RESUMEN
Loss-of-function mutations of the CFTR gene cause the life-shortening genetic disease cystic fibrosis (CF), whereas overactivity of CFTR may lead to secretory diarrhea and polycystic kidney disease. While effective drugs targeting the CFTR protein have been developed for the treatment of CF, little progress has been made for diseases caused by hyper-activated CFTR. Here, we solve the cryo-EM structure of CFTR in complex with CFTRinh-172 (Inh-172), a CFTR gating inhibitor with promising potency and efficacy. We find that Inh-172 binds inside the pore of CFTR, interacting with amino acid residues from transmembrane segments (TMs) 1, 6, 8, 9, and 12 through mostly hydrophobic interactions and a salt bridge. Substitution of these residues lowers the apparent affinity of Inh-172. The inhibitor-bound structure reveals re-orientations of the extracellular segment of TMs 1, 8, and 12, supporting an allosteric modulation mechanism involving post-binding conformational changes. This allosteric inhibitory mechanism readily explains our observations that pig CFTR, which preserves all the amino acid residues involved in Inh-172 binding, exhibits a much-reduced sensitivity to Inh-172 and that the apparent affinity of Inh-172 is altered by the CF drug ivacaftor (i.e., VX-770) which enhances CFTR's activity through binding to a site also comprising TM8.
Asunto(s)
Microscopía por Crioelectrón , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Regulación Alostérica , Activación del Canal Iónico/efectos de los fármacos , Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Animales , Unión Proteica , Aminofenoles/farmacología , Aminofenoles/química , Aminofenoles/metabolismo , Benzodioxoles/farmacología , MutaciónRESUMEN
Impaired activity of the chloride channel CFTR is the cause of cystic fibrosis. 14-3-3 proteins have been shown to stabilize CFTR and increase its biogenesis and activity. Here, we report the identification and mechanism of action of a macrocycle stabilizing the 14-3-3/CFTR complex. This molecule rescues plasma membrane localization and chloride transport of F508del-CFTR and works additively with the CFTR pharmacological chaperone corrector lumacaftor (VX-809) and the triple combination Trikafta®. This macrocycle is a useful tool to study the CFTR/14-3-3 interaction and the potential of molecular glues in cystic fibrosis therapeutics.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Aminofenoles/metabolismo , Aminopiridinas/metabolismo , Aminopiridinas/farmacología , Membrana Celular/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , MutaciónRESUMEN
The cation-independent mannose-6-phosphate receptor (CI-M6PR, aka insulin-like growth factor II receptor or IGFIIR) is a membrane protein that plays a central role in the trafficking of lysosomal acid hydrolases into lysosomes via mannose-6-phosphate (M6P) binding domains. In order to maintain cellular metabolic/catabolic homeostasis, newly synthesized lysosomal acid hydrolases are required to bind to M6PR for transit. Acid hydrolases secreted by cells can also be internalized via M6PR residing on the cell membrane and are transported to the lysosomes, a feature that enables enzyme replacement therapy for the treatment of several lysosomal storage disorders. Therefore, a thorough characterization of this receptor is critical to the development of lysosomal enzyme-based therapeutics that utilize M6PR for drug delivery to the lysosome. However, the extracellular domain (ECD) of M6PR is highly complex, containing 15-mannose receptor homology (MRH) domains. In addition, homodimerization of the receptor can occur at the membrane, making its characterization challenging. In this study, a novel human M6PR (hM6PR)-overexpressing cell line originally established for hM6PR cellular uptake assay was utilized for production of hM6PR-ECD, and a novel small molecule biomimetic (aminophenyl-M6P) affinity resin was developed for the purification of M6PR-ECD. The affinity-purified hM6PR-ECD was monomeric, contained 14 intact MRH domains (1-14) and a partial MRH domain 15, and was successfully employed in ELISA-based and surface plasmon resonance-based binding assays to demonstrate its ligand-binding functionality, making it suitable for the evaluation of biotherapeutics that utilize M6PR for cellular internalization.
