RESUMO
Triacsins are an intriguing class of specialized metabolites possessing a conserved N-hydroxytriazene moiety not found in any other known natural products. Triacsins are notable as potent acyl-CoA synthetase inhibitors in lipid metabolism, yet their biosynthesis has remained elusive. Through extensive mutagenesis and biochemical studies, we here report all enzymes required to construct and install the N-hydroxytriazene pharmacophore of triacsins. Two distinct ATP-dependent enzymes were revealed to catalyze the two consecutive N-N bond formation reactions, including a glycine-utilizing, hydrazine-forming enzyme (Tri28) and a nitrite-utilizing, N-nitrosating enzyme (Tri17). This study paves the way for future mechanistic interrogation and biocatalytic application of enzymes for N-N bond formation.
Assuntos
Coenzima A Ligases/metabolismo , Streptomyces aureofaciens/enzimologia , Streptomyces aureofaciens/genética , Triazenos/metabolismo , Biocatálise , Escherichia coli/genética , Glicina/química , Hidrazinas/química , Metabolismo dos Lipídeos , Lipídeos/química , Nitritos/química , Triazenos/químicaRESUMO
Natural products provide an unparalleled diversity of small molecules to fuel drug screening efforts, but deconvoluting the pharmacological activity of natural product mixtures to identify key bioactive compounds remains a vexing and labor-intensive process. Therefore, we have developed a new platform to probe the non-specific pharmacological potential of compounds present in common dietary supplements via shotgun derivatization with isotopically labeled propanoic acid, a live cell affinity assay, which was used to selectively recognize the population of compounds which bind tightly to HeLa cells in culture, and a computational LC-MS data analysis of isotopically labeled compounds from cell lysate. The data analysis showed that hundreds of compounds were successfully derivatized in each extract, and dozens of those compounds showed high affinity for HeLa cells. In total, over a thousand isotopically labeled compounds were screened for cell affinity across three separate experiments, resulting in the identification of several known bioactive compounds with specific protein targets and six previously unreported structures. The new natural products include three tulsinol compounds which were isolated from Ocimum tenuiflorum and three valeraninium alkaloids from Valeriana officinalis. The valeraninium alkaloids constitute a distinct new family of alkaloids from valerian, which may have previously undescribed bioactivity. These results collectively demonstrate the tag and snag workflow's viability as a drug discovery method.
Assuntos
Alcaloides , Produtos Biológicos , Humanos , Produtos Biológicos/química , Células HeLa , Alcaloides/farmacologia , Descoberta de Drogas/métodos , Espectrometria de MassasRESUMO
Diamine-appended Mg2(dobpdc) (dobpdc4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate) metal-organic frameworks are a promising class of CO2 adsorbents, although their stability to SO2âa trace component of industrially relevant exhaust streamsâremains largely untested. Here, we investigate the impact of SO2 on the stability and CO2 capture performance of dmpn-Mg2(dobpdc) (dmpn = 2,2-dimethyl-1,3-propanediamine), a candidate material for carbon capture from coal flue gas. Using SO2 breakthrough experiments and CO2 isobar measurements, we find that the material retains 91% of its CO2 capacity after saturation with a wet simulated flue gas containing representative levels of CO2 and SO2, highlighting the robustness of this framework to SO2 under realistic CO2 capture conditions. Initial SO2 cycling experiments suggest dmpn-Mg2(dobpdc) may achieve a stable operating capacity in the presence of SO2 after initial passivation. Evaluation of several other diamine-Mg2(dobpdc) variants reveals that those with primary,primary (1°,1°) diamines, including dmpn-Mg2(dobpdc), are more robust to humid SO2 than those featuring primary,secondary (1°,2°) or primary,tertiary (1°,3°) diamines. Based on the solid-state 15N NMR spectra and density functional theory calculations, we find that under humid conditions, SO2 reacts with the metal-bound primary amine in 1°,2° and 1°,3° diamine-appended Mg2(dobpdc) to form a metal-bound bisulfite species that is charge balanced by a primary ammonium cation, thereby facilitating material degradation. In contrast, humid SO2 reacts with the free end of 1°,1° diamines to form ammonium bisulfite, leaving the metal-diamine bond intact. This structure-property relationship can be used to guide further optimization of these materials for CO2 capture applications.
