RESUMEN
The dodecameric vanadium-dependent bromoperoxidase from Corallina officinalis has been cloned and over-expressed in Escherichia coli. However, the enzyme was found to be predominantly in the form of inclusion bodies. This protein presents a challenging target for refolding, both due to the size (768kDa) and quaternary structure (12x64kDa). Successful refolding conditions have been established which result in an increase in the final yield of active bromoperoxidase from 0.5mg to 40mg per litre of culture. The refolded protein has been characterised and compared to the native enzyme and was shown to be stable at temperatures of 80 degrees C, over a pH range 5.5-10 and in organic solvents such as ethanol, acetonitrile, methanol, and acetone. The novel refolding approach reported in this paper opens up the full potential of this versatile enzyme for use in large scale biotransformation studies.
Asunto(s)
Eucariontes/enzimología , Yoduro Peroxidasa/química , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Combinación de Medicamentos , Escherichia coli/genética , Expresión Génica , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , Biología Marina , Datos de Secuencia Molecular , Aceites , Fenoles , Polímeros , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
Sema7A is a recently described member of the semaphorin family that is associated with the cell surface via a glycophosphatidylinositol linkage. This study examined the mRNA expression and biological properties of this protein. Although the expression of Sema7A was demonstrated in lymphoid and myeloid cells, no stimulation of cytokine production or proliferation was evident in B or T cells. In contrast, Sema7A is an extremely potent monocyte activator, stimulating chemotaxis at 0.1 pm and inflammatory cytokine production (interleukin-1 (IL-1beta), tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-8) and superoxide release at 1-10 pm. Sema7A is less effective at stimulating neutrophils. Sema7A also significantly increases granulocyte-macrophage colony-stimulating factor (GM-CSF) production from monocytes but has no consistent effect on IL-10, IL-12 or IL-18. Sema7A can also induce monocytes toward a dendritic cell morphology. Sema7A is expressed in monocytes and probably released through proteolysis and acts as a very potent autocrine activator of these cells.
Asunto(s)
Antígenos CD/farmacología , Glicoproteínas/farmacología , Lipoproteínas/farmacología , Monocitos/inmunología , Semaforinas , Animales , Antígenos CD/análisis , Antígenos CD/genética , Células CHO , Células Cultivadas , Quimiotaxis de Leucocito , Clonación Molecular , Cricetinae , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Proteínas Ligadas a GPI , Glicoproteínas/genética , Humanos , Lipoproteínas/genética , Monocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , ARN Mensajero/biosíntesis , Superóxidos/metabolismo , Polimerasa Taq/metabolismoRESUMEN
Bacterial response regulators are attractive targets for antibacterial drug development, yet random screening against these targets has failed as yet to identify chemicals that constitute viable leads. Alternative methods to provide leads for drug development based on identification and optimization of low affinity ligands from NMR screens have been described. However, leads from these processes still require verification in a bioassay, which is often problematic if compounds have unfavorable optical and solubility properties. A simple method, based on using NMR to observe the activity of the target, is described. It has the advantages of being able to characterize both low affinity leads and a wider selection of compounds in a structure activity relationships series, without the problems affecting a fluorescence assay. In this example we use (31)P to monitor the turnover of a bacterial response regulator, but the generic approach could be applied to other nuclei and thus a range of biological systems.
Asunto(s)
Proteínas Bacterianas/metabolismo , Calcio/metabolismo , Magnesio/metabolismo , Proteínas de la Membrana/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/aislamiento & purificación , Catálisis , Ácido Edético/metabolismo , Ligandos , Proteínas de la Membrana/aislamiento & purificación , Proteínas Quimiotácticas Aceptoras de Metilo , Peso Molecular , Isótopos de Fósforo/metabolismo , Fosforilación/efectos de los fármacosRESUMEN
SB-219383 and its analogues are a class of potent and specific inhibitors of bacterial tyrosyl-tRNA synthetases. Crystal structures of these inhibitors have been solved in complex with the tyrosyl-tRNA synthetase from Staphylococcus aureus, the bacterium that is largely responsible for hospital-acquired infections. The full-length enzyme yielded crystals that diffracted to 2.8 A resolution, but a truncated version of the enzyme allowed the resolution to be extended to 2.2 A. These inhibitors not only occupy the known substrate binding sites in unique ways, but also reveal a butyl binding pocket. It was reported that the Bacillus stearothermophilus TyrRS T51P mutant has much increased catalytic activity. The S. aureus enzyme happens to have a proline at position 51. Therefore, our structures may contribute to the understanding of the catalytic mechanism and provide the structural basis for designing novel antimicrobial agents.
Asunto(s)
Inhibidores Enzimáticos/química , Staphylococcus aureus/enzimología , Tirosina-ARNt Ligasa/química , Secuencia de Aminoácidos , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Cristalización , Cristalografía por Rayos X , Dipéptidos/química , Dipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Furanos/química , Furanos/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Piperidinas/química , Piperidinas/farmacología , Conformación Proteica , Homología de Secuencia de Aminoácido , Tirosina-ARNt Ligasa/antagonistas & inhibidoresRESUMEN
An SAR study of a screening lead has led to the identification of 2,9-disubstituted 1,2,3,4-tetrahydropyrido[3,4-b]indoles as inhibitors of Staphylococcus aureus enoyl acyl carrier protein reductase (FabI).
