RESUMEN
The great variety and fascinating complexity of terpenoid skeletons are achieved through different cyclizations catalyzed by terpene cyclases. Here, we report a sesquiterpene cyclase (MfdS) from Aspergillus ustus for the formation of malfilanol D, a member of the group of biochemically less investigated sesquiterpenes with a bicyclo[5.4.0]undecane skeleton. Feeding 13C-labeled acetates in Aspergillus nidulans with the mfdS sequence provides evidence for a C-1 to C-10 cyclization with subsequent 1,2-alkyl and 1,2-hydride shifts in the formation of the 6/7-fused rings.
Asunto(s)
Aspergillus , Sesquiterpenos , Aspergillus/química , Aspergillus/metabolismo , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Estructura Molecular , Ciclización , Alcanos/química , Alcanos/metabolismo , Aspergillus nidulans/metabolismo , Aspergillus nidulans/química , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/metabolismoRESUMEN
Medicinal compounds from plants include bicyclo[3.3.1]nonane derivatives, the majority of which are polycyclic polyprenylated acylphloroglucinols (PPAPs). Prototype molecules are hyperforin, the antidepressant constituent of St. John's wort, and garcinol, a potential anticancer compound. Their complex structures have inspired innovative chemical syntheses, however, their biosynthesis in plants is still enigmatic. PPAPs are divided into two subclasses, named type A and B. Here we identify both types in Hypericum sampsonii plants and isolate two enzymes that regiodivergently convert a common precursor to pivotal type A and B products. Molecular modelling and substrate docking studies reveal inverted substrate binding modes in the two active site cavities. We identify amino acids that stabilize these alternative binding scenarios and use reciprocal mutagenesis to interconvert the enzymatic activities. Our studies elucidate the unique biochemistry that yields type A and B bicyclo[3.3.1]nonane cores in plants, thereby providing key building blocks for biotechnological efforts to sustainably produce these complex compounds for preclinical development.
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Hypericum , Hypericum/metabolismo , Hypericum/genética , Hypericum/química , Compuestos Bicíclicos con Puentes/metabolismo , Compuestos Bicíclicos con Puentes/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Simulación del Acoplamiento Molecular , Floroglucinol/metabolismo , Floroglucinol/análogos & derivados , Floroglucinol/química , Alcanos/metabolismo , Alcanos/química , Dominio Catalítico , Terpenos/metabolismo , Terpenos/química , Modelos MolecularesRESUMEN
Biosafety of engineered bacteria as living therapeutics requires a tight regulation to control the specific delivery of protein effectors, maintaining minimum leakiness in the uninduced (OFF) state and efficient expression in the induced (ON) state. Here, we report a three repressors (3R) genetic circuit that tightly regulates the expression of multiple tac promoters (Ptac) integrated in the chromosome of E. coli and drives the expression of a complex type III secretion system injectisome for therapeutic protein delivery. The 3R genetic switch is based on the tetracycline repressor (TetR), the non-inducible lambda repressor cI (ind-) and a mutant lac repressor (LacIW220F ) with higher activity. The 3R switch was optimized with different protein translation and degradation signals that control the levels of LacIW220F . We demonstrate the ability of an optimized switch to fully repress the strong leakiness of the Ptac promoters in the OFF state while triggering their efficient activation in the ON state with anhydrotetracycline (aTc), an inducer suitable for in vivo use. The implementation of the optimized 3R switch in the engineered synthetic injector E. coli (SIEC) strain boosts expression of injectisomes upon aTc induction, while maintaining a silent OFF state that preserves normal growth in the absence of the inducer. Since Ptac is a commonly used promoter, the 3R switch may have multiple applications for tight control of protein expression in E. coli. In addition, the modularity of the 3R switch may enable its tuning for the control of Ptac promoters with different inducers.