Asunto(s)
Aminofenoles/química , Materiales Biomiméticos/química , Membrana Celular/enzimología , Manosafosfatos/química , Receptor IGF Tipo 2/aislamiento & purificación , Secuencia de Aminoácidos , Aminofenoles/metabolismo , Materiales Biomiméticos/metabolismo , Línea Celular Tumoral , Membrana Celular/química , Cromatografía de Afinidad , Pruebas de Enzimas , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/química , Fibroblastos/enzimología , Expresión Génica , Humanos , Cinética , Manosafosfatos/metabolismo , Dominios Proteicos , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Resonancia por Plasmón de SuperficieRESUMEN
Covering: up to July 2019 Terpene synthases (TSs) are responsible for generating much of the structural diversity found in the superfamily of terpenoid natural products. These elegant enzymes mediate complex carbocation-based cyclization and rearrangement cascades with a variety of electron-rich linear and cyclic substrates. For decades, two main classes of TSs, divided by how they generate the reaction-triggering initial carbocation, have dominated the field of terpene enzymology. Recently, several novel and unconventional TSs that perform TS-like reactions but do not resemble canonical TSs in sequence or structure have been discovered. In this review, we identify 12 families of non-canonical TSs and examine their sequences, structures, functions, and proposed mechanisms. Nature provides a wide diversity of enzymes, including prenyltransferases, methyltransferases, P450s, and NAD+-dependent dehydrogenases, as well as completely new enzymes, that utilize distinctive reaction mechanisms for TS chemistry. These unique non-canonical TSs provide immense opportunities to understand how nature evolved different tools for terpene biosynthesis by structural and mechanistic characterization while affording new probes for the discovery of novel terpenoid natural products and gene clusters via genome mining. With every new discovery, the dualistic paradigm of TSs is contradicted and the field of terpene chemistry and enzymology continues to expand.
Asunto(s)
Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Aminofenoles/química , Aminofenoles/metabolismo , Cannabinoides/química , Cannabinoides/metabolismo , Ciclización , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Dimetilaliltranstransferasa/química , Dimetilaliltranstransferasa/metabolismo , Iridoides/química , Iridoides/metabolismo , Toxinas de Lyngbya/química , Toxinas de Lyngbya/metabolismo , Estructura Molecular , Fenazinas/química , Fenazinas/metabolismo , Compuestos Policíclicos/química , Compuestos Policíclicos/metabolismo , Conformación Proteica , Terpenos/química , Terpenos/metabolismoRESUMEN
Elucidation of the structure and function of biomolecules provides us knowledge that can be transferred into the generation of new materials and eventually applications in e.g., catalysis or bioassays. The main problems, however, concern the complexity of the natural systems and their limited availability, which necessitates utilization of simple biomimetic analogues that are, to a certain degree, similar in terms of structure and thus behaviour. We have, therefore, devised a small library of six tridentate N-heterocyclic coordinating agents (L1-L6), which, upon complexation, form two groups of artificial, monometallic non-heme iron species. Utilization of iron(III) chloride leads to the formation of the 1:1 (Fe:Ln) 'open' complexes, whereas iron(II) trifluoromethanosulfonate allows for the synthesis of 1:2 (M:Ln) 'closed' systems. The structural differences between the individual complexes are a result of the information encoded within the metallic centre and the chosen counterion, whereas the organic scaffold influences the observed properties. Indeed, the number and nature of the external hydrogen bond donors coming from the presence of (benz)imidazole moieties in the ligand framework are responsible for the observed biological behaviour in terms of mimicking phenoxazinone synthase activity and interaction with DNA.
Asunto(s)
Bencimidazoles/química , Materiales Biomiméticos/química , ADN/metabolismo , Hierro/química , Oxidorreductasas/metabolismo , Bases de Schiff/química , Aminofenoles/metabolismo , Animales , Unión Competitiva , Catálisis , Bovinos , Fluorescencia , Imidazoles , Cinética , Ligandos , Oxazinas , Oxidación-Reducción , Bases de Schiff/síntesis química , Elementos de Transición/metabolismoRESUMEN
Nonheme diiron monooxygenases make up a rapidly growing family of oxygenases that are rarely identified in secondary metabolism. Herein, we report the in vivo, in vitro, and structural characterizations of a nonheme diiron monooxygenase, PtmU3, that installs a C-5 ß-hydroxyl group in the unified biosynthesis of platensimycin and platencin, two highly functionalized diterpenoids that act as potent and selective inhibitors of bacterial and mammalian fatty acid synthases. This hydroxylation sets the stage for the subsequent A-ring cleavage step key to the unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 adopts an unprecedented triosephosphate isomerase (TIM) barrel structural fold for this class of enzymes and possesses a noncanonical diiron active site architecture with a saturated six-coordinate iron center lacking a µ-oxo bridge. This study reveals the first member of a previously unidentified superfamily of TIM-barrel-fold enzymes for metal-dependent dioxygen activation, with the majority predicted to act on CoA-linked substrates, thus expanding our knowledge of nature's repertoire of nonheme diiron monooxygenases and TIM-barrel-fold enzymes.
