of phenol positions in phenazine-based molecules, further expanding the limits of computational methods for the structure elucidation of complex natural products.
Asunto(s)
Productos Biológicos , Estructura Molecular , Productos Biológicos/química , Fenol , Espectroscopía de Resonancia MagnéticaRESUMEN
Nef is an HIV-encoded accessory protein that enhances pathogenicity by down-regulating major histocompatibility class I (MHC-I) expression to evade killing by cytotoxic T lymphocytes (CTLs). A potent Nef inhibitor that restores MHC-I is needed to promote immune-mediated clearance of HIV-infected cells. We discovered that the plecomacrolide family of natural products restored MHC-I to the surface of Nef-expressing primary cells with variable potency. Concanamycin A (CMA) counteracted Nef at subnanomolar concentrations that did not interfere with lysosomal acidification or degradation and were nontoxic in primary cell cultures. CMA specifically reversed Nef-mediated down-regulation of MHC-I, but not CD4, and cells treated with CMA showed reduced formation of the Nef:MHC-I:AP-1 complex required for MHC-I down-regulation. CMA restored expression of diverse allotypes of MHC-I in Nef-expressing cells and inhibited Nef alleles from divergent clades of HIV and simian immunodeficiency virus, including from primary patient isolates. Lastly, we found that restoration of MHC-I in HIV-infected cells was accompanied by enhanced CTL-mediated clearance of infected cells comparable to genetic deletion of Nef. Thus, we propose CMA as a lead compound for therapeutic inhibition of Nef to enhance immune-mediated clearance of HIV-infected cells.
Asunto(s)
VIH-1 , Interacciones Huésped-Patógeno , Macrólidos , Linfocitos T Citotóxicos , Células Cultivadas , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/inmunología , Humanos , Macrólidos/inmunología , Macrólidos/farmacología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virología , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Alzheimer's disease poses a global health concern with unmet demand requiring creative approaches to discover new medications. In this study, we investigated the chemical composition and the anticholinesterase activity of Aspergillus niveus Fv-er401 isolated from Foeniculum vulgare (Apiaceae) roots. Fifty-eight metabolites were identified using UHPLC-MS/MS analysis of the crude extract. The fungal extract showed acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory effects with IC50 53.44 ± 1.57 and 48.46 ± 0.41 µg/mL, respectively. Two known metabolites were isolated, terrequinone A and citrinin, showing moderate AChE and BuChE inhibitory activity using the Ellman's method (IC50 = 11.10 ± 0.38 µg/mL and 5.06 ± 0.15 µg/mL, respectively for AChE, and IC50 15.63 ± 1.27 µg/mL and 8.02 ± 0.08 µg/mL, respectively for BuChE). As evidenced by molecular docking, the isolated compounds and other structurally related metabolites identified by molecular networking had the required structural features for AChE and BuChE inhibition. Where varioxiranol G (-9.76 and -10.36 kcal/mol), penicitrinol B (-9.50 and -8.02 kcal/mol), dicitrinol A (-8.53 and -7.98 kcal/mol) and asterriquinone CT5 (-8.02 and -8.25 kcal/mol) showed better binding scores as AChE and BuChE inhibitors than the co-crystallized inhibitor (between -7.89 and 7.82 kcal/mol) making them promising candidates for the development of new drugs to treat Alzheimer's.
Asunto(s)
Enfermedad de Alzheimer , Inhibidores de la Colinesterasa , Inhibidores de la Colinesterasa/química , Butirilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Simulación del Acoplamiento Molecular , Espectrometría de Masas en Tándem , Enfermedad de Alzheimer/tratamiento farmacológico , Metabolómica , Hongos/metabolismoRESUMEN
Root caries in geriatric patients is a growing problem as more people are maintaining their natural teeth into advanced age. We determined the levels of various bacterial species previously implicated in root caries disease or health using quantitative real-time PCR in a pilot study of 7 patients with 1 to 4 root caries lesions per person. Levels of 12 different species on diseased roots compared to healthy (contralateral control) roots were measured. Four species were found at significantly higher levels on diseased roots (Streptococcus mutans, Veillonella parvula/dispar, Actinomyces naeslundii/viscosus, and Capnocytophaga granulosa) compared across all plaque samples. The level of colonization by these species varied dramatically (up to 1,000-fold) between patients, indicating different patients have different bacteria contributing to root caries disease. Neither of the two species previously reported to correlate with healthy roots (C. granulosa and Delftia acidovorans) showed statistically significant protective roles in our population, although D. acidovorans showed a trend toward higher levels on healthy teeth (P = 0.08). There was a significant positive correlation between higher levels of S. mutans and V. parvula/dispar on the same diseased teeth. In vitro mixed biofilm studies demonstrated that co-culturing S. mutans and V. parvula leads to a 50 to 150% increase in sucrose-dependent biofilm mass compared to S. mutans alone, depending on the growth conditions, while V. parvula alone did not form in vitro biofilms. The presence of V. parvula also decreased the acidification of S. mutans biofilms when grown in artificial saliva and enhanced the health of mixed biofilms.