Asunto(s)
Compuestos Bicíclicos con Puentes , Escherichia coli , Tiadiazoles , Escherichia coli/genética , Escherichia coli/metabolismo , Regiones Promotoras Genéticas , Compuestos Bicíclicos con Puentes/metabolismo , Represoras Lac/genética , Represoras Lac/metabolismoRESUMEN
Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) has emerged as a topic of great interest. Despite the presence of O-GlcNAc on hundreds of proteins within cells, only two enzymes regulate this modification. One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase that has a deep active site cleft in which diverse substrates are accommodated. Chemical tools to control OGA are emerging as essential resources for helping to decode the biochemical and cellular functions of the O-GlcNAc pathway. Here we describe rationally designed bicyclic thiazolidine inhibitors that exhibit superb selectivity and picomolar inhibition of human OGA. Structures of these inhibitors in complex with human OGA reveal the basis for their exceptional potency and show that they extend out of the enzyme active site cleft. Leveraging this structure, we create a high affinity chemoproteomic probe that enables simple one-step purification of endogenous OGA from brain and targeted proteomic mapping of its post-translational modifications. These data uncover a range of new modifications, including some that are less-known, such as O-ubiquitination and N-formylation. We expect that these inhibitors and chemoproteomics probes will prove useful as fundamental tools to decipher the mechanisms by which OGA is regulated and directed to its diverse cellular substrates. Moreover, the inhibitors and structures described here lay out a blueprint that will enable the creation of chemical probes and tools to interrogate OGA and other carbohydrate active enzymes.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Compuestos Bicíclicos con Puentes/química , Inhibidores Enzimáticos/química , Histona Acetiltransferasas/metabolismo , Hialuronoglucosaminidasa/metabolismo , Secuencia de Aminoácidos , Encéfalo/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/metabolismo , Histona Acetiltransferasas/antagonistas & inhibidores , Humanos , Hialuronoglucosaminidasa/antagonistas & inhibidores , Espectrometría de Masas , Péptidos/análisis , Péptidos/química , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Relación Estructura-Actividad , Tiazolidinas/química , Tiazolidinas/metabolismo , Cadena alfa de beta-Hexosaminidasa/antagonistas & inhibidores , Cadena alfa de beta-Hexosaminidasa/metabolismoRESUMEN
Bacterial infections can cause serious problems that threaten public health over a long period of time. Moreover, the continuous emergence of drug-resistant bacteria necessitates the development of novel antibacterial agents. D-alanyl-D-alanine ligase (Ddl) is an indispensable adenosine triphosphate-dependent bacterial enzyme involved in the biosynthesis of peptidoglycan precursor, which catalyzes the ligation of two D-alanine molecules into one D-alanyl-D-alanine dipeptide. This dipeptide is an essential component of the intracellular peptidoglycan precursor, uridine diphospho-N-acetylmuramic acid (UDP-MurNAc)-pentapeptide, that maintains the integrity of the bacterial cell wall by cross-linking the peptidoglycan chain, and is crucial for the survival of pathogens. Consequently, Ddl is expected to be a promising target for the development of antibacterial agents. In this review, we present a brief introduction regarding the structure and function of Ddl, as well as an overview of the various Ddl inhibitors currently being used as antibacterial agents, specifically highlighting their inhibitory activities, structure-activity relationships and mechanisms of action.
Asunto(s)
Antibacterianos/química , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Péptido Sintasas/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Compuestos Bicíclicos con Puentes/química , Compuestos Bicíclicos con Puentes/metabolismo , Compuestos Bicíclicos con Puentes/farmacología , Inhibidores Enzimáticos/metabolismo , Compuestos Heterocíclicos con 1 Anillo/química , Compuestos Heterocíclicos con 1 Anillo/metabolismo , Compuestos Heterocíclicos con 1 Anillo/farmacología , Péptido Sintasas/metabolismo , Peptidoglicano/biosíntesis , Relación Estructura-ActividadRESUMEN
Gabapentinoids such as gabapentin and pregabalin, which bind specifically to the α2δ subunit of voltage-gated Ca2+ channels, are used for first-line treatment of neuropathic pain. Here, we examined the analgesic effect of mirogabalin besilate (referred to simply as mirogabalin), a novel gabapentinoid, focusing on its action on the spinal cord and the descending noradrenergic pain inhibitory system. When administered systemically (10 and 30 mg/kg, intraperitoneally (i.p.)) and locally (10 and 30 µg, intracerebroventricularly (i.c.v.) or intrathecally (i.t.)) to mice, mirogabalin was found to exert analgesic effects on thermal (plantar test) and mechanical (von Frey test) hypersensitivity developing after partial sciatic nerve ligation. Notably, its analgesic effects (30 mg/kg, i.p. and 30 µg, i.c.v.) disappeared in mice pretreated with yohimbine hydrochloride (3 µg, i.t.). Moreover, in mice harboring a mutation in the α2δ-1 subunit resulting in substitution of arginine at position 217 with alanine to prevent gabapentinoid binding (R217A mutant mice), the analgesic effects of pregabalin and mirogabalin (30 µg, i.c.v., respectively) on mechanical hypersensitivity were almost completely suppressed. These results clearly demonstrate that mirogabalin also operates via the descending noradrenergic system, and that binding to the α2δ-1 subunit supraspinally is essential for the pain relief effect of gabapentinoids.