Asunto(s)
Adamantano/metabolismo , Aminobenzoatos/metabolismo , Aminofenoles/metabolismo , Anilidas/metabolismo , Hierro/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Compuestos Policíclicos/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Hidroxilación , Modelos MolecularesRESUMEN
Platensimycin (PTM) and platencin (PTN) are highly functionalized bacterial diterpenoids of ent-kauranol and ent-atiserene biosynthetic origin. C7 oxidation in the B-ring plays a key biosynthetic role in generating structural complexity known for ent-kaurane and ent-atisane derived diterpenoids. While all three oxidation patterns, α-hydroxyl, ß-hydroxyl, and ketone, at C7 are seen in both the ent-kaurane and ent-atisane derived diterpenoids, their biosynthetic origins remain largely unknown. We previously established that PTM and PTN are produced by a single biosynthetic machinery, featuring cryptic C7 oxidations at the B-rings that transform the ent-kauranol and ent-atiserene derived precursors into the characteristic PTM and PTN scaffolds. Here, we report a three-enzyme cascade affording C7 α-hydroxylation in PTM and PTN biosynthesis. Combining in vitro and in vivo studies, we show that PtmO3 and PtmO6 are two functionally redundant α-ketoglutarate-dependent dioxygenases that generate a cryptic C7 ß-hydroxyl on each of the ent-kauranol and ent-atiserene scaffolds, and PtmO8 and PtmO1, a pair of NAD+/NADPH-dependent dehydrogenases, subsequently work in concert to invert the C7 ß-hydroxyl to α-hydroxyl via a C7 ketone intermediate. PtmO3 and PtmO6 represent the first dedicated C7 ß-hydroxylases characterized to date and, together with PtmO8 and PtmO1, provide an account for the biosynthetic origins of all three C7 oxidation patterns that may shed light on other B-ring modifications in bacterial, plant, and fungal diterpenoid biosynthesis. Given their unprecedented activities in C7 oxidations, PtmO3, PtmO6, PtmO8, and PtmO1 enrich the growing toolbox of novel enzymes that could be exploited as biocatalysts to rapidly access complex diterpenoid natural products.
Asunto(s)
Adamantano/metabolismo , Aminobenzoatos/metabolismo , Aminofenoles/metabolismo , Anilidas/metabolismo , Compuestos Policíclicos/metabolismo , Adamantano/química , Aminobenzoatos/química , Aminofenoles/química , Anilidas/química , Hidroxilación , Conformación Molecular , Oxidación-Reducción , Compuestos Policíclicos/química , EstereoisomerismoRESUMEN
The α,ß-tubulin is the building block of microtubules, which is associated with and dissociated from the microtubular architecture complying with the dynamic instability of the microtubules. This dynamic instability has a direct relation with the spindle formation by the microtubules and cell division kinetics. E7010 is one of the promising ligands of an α,ß-tubulin protein that binds at the core of this protein and can diminish the protein's ability to fit to a growing microtubule, thus frustrating cell division. Although X-ray crystallography has reported a specific binding conformation of E7010 in PDB, molecular dynamics (MD) simulations have revealed two other conformational states of the ligand capable of binding to tubulin with stabilities close to that state reported in PDB. To rationalize this quasidegeneracy of ligand binding modes, MD simulations have further revealed that the understanding of the mechanism of E7010-tubulin binding remains incomplete unless the role of water molecules to bridge this interaction is taken into consideration, a very critical insight that was not visible from the PDB structure. Further, these water molecules differ from the standard examples of "bridging" waters which generally exist as isolated water molecules between the receptor and the ligand. In the present case, the water molecules sandwiched between ligand and protein, sequestered from the bulk solvent, integrate with each other by an H-bonds network forming a group, which appear as microclusters of water. The structural packing with the ligand binding pocket and the bridging interactions between protein and ligand take place through such clusters. The presence of this microcluster of water is not just cosmetic, instead they have a crucial impact on the ligand binding thermodynamics. Only with the explicit consideration of these water clusters in the binding energy calculations (MMGBSA) is the stability of the native mode of ligand binding reported in PDB rationalized. At the same time, two other binding modes are elucidated to be quasi-degenerate with the native state and that indicates the further possibility in gaining more entropic stabilization of the complex. The role of such "bridging" water clusters to enhance the protein-ligand interaction will be insightful for designing the next generation prospective compounds in the field of cancer therapeutics.
