RESUMEN
Macrolides are a group of antibiotics structurally characterized by a macrocyclic lactone to which one or several deoxy-sugar moieties are attached. The sugar moieties are transferred to the different aglycones by glycosyltransferases (GTF). The OleI GTF of an oleandomycin producer, Streptomyces antibioticus, catalyzes the inactivation of this macrolide by glycosylation. The product of this reaction was isolated and its structure elucidated. The donor substrate of the reaction was UDP-alpha-D-glucose, but the reaction product showed a beta-glycosidic linkage. The inversion of the anomeric configuration of the transferred sugar and other data about the kinetics of the reaction and primary structure analysis of several GTFs are compatible with a reaction mechanism involving a single nucleophilic substitution at the sugar anomeric carbon in the catalytic center of the enzyme.
Asunto(s)
Antibacterianos/antagonistas & inhibidores , Antibacterianos/química , Glucosiltransferasas/metabolismo , Oleandomicina/antagonistas & inhibidores , Oleandomicina/química , Secuencia de Aminoácidos , Antibacterianos/metabolismo , Conformación de Carbohidratos , Glucosa/química , Glucosiltransferasas/genética , Glicosilación , Espectroscopía de Resonancia Magnética , Modelos Químicos , Oleandomicina/metabolismo , Homología de Secuencia de Aminoácido , Streptomyces antibioticus/enzimología , Streptomyces antibioticus/genéticaRESUMEN
By homology to the mgt gene (encoding a macrolide glycosyltransferase) from Streptomyces lividans, a 3.3-kb DNA fragment from the oleandomycin producer, Streptomyces antibioticus, was cloned and sequenced. Analysis of the sequence revealed the presence of the 3' end of a gene (ORF1) and two complete ORFs (ORF2 and oleD), all of them translationally coupled. The deduced product of the sequenced region of ORF1 contained the typical signature of integral membrane proteins responsible for the translocation of substrates across the membrane. The ORF2 product did not show significant similarity with proteins in databases, but contains an N-terminus leader peptide region characteristic of secreted proteins, and a lipid attachment site motif characteristic of membrane lipoproteins synthesized with a precursor signal peptide. The oleD product showed clear similarity with several UDP-glucuronosyl- and UDP-glycosyl-transferases from different origins and particularly with the mgt gene from S. lividans, and might encode a glycosyltransferase activity capable of inactivating macrolides. It is proposed that these three genes could participate in the intracellular glycosylation of oleandomycin and its secretion during antibiotic production.
Asunto(s)
Genes Bacterianos , Glicosiltransferasas/genética , Familia de Multigenes , Oleandomicina/antagonistas & inhibidores , Streptococcus/genética , Glicosiltransferasas/metabolismo , Datos de Secuencia MolecularRESUMEN
The resistance mechanism of Escherichia coli BM2506 to macrolides was found to be due to inactivation. Inactivated oleandomycin was identified as oleandomycin 2'-phosphate by thin-layer chromatography. A new type of macrolide-phosphorylating enzyme, macrolide 2'-phosphotransferase type II (MPH(2')II), was detected, purified 95-fold and its enzymological properties investigated. MPH(2')II was a constitutive intracellular enzyme which showed high levels of activity with both 14-member-ring and 16-member-ring macrolides. The optimum pH for the inactivation of oleandomycin was 8.2 and the optimum temperature of the reaction was 40 degrees C. Enzyme activity was lost by heat treatment at 60 degrees C for 1 min. The isoelectric point and M(r) of the enzyme were 5.3 and 48,000, respectively. Purine nucleotides, such as ITP, GTP and ATP, were effective as cofactors in the inactivation of macrolides. An inhibitory effect of iodine, EDTA, or divalent cations on MPH(2')II activity was observed.
Asunto(s)
Escherichia coli/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol) , Fosfotransferasas/aislamiento & purificación , Antibacterianos/antagonistas & inhibidores , Antibacterianos/farmacología , Farmacorresistencia Microbiana , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Humanos , Pruebas de Sensibilidad Microbiana , Oleandomicina/antagonistas & inhibidores , Oleandomicina/farmacología , Fosfotransferasas/química , Fosfotransferasas/metabolismo , Especificidad por SustratoRESUMEN
Strains, producers of oleandomycin, with different level of antibiotic-formation have been studied for their resistance to their own antibiotic. The obtained highly active strain possesses double resistance to oleandomycin and 50% higher activity. Identity of oleandomycin phosphate substances synthesized by initial and produced highly active strains is shown by the HELC method.