Asunto(s)
Analgésicos , Compuestos Bicíclicos con Puentes/administración & dosificación , Compuestos Bicíclicos con Puentes/farmacología , Canales de Calcio/metabolismo , Neuralgia/tratamiento farmacológico , Norepinefrina/metabolismo , Animales , Compuestos Bicíclicos con Puentes/metabolismo , Canales de Calcio/genética , Vías de Administración de Medicamentos , Femenino , Masculino , Ratones , Ratones Endogámicos , Ratones Mutantes , Mutación , Unión Proteica/efectos de los fármacosRESUMEN
Photosensitization is a novel environmentally friendly technology with promising applications in the food industry to extend food shelf life. In this study, the natural food dye curcumin, when combined with visible light (430 nm), was shown to be an effective photosensitizer against the common phytopathogenic fungi Botrytis cinerea (the cause of grey mould). Production of the associated phytotoxic metabolites botrydial and dihydrobotrydial was measured by our newly developed and validated HRAM UPLC-MS/MS method, and was also shown to be reduced by this treatment. With a light dose of 120 J/cm2, the reduction in spore viability was directly proportional to curcumin concentrations, and the overall concentration of both botrydial and dihydrobotrydial also decreased with increasing curcumin concentration above 200 µM. With curcumin concentrations above 600 µM, the percentage reduction in fungal spores was close to 100%. When the dye concentration was increased to 800 µM, the spores were completely inactive and neither botrydial nor dihydrobotrydial could be detected. These results suggest that curcumin-mediated photosensitization is a potentially effective method to control B. cinerea spoilage, and also to reduce the formation of these phytotoxic botryane secondary metabolites.
Asunto(s)
Botrytis/efectos de los fármacos , Curcumina/farmacología , Fragaria/microbiología , Frutas/microbiología , Fungicidas Industriales/farmacología , Fármacos Fotosensibilizantes/farmacología , Esporas Fúngicas/efectos de los fármacos , Aldehídos/metabolismo , Aldehídos/toxicidad , Botrytis/crecimiento & desarrollo , Botrytis/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Compuestos Bicíclicos con Puentes/toxicidad , Cromatografía Liquida , Microbiología de Alimentos , Conservación de Alimentos , Metabolismo Secundario , Espectrometría de Masa por Ionización de Electrospray , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/metabolismo , Espectrometría de Masas en TándemRESUMEN
The dissipation behaviors and exposure risks of individual and joint application of procymidone and carbendazim in greenhouse strawberries were studied. The initial concentrations were similar after individual or joint applications, while the dissipation half-lives and finial concentrations were significantly different. After joint application, the dissipation half-lives of procymidone and carbendazim were 12.9 and 16.0 days, respectively, which were about 1.8 times higher than those after individual application. Furthermore, the final residues under joint application condition were 1.8-3.5 times higher than those under individual application condition. The joint application decreased the dissipation rates of procymidone or carbendazim in strawberries, and increased the final residue concentrations. The dietary intake risks of procymidone and carbendazim (whether applied individually or jointly) were no higher than 0.12, which were acceptable for human health. This work would shed a light for the guidance of the joint application and risk assessment of the typical fungicides in strawberry.