Asunto(s)
Aminofenoles/química , Simulación de Dinámica Molecular , Sulfonamidas/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Agua/química , Aminofenoles/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Microtúbulos/metabolismo , Conformación Proteica , Sulfonamidas/metabolismoRESUMEN
Kynurenines, the products of tryptophan oxidative degradation, are involved in multiple neuropathologies, such as Huntington's chorea, Parkinson's disease, senile dementia, etc. The major cause for hydroxykynurenines's neurotoxicity is the oxidative stress induced by the reactive oxygen species (ROS), the by-products of L-3-hydroxykynurenine (L-3HOK) and 3-hydroxyanthranilic acid (3HAA) oxidative self-dimerization. 2-aminophenol (2AP), a structural precursor of L-3HOK and 3HAA, undergoes the oxidative conjugation to form 2-aminophenoxazinone. There are several modes of 2AP dimerization, including both enzymatic and non-enzymatic stages. In this study, the free energies for 2AP, L-3HOK and 3HAA dimerization stages have been calculated at B3LYP/6-311G(d,p)//6-311+(O)+G(d) level, both in the gas phase and in heptane or water solution. For the intermediates, ionization potentials and electron affinities were calculated, as well as free energy and kinetics of molecular oxygen interaction with several non-enzymatically formed dimers. H-atom donating power of the intermediates increases upon the progress of the oxidation, making possible generation of hydroperoxyl radical or hydrogen peroxide from O2 at the last stages. Among the dimerization intermediates, 2-aminophenoxazinole derivatives have the lowest ionization potential and can reduce O2 to superoxide anion. The rate for O-H homolytic bond dissociation is significantly higher than that for C-H bond in non-enzymatic quinoneimine conjugate. However, the last reaction passes irreversibly, reducing O2 to hydroperoxyl radical. The inorganic ferrous iron and the heme group of Drosophila phenoxazinone synthase significantly reduce the energy cost of 2AP H-atom abstraction by O2. We have also shown experimentally that total antioxidant capacity decreases in Drosophila mutant cardinal with L-3HOK excess relative to the wild type Canton-S, and lipid peroxidation decreases in aged cardinal. Taken together, our data supports the conception of hydroxykynurenines' dual role in neurotoxicity: serving as antioxidants themselves, blocking lipid peroxidation by H-atom donation, they also can easily generate ROS upon dimerization, leading to the oxidative stress development.
Asunto(s)
Quinurenina/química , Quinurenina/metabolismo , Modelos Biológicos , Aminofenoles/química , Aminofenoles/metabolismo , Animales , Antioxidantes/metabolismo , Biología Computacional , Dimerización , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Quinurenina/toxicidad , Redes y Vías Metabólicas , Modelos Moleculares , Conformación Molecular , Síndromes de Neurotoxicidad/etiología , Síndromes de Neurotoxicidad/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Oxígeno/química , Oxígeno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Termodinámica , Triptófano/metabolismoRESUMEN
Thiocarboxylic acid-containing natural products are rare and their biosynthesis and biological significance remain unknown. Thioplatensimycin (thioPTM) and thioplatencin (thioPTN), thiocarboxylic acid congeners of the antibacterial natural products platensimycin (PTM) and platencin (PTN), were recently discovered. Here we report the biosynthetic origin of the thiocarboxylic acid moiety in thioPTM and thioPTN. We identify a thioacid cassette encoding two proteins, PtmA3 and PtmU4, responsible for carboxylate activation by coenzyme A and sulfur transfer, respectively. ThioPTM and thioPTN bind tightly to ß-ketoacyl-ACP synthase II (FabF) and retain strong antibacterial activities. Density functional theory calculations of binding and solvation free energies suggest thioPTM and thioPTN bind to FabF more favorably than PTM and PTN. Additionally, thioacid cassettes are prevalent in the genomes of bacteria, implicating that thiocarboxylic acid-containing natural products are underappreciated. These results suggest that thiocarboxylic acid, as an alternative pharmacophore, and thiocarboxylic acid-containing natural products may be considered for future drug discovery.