Asunto(s)
Caries Dental , Caries Radicular , Humanos , Anciano , Streptococcus mutans , Caries Radicular/microbiología , Saliva Artificial , Proyectos Piloto , Veillonella , Biopelículas , SacarosaRESUMEN
Cyanobacterial harmful algal blooms (cyanoHABs) degrade freshwater ecosystems globally. Microcystis aeruginosa often dominates cyanoHABs and produces microcystin (MC), a class of hepatotoxins that poses threats to human and animal health. Microcystin toxicity is influenced by distinct structural elements across a diversity of related molecules encoded by variant mcy operons. However, the composition and distribution of mcy operon variants in natural blooms remain poorly understood. Here, we characterized the variant composition of mcy genes in western Lake Erie Microcystis blooms from 2014 and 2018. Sampling was conducted across several spatial and temporal scales, including different bloom phases within 2014, extensive spatial coverage on the same day (2018), and frequent, autonomous sampling over a 2-week period (2018). Mapping of metagenomic and metatranscriptomic sequences to reference sequences revealed three Microcystis mcy genotypes: complete (all genes present [mcyA-J]), partial (truncated mcyA, complete mcyBC, and missing mcyD-J), and absent (no mcy genes). We also detected two different variants of mcyB that may influence the production of microcystin congeners. The relative abundance of these genotypes was correlated with pH and nitrate concentrations. Metatranscriptomic analysis revealed that partial operons were, at times, the most abundant genotype and expressed in situ, suggesting the potential biosynthesis of truncated products. Quantification of genetic divergence between genotypes suggests that the observed strains are the result of preexisting heterogeneity rather than de novo mutation during the sampling period. Overall, our results show that natural Microcystis populations contain several cooccurring mcy genotypes that dynamically shift in abundance spatiotemporally via strain succession and likely influence the observed diversity of the produced congeners. IMPORTANCE Cyanobacteria are responsible for producing microcystins (MCs), a class of potent and structurally diverse toxins, in freshwater systems around the world. While microcystins have been studied for over 50 years, the diversity of their chemical forms and how this variation is encoded at the genetic level remain poorly understood, especially within natural populations of cyanobacterial harmful algal blooms (cyanoHABs). Here, we leverage community DNA and RNA sequences to track shifts in mcy genes responsible for producing microcystin, uncovering the relative abundance, expression, and variation of these genes. We studied this phenomenon in western Lake Erie, which suffers annually from cyanoHAB events, with impacts on drinking water, recreation, tourism, and commercial fishing.
Asunto(s)
Cianobacterias , Microcystis , Cianobacterias/genética , Ecosistema , Genotipo , Lagos/microbiología , Microcistinas/genética , Microcistinas/metabolismo , Microcystis/genética , Microcystis/metabolismo , OperónRESUMEN
The rapid spread of bacterial infection caused by Staphylococcus aureus has become a problem to public health despite the presence of past trials devoted to controlling the infection. Thus, the current study aimed to explore the chemical composition of the extract of endophytic fungus Aspergillus fumigatus, isolated from Albizia lucidior leaves, and investigate the antimicrobial activity of isolated metabolites and their probable mode of actions. The chemical investigation of the fungal extract via UPLC/MS/MS led to the identification of at least forty-two metabolites, as well as the isolation and complete characterization of eight reported metabolites. The antibacterial activities of isolated metabolites were assessed against S. aureus using agar disc diffusion and microplate dilution methods. Compounds ergosterol, helvolic acid and monomethyl sulochrin-4-sulphate showed minimal inhibitory concentration (MIC) values of 15.63, 1.95 and 3.90 µg/mL, respectively, compared to ciprofloxacin. We also report the inhibitory activity of the fungal extract on DNA gyrase and topoisomerase IV, which led us to perform molecular docking using the three most active compounds isolated from the extract against both enzymes. These active compounds had the required structural features for S. aureus DNA gyrase and topoisomerase IV inhibition, evidenced via molecular docking.