Asunto(s)
Bencimidazoles/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Carbamatos/metabolismo , Fragaria/química , Bencimidazoles/análisis , Compuestos Bicíclicos con Puentes/análisis , Carbamatos/análisis , Cromatografía Líquida de Alta Presión , Fragaria/metabolismo , Efecto Invernadero , Semivida , Humanos , Residuos de Plaguicidas/análisis , Medición de Riesgo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Multidrug-resistant (MDR) pathogens pose a significant public health threat. A major mechanism of resistance expressed by MDR pathogens is ß-lactamase-mediated degradation of ß-lactam antibiotics. The diazabicyclooctane (DBO) compounds zidebactam and WCK 5153, recognized as ß-lactam "enhancers" due to inhibition of Pseudomonas aeruginosa penicillin-binding protein 2 (PBP2), are also class A and C ß-lactamase inhibitors. To structurally probe their mode of PBP2 inhibition as well as investigate why P. aeruginosa PBP2 is less susceptible to inhibition by ß-lactam antibiotics compared to the Escherichia coli PBP2, we determined the crystal structure of P. aeruginosa PBP2 in complex with WCK 5153. WCK 5153 forms an inhibitory covalent bond with the catalytic S327 of PBP2. The structure suggests a significant role for the diacylhydrazide moiety of WCK 5153 in interacting with the aspartate in the S-X-N/D PBP motif. Modeling of zidebactam in the active site of PBP2 reveals a similar binding mode. Both DBOs increase the melting temperature of PBP2, affirming their stabilizing interactions. To aid in the design of DBOs that can inhibit multiple PBPs, the ability of three DBOs to interact with P. aeruginosa PBP3 was explored crystallographically. Even though the DBOs show covalent binding to PBP3, they destabilized PBP3. Overall, the studies provide insights into zidebactam and WCK 5153 inhibition of PBP2 compared to their inhibition of PBP3 and the evolutionarily related KPC-2 ß-lactamase. These molecular insights into the dual-target DBOs advance our knowledge regarding further DBO optimization efforts to develop novel potent ß-lactamase-resistant, non-ß-lactam PBP inhibitors.IMPORTANCE Antibiotic resistance is a significant clinical problem. Developing novel antibiotics that overcome known resistance mechanisms is highly desired. Diazabicyclooctane inhibitors such as zidebactam possess this potential as they readily inactivate penicillin-binding proteins, yet cannot be degraded by ß-lactamases. In this study, we characterized the inhibition by diazabicyclooctanes of penicillin-binding proteins PBP2 and PBP3 from Pseudomonas aeruginosa using protein crystallography and biophysical analyses. These structures and analyses help define the antibiotic properties of these inhibitors, explain the decreased susceptibility of P. aeruginosa PBP2 to be inhibited by ß-lactam antibiotics, and provide insights that could be used for further antibiotic development.
Asunto(s)
Antibacterianos/farmacología , Compuestos de Azabiciclo/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Ciclooctanos/farmacología , Octanos/farmacología , Proteínas de Unión a las Penicilinas/química , Proteínas de Unión a las Penicilinas/metabolismo , Piperidinas/farmacología , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/metabolismo , Compuestos de Azabiciclo/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Cristalización , Ciclooctanos/metabolismo , Pruebas de Sensibilidad Microbiana , Octanos/metabolismo , Proteínas de Unión a las Penicilinas/antagonistas & inhibidores , Piperidinas/metabolismo , Unión Proteica , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Inhibidores de beta-Lactamasas/farmacologíaRESUMEN
Classical terpenoid biosynthesis involves the cyclization of the linear prenyl pyrophosphate precursors geranyl-, farnesyl-, or geranylgeranyl pyrophosphate (GPP, FPP, GGPP) and their isomers, to produce a huge number of natural compounds. Recently, it was shown for the first time that the biosynthesis of the unique homo-sesquiterpene sodorifen by Serratia plymuthica 4Rx13 involves a methylated and cyclized intermediate as the substrate of the sodorifen synthase. To further support the proposed biosynthetic pathway, we now identified the cyclic prenyl pyrophosphate intermediate pre-sodorifen pyrophosphate (PSPP). Its absolute configuration (6R,7S,9S) was determined by comparison of calculated and experimental CD-spectra of its hydrolysis product and matches with those predicted by semi-empirical quantum calculations of the reaction mechanism. In silico modeling of the reaction mechanism of the FPP C-methyltransferase (FPPMT) revealed a SN2 mechanism for the methyl transfer followed by a cyclization cascade. The cyclization of FPP to PSPP is guided by a catalytic dyad of H191 and Y39 and involves an unprecedented cyclopropyl intermediate. W46, W306, F56, and L239 form the hydrophobic binding pocket and E42 and H45 complex a magnesium cation that interacts with the diphosphate moiety of FPP. Six additional amino acids turned out to be essential for product formation and the importance of these amino acids was subsequently confirmed by site-directed mutagenesis. Our results reveal the reaction mechanism involved in methyltransferase-catalyzed cyclization and demonstrate that this coupling of C-methylation and cyclization of FPP by the FPPMT represents an alternative route of terpene biosynthesis that could increase the terpenoid diversity and structural space.