Asunto(s)
Productos Biológicos , Streptomyces/metabolismo , Adamantano/metabolismo , Aminobenzoatos/metabolismo , Aminofenoles/metabolismo , Anilidas/metabolismo , Productos Biológicos/química , Escherichia coli , Familia de Multigenes , Compuestos Policíclicos/metabolismo , Streptomyces/genética , Azufre/metabolismoRESUMEN
The pentafluorosulfanyl (SF5-) substituent conveys properties that are beneficial to drugs and agrochemicals. As synthetic methodologies improve the number of compounds containing this group will expand and these chemicals may be viewed as emerging pollutants. As many microorganisms can degrade aromatic xenobiotics, we investigated the catabolism of SF5-substituted aminophenols by bacteria and found that some Pseudomonas spp. can utilise these compounds as sole carbon and energy sources. GC-MS analysis of the culture supernatants from cultures grown in 5-(pentafluorosulfanyl) 2-aminophenol demonstrated the presence of the N-acetylated derivative of the starting substrate and 4-(pentafluorosulfanyl)catechol. Biotransformation experiments with re-suspended cells were also conducted and fluorine-19 NMR analyses of the organic extract and aqueous fraction from suspended cell experiments revealed new resonances of SF5-substituted intermediates. Supplementation of suspended cell cultures with yeast extract dramatically improved the degradation of the substrate as well as the release of fluoride ion. 4-(Pentafluorosulfanyl)catechol was shown to be a shunt metabolite and toxic to some of the bacteria. This is the first study to demonstrate that microorganisms can biodegrade SF5-substituted aromatic compounds releasing fluoride ion, and biotransform them generating a toxic metabolite.
Asunto(s)
Aminofenoles/metabolismo , Pseudomonas/metabolismo , Compuestos de Azufre/metabolismo , Aminofenoles/química , Biodegradación Ambiental , Biotransformación , Catecoles/metabolismo , Flúor/metabolismo , Espectroscopía de Resonancia Magnética , Metaboloma , Compuestos de Azufre/químicaRESUMEN
BACKGROUND & OBJECTIVE: Since the release of ivacaftor-lumacaftor, several red-flags have been raised that highlight the clinical efficacy of this combination strategy that may be limited due to antagonistic drug-drug interactions. METHOD: The effect of ivacaftor, its major metabolites M1 and M6, lumacaftor and the novel cystic fibrosis transmembrane conductance regulator (CFTR) modulator tezacaftor at 10 µg/mL on the enzymatic activity of the major xenobiotic metabolizing enzymes CYP1A2 and CYP3A4 as well as the minor enzymes CYP2B6 and CYP2C9 was assayed. RESULTS: Lumacaftor (3.74 x 105 ± 3.11 x 104 RLU), and ivacaftor-M6 (3.43 x 105 ± 7.61 x 103 RLU) markedly induced the activity of CYP3A4. Ivacaftor (2.22 x 105 ± 3.94 x 104 RLU) showed a lower relative ratio of luminescence units compared to chloramphenicol (3.17 x 105 ± 1.55 x 104 RLU). Interestingly, ivacaftor-M1 (6.74 x 104 ± 3.09 x 104 RLU) and the novel CFTR modulator tezacaftor (2.40 x 104 ± 8.14 x 104 RLU) did not show CYP3A4 induction. In the CYP1A2 and CYP2C9 assay, all metabolites showed a decrease in the ratio of luminescence units compared to the controls. Ivacaftor, its major metabolites, lumacaftor and tezacaftor all showed a slight increase in the ratio of luminescence units compared to the control rifampin with CYP2B6. CONCLUSION: All in all, present findings would suggest that lumacaftor and ivacaftor-M6 are strong inducers of CYP3A4, potentially reducing ivacaftor concentrations; ivacaftor itself induces CYP3A4 to some extent.