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Antibacterianos/química , Enoil-ACP Reductasa (NADH) , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli , Acido Graso Sintasa Tipo II , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Triclosán/farmacologíaRESUMEN
1,4-Disubstituted imidazole inhibitors of Staphylococcus aureus and Escherichia coli enoyl acyl carrier protein reductase (FabI) have been identified. Crystal structure data shows the inhibitor 1 bound in the enzyme active site of E. coli FabI.
Asunto(s)
Antibacterianos/farmacología , Imidazoles/farmacología , Oxidorreductasas/antagonistas & inhibidores , Antibacterianos/síntesis química , Antibacterianos/química , Enoil-ACP Reductasa (NADH) , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli , Acido Graso Sintasa Tipo II , Imidazoles/síntesis química , Imidazoles/química , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/efectos de los fármacosRESUMEN
Phenylalanyl-tRNA synthetase (pheRS) is unique among aminoacyl tRNA synthetases in that it is a heterotetrameric enzyme composed of two alpha-subunits and two larger beta-subunits. In prokaryotes, the alpha- and beta-subunits of pheRS are encoded by the genes pheS and pheT, respectively. In this report we describe the isolation of a DNA fragment (3.52 kb) containing the pheS and pheT genes from a Staphylococcus aureus (WCUH29) genomic DNA library. Both genes, found as a part of transcriptional operon, were predicted to encode polypeptides which showed strong primary and structural similarity to prokaryotic phenylalanyl-tRNA synthetase alpha- and beta- subunits. We describe the high-level overexpression and purification of recombinant S. aureus pheRS using pheS and pheT genes as part of an artificial operon in Escherichia coli. For comparative analysis we also report a procedure for the purification of native pheRS from S. aureus (Oxford Strain) and demonstrate that Michaelis-Menten parameters for both recombinant and native enzyme, at least for phenylalanine tRNA aminoacylation are comparable.
Asunto(s)
Fenilalanina-ARNt Ligasa/genética , Fenilalanina-ARNt Ligasa/metabolismo , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Haemophilus influenzae/enzimología , Cinética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Operón/genética , Fenilalanina-ARNt Ligasa/química , Fenilalanina-ARNt Ligasa/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Staphylococcus aureus/genética , Streptococcus pneumoniae/enzimología , Relación Estructura-Actividad , Thermus thermophilus/enzimologíaRESUMEN
Five imipenem resistant clinical isolates of Bacteroides fragilis isolated before 1987, were examined to determine if they produced metallo-beta-lactamases. The beta-lactamases produced by the clinical isolates all focused as doublet bands at pl 4.8/4.9, characteristic of the B. fragilis CfiA type metallo-beta-lactamase. Each enzyme had a similar substrate profile and were inhibited by EDTA and activated with zinc sulphate. The sequence of the metallo-beta-lactamase gene from B. fragilis ED262 was determined and confirmed to be a CfiA type beta-lactamase. Consequently, the five isolates examined probably produce a CfiA type beta-lactamase, suggesting that metallo-beta-lactamases were present before the widespread use of carbapenems.
Asunto(s)
Bacteroides fragilis/metabolismo , Imipenem/farmacología , Metales/farmacología , Tienamicinas/farmacología , beta-Lactamasas/biosíntesis , Bacteroides fragilis/aislamiento & purificación , Secuencia de Bases , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Estudios Retrospectivos , Especificidad por Sustrato , Reino UnidoRESUMEN
Seven classes of Streptomyces clavuligerus mutants defective in clavulanic acid (CLA) biosynthesis have been identified and used to clone the chromosomal DNA encoding eight CLA biosynthetic genes. The complete sequences of three and the partial sequences of two of these biosynthetic genes are reported, together with their known or predicted functions.
Asunto(s)
Antibacterianos/biosíntesis , Ácidos Clavulánicos/biosíntesis , Streptococcus/genética , Acetiltransferasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Ácido Clavulánico , Clonación Molecular , ADN Bacteriano/genética , Genes Bacterianos , Prueba de Complementación Genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Mapeo Restrictivo , Ureohidrolasas/genéticaRESUMEN
The extended-spectrum, plasmid-borne beta-lactamase gene blaBIL-1, which was discovered in Escherichia coli, has been cloned. Unusually for a plasmid-borne beta-lactamase, blaBIL-1 encodes a novel class C enzyme and appears to have originated from the chromosomal ampC gene of Citrobacter freundii.
Asunto(s)
Escherichia coli/genética , Genes Bacterianos/genética , Plásmidos/genética , beta-Lactamasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Citrobacter freundii/efectos de los fármacos , Citrobacter freundii/genética , Clonación Molecular , Dermatoglifia del ADN , Cartilla de ADN , Escherichia coli/efectos de los fármacos , Datos de Secuencia Molecular , beta-Lactamasas/biosíntesisRESUMEN
We have cloned and sequenced the Staphylococcus aureus Oxford ileS gene which encodes isoleucyl-tRNA synthetase (Ile-RS), the target for the antibiotic mupirocin. The gene was identified by hybridisation to oligodeoxyribonucleotide probes derived from internal Ile-RS amino acid (aa) sequences. The 2754-bp open reading frame encodes a 918-aa protein of 105 kDa which is homologous to other known Ile-RS from Gram- bacteria, archaebacteria, yeast and protozoa. Motifs which have been implicated in the functioning of the active site are strongly conserved. The gene was engineered for high-level expression in Escherichia coli. Ile-RS overproduction was toxic to the E. coli host, the magnitude of its observed effects being strain-dependent.