Asunto(s)
Proteínas Bacterianas/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Metiltransferasas/metabolismo , Octanos/metabolismo , Serratia/enzimología , Secuencias de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Biocatálisis , Compuestos Bicíclicos con Puentes/química , Clonación Molecular , Ciclización , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Octanos/química , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serratia/química , Serratia/genética , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Especificidad por SustratoRESUMEN
Fungi of the genus Botrytis infect >1,400 plant species and cause losses in many crops. Besides the broad host range pathogen Botrytis cinerea, most other species are restricted to a single host. Long-read technology was used to sequence genomes of eight Botrytis species, mostly pathogenic on Allium species, and the related onion white rot fungus, Sclerotium cepivorum. Most assemblies contained <100 contigs, with the Botrytis aclada genome assembled in 16 gapless chromosomes. The core genome and pan-genome of 16 Botrytis species were defined and the secretome, effector, and secondary metabolite repertoires analyzed. Among those genes, none is shared among all Allium pathogens and absent from non-Allium pathogens. The genome of each of the Allium pathogens contains 8-39 predicted effector genes that are unique for that single species, none stood out as potential determinant for host specificity. Chromosome configurations of common ancestors of the genus Botrytis and family Sclerotiniaceae were reconstructed. The genomes of B. cinerea and B. aclada were highly syntenic with only 19 rearrangements between them. Genomes of Allium pathogens were compared with ten other Botrytis species (nonpathogenic on Allium) and with 25 Leotiomycetes for their repertoire of secondary metabolite gene clusters. The pattern was complex, with several clusters displaying patchy distribution. Two clusters involved in the synthesis of phytotoxic metabolites are at distinct genomic locations in different Botrytis species. We provide evidence that the clusters for botcinic acid production in B. cinerea and Botrytis sinoallii were acquired by horizontal transfer from taxa within the same genus.
Asunto(s)
Allium/microbiología , Botrytis/genética , Genoma Fúngico , Especificidad del Huésped/genética , Filogenia , Aldehídos/metabolismo , Botrytis/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Familia de Multigenes , Policétidos/metabolismo , Metabolismo Secundario/genética , SinteníaRESUMEN
The role of the sesquiterpene botrydial in the interaction of the phytopathogenic fungus Botrytis cinerea and plant-associated bacteria was analyzed. From a collection of soil and phyllospheric bacteria, nine strains sensitive to growth-inhibition by B. cinerea were identified. B. cinerea mutants unable to produce botrydial caused no bacterial inhibition, thus demonstrating the inhibitory role of botrydial. A taxonomic analysis showed that these bacteria corresponded to different Bacillus species (six strains), Pseudomonas yamanorum (two strains) and Erwinia aphidicola (one strain). Inoculation of WT and botrydial non-producing mutants of B. cinerea along with Bacillusamyloliquefaciens strain MEP218 in soil demonstrated that both microorganisms exert reciprocal inhibitory effects; the inhibition caused by B. cinerea being dependent on botrydial production. Moreover, botrydial production was modulated by the presence of B. amyloliquefaciens MEP218 in confrontation assays in vitro. Purified botrydial in turn, inhibited growth of Bacillus strains in vitro and cyclic lipopeptide (surfactin) production by B. amyloliquefaciens MEP218. As a whole, results demonstrate that botrydial confers B. cinerea the ability to inhibit potential biocontrol bacteria of the genus Bacillus. We propose that resistance to botrydial could be used as an additional criterion for the selection of biocontrol agents of plant diseases caused by B. cinerea.