Asunto(s)
Aminofenoles/farmacología , Aminopiridinas/farmacología , Benzodioxoles/farmacología , Fibrosis Quística/tratamiento farmacológico , Citocromo P-450 CYP3A/biosíntesis , Interacciones Farmacológicas , Quinolonas/farmacología , Aminofenoles/metabolismo , Aminofenoles/uso terapéutico , Aminopiridinas/metabolismo , Aminopiridinas/uso terapéutico , Benzodioxoles/metabolismo , Benzodioxoles/uso terapéutico , Fibrosis Quística/sangre , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Citocromo P-450 CYP1A2/biosíntesis , Citocromo P-450 CYP2C9/biosíntesis , Combinación de Medicamentos , Pruebas de Enzimas , Inducción Enzimática/efectos de los fármacos , Femenino , Humanos , Indoles/farmacología , Quinolonas/metabolismo , Quinolonas/uso terapéuticoRESUMEN
Selective androgen receptor modulators (SARMs) are a class of androgen receptor drugs, which have a high potential to be performance enhancers in human and animal sports. Arylpropionamides are one of the major SARM classes and get rapidly metabolized significantly complicating simple detection of misconduct in blood or urine sample analysis. Specific drug-derived metabolites are required as references due to a short half-life of the parent compound but are generally lacking. The difficulty in metabolism studies is the determination of the correct regio and stereoselectivity during metabolic conversion processes. In this study, we have elucidated and verified the chemical structure of two major equine arylpropionamide-based SARM metabolites using a combination of chemical synthesis and liquid chromatography-mass spectrometry (LC-MS) analysis. These synthesized SARM-derived metabolites can readily be utilized as reference standards for routine mass spectrometry-based doping control analysis of at least three commonly used performance-enhancing drugs to unambigously identify misconduct.
Asunto(s)
Acetamidas/metabolismo , Amidas/metabolismo , Aminofenoles/metabolismo , Anabolizantes/metabolismo , Anilidas/metabolismo , Receptores Androgénicos/metabolismo , Acetamidas/química , Acetamidas/orina , Amidas/química , Amidas/orina , Aminofenoles/química , Aminofenoles/orina , Anabolizantes/química , Anabolizantes/orina , Anilidas/química , Anilidas/orina , Animales , Cromatografía Líquida de Alta Presión/métodos , Doping en los Deportes , Caballos , Humanos , Espectrometría de Masas/métodos , Detección de Abuso de Sustancias/métodosRESUMEN
The ast gene cluster (GenBank accession numbers KF813023.1 and KP284551) was characterized to be responsible for the biosynthesis of ansatrienins in Streptomyces sp. XZQH13, which contains astC, astF1, and astF2 genes involved in the assembly of the N-cyclohexanoyl d-alanyl side chain and the hydroxylation of C-19, respectively. Further to investigating the biosynthetic mechanism of ansatrienins, herein we constructed the mutant strains XZQH13OEΔastF2 and XZQH13OEΔastCΔastF2. Three new ansatrienin analogues, namely, ansatrienolsâ I-K (1-3), along with trienomycinol (4) and 3-O-demethyltrienomycinol (5), were isolated from the XZQH13OEΔastCΔastF2 strain, and trienomycinâ A (6) and trienomycinâ G (7) were isolated from the XZQH13OEΔastF2 strain. Their structures were determined by a combination of high-resolution MS (ESI) and 1D and 2D NMR spectroscopy. Accordingly, a pathway for the biosynthesis of these new ansatrienins was proposed.
Asunto(s)
Alanina/análogos & derivados , Aminofenoles/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Policétidos/metabolismo , Streptomyces/química , Alanina/biosíntesis , Alanina/química , Alanina/aislamiento & purificación , Aminofenoles/química , Aminofenoles/aislamiento & purificación , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Conformación Molecular , Policétidos/química , Policétidos/aislamiento & purificación , Estereoisomerismo , Streptomyces/metabolismoRESUMEN
Mating pheromones (gamone 1 and gamone 2) in the ciliate Blepharisma are biologically active substances that trigger sexual reproduction (conjugation) under starvation conditions. Gamone 1 is a glycoprotein secreted by type I cells, and gamone 2 is a tryptophan (Trp)-derivative compound secreted by type II cells. Both gamones stimulate complementary mating type cells to promote each gamone production and induce pair formation. To elucidate the biosynthetic pathway of gamone 2, we investigated the enzymes involved in the pathway and the specificity of the enzymes. An RNA-seq analysis revealed that Blepharisma stoltei (Heterotrichea) possesses four indoleamine 2,3-dioxygenase (IDO) genes showing distinct expression patterns. Along with results from real-time PCR, these findings demonstrated that each IDO gene has different expression patterns that depend on the cellular conditions. Expression of IDO-I was correlated with the intensity of gamone 2 expression, and the recombinant IDO-I protein showed catalytic activity for 5-hydroxy-L-Trp (5-HTP) but very weak activity for L-Trp. Our results indicate that IDO-I is an enzyme evolutionary specialized to gamone 2 production in Blepharisma, and that the biosynthetic pathway for gamone 2 uses 5-HTP as an intermediate.