Asunto(s)
Oleandomicina/antagonistas & inhibidores , Oleandomicina/biosíntesis , Streptomyces antibioticus/efectos de los fármacos , Streptomyces antibioticus/metabolismo , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Farmacorresistencia Microbiana , Oleandomicina/análisis , Factores de TiempoRESUMEN
The process of oleandomycin inactivation in aqueous alkaline solutions with their heating was studied by using the microbiological method of the antibiotic content assay. The initial specific rate of inactivation of crystalline oleandomycin in buffer solutions and oleandomycin in the fermentation broth filtrate was evaluated. It was shown that the inactivation was retarded by the reaction products and the components of the fermentation broth filtrate. The production rate of oleandomycin anhydro derivatives amounting to 3-40 per cent of the total mass of the inactivation product was estimated by UV spectrophotometry.
Asunto(s)
Álcalis/farmacología , Calor , Modelos Químicos , Oleandomicina/farmacocinética , Agua/farmacología , Álcalis/administración & dosificación , Tampones (Química) , Fermentación/efectos de los fármacos , Concentración de Iones de Hidrógeno , Oleandomicina/antagonistas & inhibidores , Soluciones , Agua/administración & dosificaciónRESUMEN
Oleandomycin biosynthesis by Streptomyces antibioticus is repressed by glucose added to the growth medium in the process of fermentation. Phosphotransferase involved in the synthesis of acetyl CoA and propionyl CoA (the precursors of the antibiotic macrolactone ring) is neither inhibited nor repressed, and the substrate specificity of the enzyme does not change. The content of cAMP in the mycelium of S. antibioticus does not change significantly when either glucose or sucrose is added to the medium 24 h after the inoculation whereas the content of exogenous cAMP rises abruptly 24 h after glucose addition. At the same time, the medium becomes much more acidic and the content of protein in the mycelium rises noticeably. Consequently, cAMP may be involved in the regulation of the culture growth.
Asunto(s)
Glucosa/farmacología , Oleandomicina/biosíntesis , Streptomyces antibioticus/metabolismo , Streptomyces/metabolismo , Medios de Cultivo , AMP Cíclico/metabolismo , Fermentación , Oleandomicina/antagonistas & inhibidoresRESUMEN
It was shown that addition of oleandomycin to the fermentation broth during development of Str. antibioticus inhibited the mycelium growth within the first 2 days and lowered the level of its further biosynthesis. The inhibitory effect depended on concentration of the added antibiotic and medium composition. An excess of glucose increased the inhibitory effect of oleandomycin. The antibiotic influenced both synthesis of the oleandomycin macrolide ring and methylation of the molecule.
Asunto(s)
Oleandomicina/farmacología , Streptomyces antibioticus/efectos de los fármacos , Streptomyces/efectos de los fármacos , Medios de Cultivo/metabolismo , Depresión Química , Fermentación , Oleandomicina/antagonistas & inhibidores , Oleandomicina/biosíntesis , Streptomyces antibioticus/crecimiento & desarrollo , Factores de TiempoRESUMEN
The results of the study on the effect of glucose and various carbohydrates on biosynthesis of oleandomycin by Streptomyces antibioticus are presented. It was found that glucose added at the beginning or by the 48th hour of the fermentation process on the complex medium inhibited oleandomycin biosynthesis. To investigate the mechanism of the glucose effect, a fermentation medium was developed. It provided variation of the carbohydrate composition, determination of the protein content in the culture and evaluation of the mycelium productivity. With the use of this medium it was shown that monosaccharides such as galactose, fructose and glucose significantly activated the mycelium growth as compared to lactose and sucrose. At the same time glucose completely inhibited oleandomycin biosynthesis when added either as an only carbohydrate component or in combination with galactose or fructose, while the presence of the other two monosaccharides did not prevent antibiotic production, though the mycelium productivity was lowered as compared to that with the use of the disaccharide. Therefore, the inhibitory effect of glucose on biosynthesis of oleandomycin was not connected with activation of the culture growth by it. Acidification of the medium on cultivation of the streptomycete in the presence of glucose only partially explained its inhibitory effect, since inhibition was maintained on the medium with addition of CaCO3 which stabilizes pH. Addition of 2-deoxy-D-glucose, a nonmetabolized glucose analog, to the fermentation medium retarded antibiotic production. It is possible that the inhibitory effect of glucose on biosynthesis of oleandomycin is not associated with its metabolism.