Asunto(s)
Aldehídos/farmacología , Antibiosis , Bacillus/fisiología , Fenómenos Fisiológicos Bacterianos , Botrytis/fisiología , Compuestos Bicíclicos con Puentes/farmacología , Microbiología del Suelo , Aldehídos/metabolismo , Bacillus/efectos de los fármacos , Bacillus/crecimiento & desarrollo , Bacillus amyloliquefaciens/efectos de los fármacos , Bacillus amyloliquefaciens/crecimiento & desarrollo , Bacillus amyloliquefaciens/fisiología , Bacterias/crecimiento & desarrollo , Botrytis/crecimiento & desarrollo , Compuestos Bicíclicos con Puentes/metabolismo , Lipopéptidos/metabolismo , Péptidos Cíclicos/metabolismoRESUMEN
Mitochondrial free calcium is critically linked to the regulation of cellular metabolism. Free ionic calcium concentration within these organelles is determined by the interplay between two processes: exchange across the mitochondrial inner membrane and calcium-buffering within the matrix. During stimulated calcium uptake, calcium is primarily buffered by orthophosphate, preventing calcium toxicity while allowing for well-regulated yet elevated calcium loads. However, if limited to orthophosphates only, this buffering system is expected to lead to the irreversible formation of insoluble precipitates, which are not observed in living cells, under physiological conditions. Here, we demonstrate that the regulation of free mitochondrial calcium requires the presence of free inorganic polyphosphate (polyP) within the organelle. We found that the overexpression of a mitochondrial-targeted enzyme hydrolyzing polyP leads to the loss of the cellular ability to maintain elevated calcium concentrations within the organelle, following stimulated cytoplasmic signal. We hypothesize that the presence of polyP prevents the formation of calcium-phosphate insoluble clusters, allowing for the maintenance of elevated free calcium levels, during stimulated calcium uptake.
Asunto(s)
Calcio/metabolismo , Mitocondrias/metabolismo , Polifosfatos/farmacología , Adenosina Trifosfato/farmacología , Benzoatos/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Señalización del Calcio/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cicloheptanos/metabolismo , Células HEK293 , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Modelos Biológicos , Rojo de Rutenio/metabolismo , Sesquiterpenos/metabolismoRESUMEN
Studies on the substrate selectivity of recombinant ferrous-iron- and 2-oxoglutarate-dependent proline hydroxylases (PHs) reveal that they can catalyse the production of dihydroxylated 5-, 6-, and 7-membered ring products, and can accept bicyclic substrates. Ring-substituted substrate analogues (such hydroxylated and fluorinated prolines) are accepted in some cases. The results highlight the considerable, as yet largely untapped, potential for amino acid hydroxylases and other 2OG oxygenases in biocatalysis.
Asunto(s)
Compuestos Bicíclicos con Puentes/metabolismo , Prolil Hidroxilasas/metabolismo , Biocatálisis , Compuestos Bicíclicos con Puentes/química , Estructura Molecular , Prolil Hidroxilasas/química , Especificidad por SustratoRESUMEN
Chronic inflammation plays an important role in the mechanisms underpinning the development of anesthesia-induced cognitive dysfunction. However, less is known about how anesthesia causes inflammation. One possibility is that the inflammation is related to alteration of the activity of the alpha 7 nicotinic acetylcholine receptor cholinergic anti-inflammatory pathway. This study analyzed the effect of sevoflurane administration on the cognitive function by using a novel object recognition test and Y-maze test, and on acetylcholinesterase activity and expression in hippocampal tissue by using an acetylcholinesterase assay kit and quantitative real-time PCR. This study also evaluated the effect of alpha 7 nicotinic acetylcholine receptor agonist PNU-282987 and antagonist methyllycaconitine on cognitive function and the level of hippocampal tumor necrosis factor-α in aged rats exposed to sevoflurane anesthesia. We found that 3% sevoflurane significantly impaired cognitive function and increased acetylcholinesterase activity by upregulating its expression in hippocampal tissue. Sevoflurane-induced impairment of cognitive function was significantly rescued by PNU-282987 but aggravated by methyllycaconitine. In addition to impairment of cognitive function, sevoflurane also significantly increased tumor necrosis factor-α level in plasma and hippocampal tissue. Similarly, this sevoflurane-induced change of tumor necrosis factor-α level in rats was antagonized by PNU-282987 but amplified by methyllycaconitine. In conclusion, our data show that the development of inflammation in sevoflurane-induced cognitive decline is associated with the downregulation of alpha 7 nicotinic acetylcholine receptor cholinergic anti-inflammatory pathway in aged rats.