Asunto(s)
Aminofenoles/metabolismo , Cilióforos/genética , Conjugación Genética , Lactatos/metabolismo , Feromonas/biosíntesis , Proteínas Protozoarias/genética , Cilióforos/enzimología , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Proteínas Protozoarias/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reproducción , Transcripción Genética , Triptófano Oxigenasa/genética , Triptófano Oxigenasa/metabolismoRESUMEN
We developed an electrochemical microalgal bioassay for the determination of heavy metal toxicity in water on the basis of the alkaline phosphatase (ALP) enzyme inhibition of Chlamydomonas reinhardtii. Five heavy metals were chosen as toxicants: Hg, Cd, Pb, Zn, and Cu. The induced ALP activity of C. reinhardtii was inhibited using the phosphate starvation method, and the results were evaluated by measuring the electrochemical oxidation of p-aminophenol (PAP) following the enzymatic conversion of p-aminophenyl phosphate (PAPP) as a substrate. The rapid determination of enzymatic activity was achieved using hydrodynamic voltammetry in a 50 µL micro-droplet with a rotating disk electrode (RDE). Enzymatic activity over a PAPP substrate is affected by heavy metal ions, and this phenomenon decreases the chronoamperometric current signal. The concentrations of Hg, Cd, Pb, Zn, and Cu in which the ALP activity was half that of the control (EC50) were found to be 0.017, 0.021, 0.27, 1.30, and 1.36 µM, respectively. The RDE system was demonstrated to be capable of detecting enzymatic activity by using a small amount of regent, a reaction time of only 60 s, and a detection limit of 5.4 × 10-7 U.
Asunto(s)
Fosfatasa Alcalina/antagonistas & inhibidores , Bioensayo/métodos , Contaminantes Ambientales/análisis , Metales Pesados/toxicidad , Microalgas/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Aminofenoles/metabolismo , Chlamydomonas reinhardtii/enzimología , Chlamydomonas reinhardtii/metabolismo , Electroquímica/métodos , Contaminantes Ambientales/toxicidad , Hidrodinámica , Metales Pesados/análisis , Oxidación-ReducciónRESUMEN
Diseases caused by enterotoxicogenic Escherichia coli F4 (K88) (ETEC F4) are a problem in swine production establishments. Due to the high rate of mortality and morbidity of E. coli infections, a rapid and accurate diagnosis is important in order to choose an appropriate treatment to reduce the economic impact. Therefore, an electrochemical magneto-immunosensor (EMI) was developed to detect and quantify ETEC F4 in swine feces samples through a direct non-competitive immunoassay. ETEC F4 was selectively captured by immunomagnetic separation. The detection principle was based on the activity of ß-galactosidase endogenous enzyme (ß-gal), which hydrolyses the p-aminophenyl-ß-D-galactopyranoside (p-APG) producing p-aminophenol (p-AP), which was oxidized on a carbon screen printed electrode (CSPE) using square wave voltammetry (SWV). All parameters related to construction and electrochemical responses were optimized. The total analysis time to quantify ETEC F4 using the EMI was less than 2h and the limit of detection (LOD) was 33CFUmL-1. The perceptual relative error (%Er) was 20%. The magneto-immunosensor was validated versus conventional method of culture and plate count, obtaining a very good agreement. The EMI is simple, fast and economical to detect and quantify ETEC F4 in swine feces samples, being thus a valuable tool in swine production.