Assuntos
Diaminas , Dióxido de Enxofre , Dióxido de Carbono , Aminas , CarbonoRESUMO
The isonitrile moiety is an electron-rich functionality that decorates various bioactive natural products isolated from diverse kingdoms of life. Isonitrile biosynthesis was restricted for over a decade to isonitrile synthases, a family of enzymes catalyzing a condensation reaction between l-Trp/l-Tyr and ribulose-5-phosphate. The discovery of ScoE, a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase, demonstrated an alternative pathway employed by nature for isonitrile installation. Biochemical, crystallographic, and computational investigations of ScoE have previously been reported, yet the isonitrile formation mechanism remains obscure. In the present work, we employed in vitro biochemistry, chemical synthesis, spectroscopy techniques, and computational simulations that enabled us to propose a plausible molecular mechanism for isonitrile formation. Our findings demonstrate that the ScoE reaction initiates with C5 hydroxylation of (R)-3-((carboxymethyl)amino)butanoic acid to generate 1, which undergoes dehydration, presumably mediated by Tyr96 to synthesize 2 in a trans configuration. (R)-3-isocyanobutanoic acid is finally generated through radical-based decarboxylation of 2, instead of the common hydroxylation pathway employed by this enzyme superfamily.
Assuntos
Carboxiliases , Oxirredutases , Carboxiliases/química , Compostos Ferrosos/química , Ferro/química , Ácidos Cetoglutáricos/metabolismoRESUMO
Mycobacterium tuberculosis spontaneously grows at the air-medium interface, forming pellicle biofilms, which harbor more drug-tolerant persisters than planktonic cultures. The underlying basis for increased persisters in M. tuberculosis biofilms is unknown. Using a transposon sequencing (Tn-seq) approach, we show here that multiple genes that are necessary for fitness of M. tuberculosis cells within biofilms, but not in planktonic cultures, are also implicated in tolerance of bacilli to a diverse set of stressors and antibiotics. Thus, development of M. tuberculosis biofilms appears to be associated with an enrichment of population, in which challenging growth conditions within biofilm architecture select for cells that maintain intrinsic tolerance to exogenous stresses, including antibiotic exposure. We further observed that the intrinsic drug tolerance of constituent cells of a biofilm determines the frequency of persisters. These findings together allow us to propose that the selection of elite cells during biofilm development promotes the frequency of persisters. Furthermore, probing the possibility that the population enrichment is an outcome of unique environment within biofilms, we demonstrate biofilm-specific induction in the synthesis of isonitrile lipopeptide (INLP). Mutation analysis indicates that INLP is necessary for the architecture development of M. tuberculosis biofilms. In summary, this study offers an insight into persistence of M. tuberculosis biofilms under antibiotic exposure, while identifying INLP as a potential biomarker for further investigation of this phenomenon.
Assuntos
Antituberculosos/farmacologia , Biofilmes/crescimento & desenvolvimento , Mycobacterium tuberculosis/crescimento & desenvolvimento , Adaptação Fisiológica/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genéticaRESUMO
The ability to design and construct combinatorial synthetic metabolic pathways has far exceeded our capacity for efficient screening and selection of the resulting microbial strains. The need for high-throughput rapid screening techniques is of upmost importance for the future of synthetic biology and metabolic engineering. Here we describe the development of an RNA riboswitch-based biosensor module with dual fluorescent reporters, and demonstrate a high-throughput flow cytometry-based screening method for identification of naringenin over producing Escherichia coli strains in co-culture. Our efforts helped identify a number of key operating parameters that affect biosensor performance, including the selection of promoter and linker elements within the sensor-actuator domain, and the effect of host strain, fermentation time, and growth medium on sensor dynamic range. The resulting biosensor demonstrates a high correlation between specific fluorescence of the biosensor strain and naringenin titer produced by the second member of the synthetic co-culture system. This technique represents a novel application for synthetic microbial co-cultures and can be expanded from naringenin to any metabolite if a suitable riboswitch is identified. The co-culture technique presented here can be applied to a variety of target metabolites in combination with the SELEX approach for aptamer design. Due to the compartmentalization of the two genetic constructs responsible for production and detection into separate cells and application as independent modules of a synthetic microbial co-culture we have subsequently reduced the need for re-optimization of the producer module when the biosensor is replaced or removed. Biotechnol. Bioeng. 2017;114: 2235-2244. © 2017 Wiley Periodicals, Inc.
Assuntos
Técnicas Biossensoriais/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Flavanonas/farmacologia , Riboswitch/genética , Espectrometria de Fluorescência/métodos , Técnicas de Cocultura/métodos , Engenharia Metabólica/métodos , Técnicas de Sonda MolecularRESUMO
The discovery of bioactive natural products lies at the forefront of human medicine. The continued discovery of these molecules is imperative in the fight against infection and disease. While natural products have historically dominated the drug market, discovery in recent years has slowed significantly, partly due to limitations in current discovery methodologies. This work demonstrates a new workflow, deuterium adduct bioactivity screening (DABS), which pairs untargeted isotope labeling with whole cell binding assays for bioactive natural product discovery. DABS was validated and led to the discovery of a new isoprenyl guanidine alkaloid, zillamycin, which showed anti-cancer and anti-microbial activities. DABS thus represents a new workflow to accelerate discovery of natural products with a wide range of bioactive potentials.