Asunto(s)
Inflamación/fisiopatología , Neuroinmunomodulación/efectos de los fármacos , Sevoflurano/farmacología , Acetilcolinesterasa/metabolismo , Anestésicos por Inhalación/farmacología , Animales , Benzamidas/metabolismo , Encéfalo/efectos de los fármacos , Compuestos Bicíclicos con Puentes/metabolismo , Cognición/fisiología , Disfunción Cognitiva/metabolismo , Hipocampo/efectos de los fármacos , Inflamación/inmunología , Masculino , Éteres Metílicos/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
BACKGROUND: Serratia plymuthica WS3236 was selected for whole genome sequencing based on preliminary genetic and chemical screening indicating the presence of multiple natural product pathways. This led to the identification of a putative sodorifen biosynthetic gene cluster (BGC). The natural product sodorifen is a volatile organic compound (VOC) with an unusual polymethylated hydrocarbon bicyclic structure (C16H26) produced by selected strains of S. plymuthica. The BGC encoding sodorifen consists of four genes, two of which (sodA, sodB) are homologs of genes encoding enzymes of the non-mevalonate pathway and are thought to enhance the amounts of available farnesyl pyrophosphate (FPP), the precursor of sodorifen. Proceeding from FPP, only two enzymes are necessary to produce sodorifen: an S-adenosyl methionine dependent methyltransferase (SodC) with additional cyclisation activity and a terpene-cyclase (SodD). Previous analysis of S. plymuthica found sodorifen production titers are generally low and vary significantly among different producer strains. This precludes studies on the still elusive biological function of this structurally and biosynthetically fascinating bacterial terpene. RESULTS: Sequencing and mining of the S. plymuthica WS3236 genome revealed the presence of 38 BGCs according to antiSMASH analysis, including a putative sodorifen BGC. Further genome mining for sodorifen and sodorifen-like BGCs throughout bacteria was performed using SodC and SodD as queries and identified a total of 28 sod-like gene clusters. Using direct pathway cloning (DiPaC) we intercepted the 4.6 kb candidate sodorifen BGC from S. plymuthica WS3236 (sodA-D) and transformed it into Escherichia coli BL21. Heterologous expression under the control of the tetracycline inducible PtetO promoter firmly linked this BGC to sodorifen production. By utilizing this newly established expression system, we increased the production yields by approximately 26-fold when compared to the native producer. In addition, sodorifen was easily isolated in high purity by simple head-space sampling. CONCLUSIONS: Genome mining of all available genomes within the NCBI and JGI IMG databases led to the identification of a wealth of sod-like pathways which may be responsible for producing a range of structurally unknown sodorifen analogs. Introduction of the S. plymuthica WS3236 sodorifen BGC into the fast-growing heterologous expression host E. coli with a very low VOC background led to a significant increase in both sodorifen product yield and purity compared to the native producer. By providing a reliable, high-level production system, this study sets the stage for future investigations of the biological role and function of sodorifen and for functionally unlocking the bioinformatically identified putative sod-like pathways.
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Compuestos Bicíclicos con Puentes/metabolismo , Escherichia coli/metabolismo , Familia de Multigenes , Octanos/metabolismo , Serratia/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Clonación Molecular , Biología Computacional , Escherichia coli/genética , Genoma Bacteriano , Pirofosfatasas/metabolismoRESUMEN
Microbial consortia, which degrade branched, long-chain hydrocarbons, can be regarded as a promising source of novel enzymes for the stereo- and regio-selective oxyfunctionalization of hydrocarbons. The hydrocarbon-degrading bacterium Pimelobacter sp. Bb-B was isolated from the consortium associated with the colonial hydrocarbon-excreting microalga Botryococcus braunii. Three new type II flavoprotein monooxygenases (type II FMOs) from this bacterium have been made available in recombinant form through cloning and overexpression in an E. coli host organism. These enzymes (PsFMO_A-C) were characterized in terms of their capability of catalyzing Baeyer-Villiger oxidations with distinct substrates. The highest activity was detected when utilizing camphor and bicyclo[3.2.0]hept-2-en-6-one as substrate in combination with PsFMO_A as the most promising enzyme. Furthermore, synthetic biotransformations with 5 mM of the substrate bicyclo[3.2.0]hept-2-en-6-one, formate and formate dehydrogenase for in situ-cofactor recycling were conducted with this enzyme, leading to a substrate consumption of 85% after 66 h and excellent enantioselectivity of 99% ee for the (1R,5S)-enantiomer. Additionally, an alternative in situ-cofactor recycling based on the use of microalgae for in situ-production of formate from carbon dioxide, water and light together with a formate dehydrogenase was combined successfully with the enzyme PsFMO_A, leading to a substrate consumption of 94% and an enantioselectivity of >99% ee for the so-called "normal lactone"-enantiomer with the absolute configuration 1R,5S.