Asunto(s)
Técnicas Biosensibles/métodos , Escherichia coli Enterotoxigénica/aislamiento & purificación , Heces/microbiología , Inmunoensayo/métodos , Separación Inmunomagnética , beta-Galactosidasa/metabolismo , Aminofenoles/química , Aminofenoles/metabolismo , Animales , Calibración , Carbono/química , Electroquímica , Electrodos , Oxidación-Reducción , Permeabilidad , PorcinosRESUMEN
Nitric oxide (NO) has been known as a gaseous chemical mediator, which modulates several physiological functions. Spatial and temporal control of NO release facilitates further study and medical application of NO. Herein, we report design and synthesis of a novel NO donor, NO-Rosa. NO-Rosa has a rosamine moiety, which absorbs yellowish green light. Upon irradiation with yellowish green light (530-590 nm), NO is released from NO-Rosa, presumably via photoinduced electron transfer from the N-nitrosoaminophenol moiety to the rosamine moiety. NO release from NO-Rosa was detected by ESR spin trapping and a NO fluorescent probe. Cellular NO release control was achieved in HEK293 cells using a NO fluorescent probe, DAF-FM DA. Furthermore, temporally controlled NO-induced vasodilation was demonstrated by treatment of a rat aortic strip with NO-Rosaex vivo and irradiation by yellowish green light. NO-Rosa is expected to be utilized for further study of NO-related physiological functions, utilizing its ability of spatiotemporal release of NO as a photocontrollable compound with harmless yellowish-green light.
Asunto(s)
Aminofenoles/metabolismo , Luz , Donantes de Óxido Nítrico/metabolismo , Compuestos Nitrosos/metabolismo , Aminofenoles/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Estructura Molecular , Donantes de Óxido Nítrico/química , Compuestos Nitrosos/química , Procesos FotoquímicosRESUMEN
In this study, an enzyme-based electrochemical method was developed for the detection of Escherichia coli (E. coli) using the T7 bacteriophages engineered with lacZ operon encoding for beta-galactosidase (ß-gal). The T7lacZ phages can infect E. coli, and have the ability to trigger the overexpression of ß-gal during the infection of E. coli. The use of the engineered phages resulted in a more sensitive detection of E. coli by (1) overexpression of ß-gal in E. coli during the specific infection and (2) release of the endogenous intracellular ß-gal from E. coli following infection. The endogenous and phage-induced ß-gal was detected using the electrochemical method with 4-aminophenyl-ß-galactopyranoside (PAPG) as a substrate. The ß-gal catalyzed PAPG to an electroactive species p-aminophenol (PAP) which could be monitored on an electrode. The electrochemical signal was proportional to the concentration of E. coli in the original sample. We demonstrated the application of our strategy in aqueous samples (drinking water, apple juice, and skim milk). Using this method, we were able to detect E. coli at the concentration of approximately 105 CFU/mL in these aqueous samples in 3 h and 102 CFU/mL after 7 h. This strategy has the potential to be extended to detect different bacteria using specific bacteriophages engineered with gene encoding for appropriate enzymes.
Asunto(s)
Bacteriófagos/genética , Técnicas Electroquímicas , Escherichia coli/aislamiento & purificación , Aminofenoles/química , Aminofenoles/metabolismo , Bebidas/microbiología , Electrodos , Escherichia coli/enzimología , Galactósidos/química , Galactósidos/metabolismo , Concentración de Iones de Hidrógeno , Microbiología del Agua , beta-Galactosidasa/genéticaRESUMEN
Natural products have served as the main source of drugs and drug leads, and natural products produced by microorganisms are one of the most prevalent sources of clinical antibiotics. Their unparalleled structural and chemical diversities provide a basis to investigate fundamental biological processes while providing access to a tremendous amount of chemical space. There is a pressing need for novel antibiotics with new mode of actions to combat the growing challenge of multidrug resistant pathogens. This review begins with the pioneering discovery and biological activities of platensimycin (PTM) and platencin (PTN), two antibacterial natural products isolated from Streptomyces platensis. The elucidation of their unique biochemical mode of action, structure-activity relationships, and pharmacokinetics is presented to highlight key aspects of their biological activities. It then presents an overview of how microbial genomics has impacted the field of PTM and PTN and revealed paradigm-shifting discoveries in terpenoid biosynthesis, fatty acid metabolism, and antibiotic and antidiabetic therapies. It concludes with a discussion covering the future perspectives of PTM and PTN in regard to natural products discovery, bacterial diterpenoid biosynthesis, and the pharmaceutical promise of PTM and PTN as antibiotics and for the treatment of metabolic disorders. PTM and PTN have inspired new discoveries in chemistry, biology, enzymology, and medicine and will undoubtedly continue to do so.