Asunto(s)
Albizzia/microbiología , Antibacterianos/metabolismo , Aspergillus fumigatus/metabolismo , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Aspergillus fumigatus/química , Humanos , Metaboloma , Simulación del Acoplamiento Molecular , Hojas de la Planta/química , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacosRESUMEN
SUMMARY: DDAP is a tool for predicting the biosynthetic pathways of the products of type I modular polyketide synthase (PKS) with the focus on providing a more accurate prediction of the ordering of proteins and substrates in the pathway. In this study, the module docking domain (DD) affinity prediction performance on a hold-out testing dataset reached 0.88 as measured by the area under the receiver operating characteristic (ROC) curve (AUC); the Mean Reciprocal Ranking (MRR) of pathway prediction reached 0.67. DDAP has advantages compared to previous informatics tools in several aspects: (i) it does not rely on large databases, making it a high efficiency tool, (ii) the predicted DD affinity is represented by a probability (0-1), which is more intuitive than raw scores, (iii) its performance is competitive compared to the current popular rule-based algorithm. DDAP is so far the first machine learning based algorithm for type I PKS DD affinity and pathway prediction. We also established the first database of type I modular PKSs, featuring a comprehensive annotation of available docking domains information in bacterial biosynthetic pathways. AVAILABILITY AND IMPLEMENTATION: The DDAP database is available at https://tylii.github.io/ddap. The prediction algorithm DDAP is freely available on GitHub (https://github.com/tylii/ddap) and released under the MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Vías Biosintéticas , Sintasas Poliquetidas , Algoritmos , BacteriasRESUMEN
The antibiotic-resistant bacteria-associated infections are a major global healthcare threat. New classes of antimicrobial compounds are urgently needed as the frequency of infections caused by multidrug-resistant microbes continues to rise. Recent metagenomic data have demonstrated that there is still biosynthetic potential encoded in but transcriptionally silent in cultivatable bacterial genomes. However, the culture conditions required to identify and express silent biosynthetic gene clusters that yield natural products with antimicrobial activity are largely unknown. Here, we describe a new antibiotic discovery scheme, dubbed the modified crowded plate technique (mCPT), that utilizes complex microbial interactions to elicit antimicrobial production from otherwise silent biosynthetic gene clusters. Using the mCPT as part of the antibiotic crowdsourcing educational program Tiny EarthTM, we isolated over 1400 antibiotic-producing microbes, including 62 showing activity against multidrug-resistant pathogens. The natural product extracts generated from six microbial isolates showed potent activity against vancomycin-intermediate resistant Staphylococcus aureus. We utilized a targeted approach that coupled mass spectrometry data with bioactivity, yielding a new macrolactone class of metabolite, desertomycin H. In this study, we successfully demonstrate a concept that significantly increased our ability to quickly and efficiently identify microbes capable of the silent antibiotic production.
Asunto(s)
Antibacterianos/química , Organismos Acuáticos/química , Macrólidos/química , Animales , Colaboración de las MasasRESUMEN
Serratia marcescens is a bacterium frequently found in the environment, but over the last several decades it has evolved into a concerning clinical pathogen, causing fatal bacteremia. To establish such infections, pathogens require specific nutrients; one very limited but essential nutrient is iron. We sought to characterize the iron acquisition systems in S. marcescens isolate UMH9, which was recovered from a clinical bloodstream infection. Using RNA sequencing (RNA-seq), we identified two predicted siderophore gene clusters (cbs and sch) that were regulated by iron. Mutants were constructed to delete each iron acquisition locus individually and in conjunction, generating both single and double mutants for the putative siderophore systems. Mutants lacking the sch gene cluster lost their iron-chelating ability as quantified by the chrome azurol S (CAS) assay, whereas the cbs mutant retained wild-type activity. Mass spectrometry-based analysis identified the chelating siderophore to be serratiochelin, a siderophore previously identified in Serratia plymuthica Serratiochelin-producing mutants also displayed a decreased growth rate under iron-limited conditions created by dipyridyl added to LB medium. Additionally, mutants lacking serratiochelin were significantly outcompeted during cochallenge with wild-type UMH9 in the kidneys and spleen after inoculation via the tail vein in a bacteremia mouse model. This result was further confirmed by an independent challenge, suggesting that serratiochelin is required for full S. marcescens pathogenesis in the bloodstream. Nine other clinical isolates have at least 90% protein identity to the UMH9 serratiochelin system; therefore, our results are broadly applicable to emerging clinical isolates of S. marcescens causing bacteremia.