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Actinobacteria/enzimología , Proteínas Bacterianas , Oxigenasas de Función Mixta , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Compuestos Bicíclicos con Puentes/metabolismo , Alcanfor/metabolismo , Escherichia coli/genética , Flavoproteínas , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Oxidación-Reducción , EstereoisomerismoRESUMEN
The PYR/PYL/RCAR protein families have recently emerged as receptors of the phytohormone abscisic acid (ABA, 1), which regulates plant responses to environmental stress. These families have multiple members with different physiological actions, and so selective agonists or antagonists are needed both as tools to elucidate functional differences and as lead compounds for agrochemicals. We previously identified RK460 (rac-3 a) as a PYR1-selective antagonist, and showed that it possesses five stereocenters on a 6,5-cis-bicyclo skeleton. Here, we synthesized all the stereoisomers of RK460 and evaluated their activity towards a panel of receptors. Relative stereochemistry as well as absolute stereochemistry was important for selective action.
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Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/antagonistas & inhibidores , Arabidopsis/efectos de los fármacos , Compuestos Bicíclicos con Puentes/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Reguladores del Crecimiento de las Plantas/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Bicíclicos con Puentes/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Reguladores del Crecimiento de las Plantas/síntesis química , Reguladores del Crecimiento de las Plantas/química , Receptores de Superficie Celular/metabolismo , EstereoisomerismoRESUMEN
We examined the possible role of monoterpene ß-pinene in providing protection against Cr(VI) toxicity in maize (Zea mays). Treatment with ß-pinene (10 µM) significantly alleviated Cr(VI) accumulation and recuperated Cr(VI) caused decline in root and coleoptile growth in maize. ß-Pinene addition caused a decline in Cr(VI)-induced accumulation of superoxide anion, hydroxyl ion, hydrogen peroxide and confirmed by in-situ detection of ROS using histochemical localization. It suggested that the ß-pinene quenches/neutralizes enhanced ROS generated under Cr(VI) exposure. ß-Pinene also reduced Cr(VI)-induced electrolyte leakage, thereby suggesting its role in membrane stabilization. Further, ß-pinene regulated the activity of scavenging enzymes, thereby suggesting a role in modulating Cr(VI)-induced oxidative damage. In conclusion, our results suggest that the addition of ß-pinene has a protective role against Cr(VI) stress and provides resistance to maize against Cr(VI) toxicity.
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Compuestos Bicíclicos con Puentes/metabolismo , Cromo/toxicidad , Monoterpenos/metabolismo , Zea mays/fisiología , Antioxidantes , Monoterpenos Bicíclicos , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno , Superóxidos , Zea mays/efectos de los fármacosRESUMEN
Sodorifen is the major volatile of Serratia plymuthica 4Rx13. It is assumed to be a long-distance communication signal. However, so far the emission patterns of sodorifen had been studied using mono-cultures of S. plymuthica 4Rx13 neglecting that in natura bacteria live in communities. Here, we show that the structured co-cultivation of S. plymuthica 4Rx13 and Bacillus subtilis B2g in a low-diversity model community grown under nutrient-rich conditions led to quantitative changes in sodorifen emission compared to self-paired mono-cultivations. Co-culturing revealed a decreased emission of sodorifen (50%) during exponential growth phase, whereas in the late stationary stage of growth, the amount of headspace sodorifen was increased compared to self-paired mono-cultivation (217% at 500 h of cultivation). Six other compounds that are most probably related to sodorifen or are isomers showed similar emission patterns. These data indicated that S. plymuthica 4Rx13 enhances its communication signal sodorifen as a consequence of interaction with B. subtilis B2g.