Asunto(s)
Bacteriemia/microbiología , Proteínas Bacterianas/genética , Hierro/metabolismo , Infecciones por Serratia/microbiología , Serratia marcescens/genética , Serratia marcescens/patogenicidad , Sideróforos/genética , Animales , Bacteriemia/sangre , Bacteriemia/inmunología , Bacteriemia/patología , Proteínas Bacterianas/inmunología , Unión Competitiva , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Prueba de Complementación Genética , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Transporte Iónico , Hierro/inmunología , Ratones , Ratones Endogámicos CBA , Familia de Multigenes , Unión Proteica , Infecciones por Serratia/sangre , Infecciones por Serratia/inmunología , Infecciones por Serratia/patología , Serratia marcescens/inmunología , Sideróforos/inmunología , VirulenciaRESUMEN
The fungal indole alkaloids are a unique class of complex molecules that have a characteristic bicyclo[2.2.2]diazaoctane ring and frequently contain a spiro-oxindole moiety. While various strains produce these compounds, an intriguing case involves the formation of individual antipodes by two unique species of fungi in the generation of the potent anticancer agents (+)- and (-)-notoamide A. NotI and NotI' have been characterized as flavin-dependent monooxygenases that catalyze epoxidation and semi-pinacol rearrangement to form the spiro-oxindole center within these molecules. This work elucidates a key step in the biosynthesis of the notoamides and provides an evolutionary hypothesis regarding a common ancestor for production of enantiopure notoamides.
Asunto(s)
Flavinas/metabolismo , Alcaloides Indólicos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxindoles/metabolismo , Compuestos de Espiro/metabolismo , Flavinas/química , Alcaloides Indólicos/química , Oxigenasas de Función Mixta/química , Conformación Molecular , Oxindoles/química , Compuestos de Espiro/química , EstereoisomerismoRESUMEN
Phosphopantetheine is a key structural element in biological acyl transfer reactions found embedded within coenzyme A (CoA). Phosphopantothenoylcysteine synthetase (PPCS) is responsible for installing a cysteamine group within phosphopantetheine. Therefore, it holds considerable potential as a drug target for developing new antimicrobials. In this study, we adapted a biochemical assay specific for bacterial PPCS to screen for inhibitors of CoA biosynthesis against a library of marine microbial derived natural product extracts (NPEs). Analysis of the NPE derived from Streptomyces blancoensis led to the isolation of novel antibiotics (10-12, and 14) from the adipostatin class of molecules. The most potent molecule (10) displayed in vitro activity with IC50= 0.93 µM, against S. pneumoniae PPCS. The whole cell antimicrobial assay against isolated molecules demonstrated their ability to penetrate bacterial cells and inhibit clinically relevant pathogenic strains. This establishes the validity of PPCS as a pertinent drug target, and the value of NPEs to provide new antibiotics.
RESUMEN
Cahuitamycins are biofilm inhibitors assembled by a convergent nonribosomal peptide synthetase pathway. Previous genetic analysis indicated that a discrete enzyme, CahJ, serves as a gatekeeper for cahuitamycin structural diversification. Here, the CahJ protein was probed structurally and functionally to guide the formation of new analogues by mutasynthetic studies. This analysis enabled the in vivo production of a new cahuitamycin congener through targeted precursor incorporation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Oligopéptidos/metabolismo , Péptido Sintasas/metabolismo , Streptomyces/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Vías Biosintéticas , Simulación del Acoplamiento Molecular , Oligopéptidos/química , Péptido Sintasas/química , Conformación Proteica , Streptomyces/química , Especificidad por SustratoRESUMEN
Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from both Malbranchea aurantiaca and Malbranchea graminicola that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. In this study, we characterized the two flavin-dependent halogenases involved in the late-stage halogenation of malbrancheamide in two different fungal strains. MalA and MalA' catalyze the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogues were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA' in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases and provides new insights into a potentially unique reaction mechanism.
Asunto(s)
Ascomicetos/enzimología , Proteínas Fúngicas/metabolismo , Alcaloides Indólicos/metabolismo , Ascomicetos/química , Ascomicetos/metabolismo , Vías Biosintéticas , Proteínas Fúngicas/química , Halogenación , Alcaloides Indólicos/química , Cinética , Modelos MolecularesRESUMEN
Indole alkaloids are a diverse class of natural products known for their wide range of biological activities and complex chemical structures. Rarely observed in this class are indolic nitrones, such as avrainvillamide and waikialoid, which possess potent bioactivities. Herein the oxa gene cluster from the marine-derived fungus Penicillium oxalicum F30 is described along with the characterization of OxaD, a flavin-dependent oxidase that generates roquefortine L, a nitrone-bearing intermediate in the biosynthesis of oxaline. Nitrone functionality in roquefortine L was confirmed by spectroscopic methods and 1,3-dipolar cycloaddition with methyl acrylate. OxaD is a versatile biocatalyst that converts an array of semisynthetic roquefortine C derivatives bearing indoline systems to their respective nitrones. This work describes the first implementation of a nitrone synthase as a biocatalyst and establishes a novel platform for late-stage diversification of a range of complex natural products.