RESUMEN
The genetic engineering of antibiotic-producing Streptomyces strains is an approach that is emerging and ready to become established as a successful methodology in developing analogues of the original, pharmaceutically important, natural products obtained from the organisms. The current report highlights this succes by demonstrating the high-level production of novel anthracyclines. The biosynthetic pathways of the nogalamycin-producing Streptomyces nogalater and the aclacinomycin-producing S. galilaeus were combined by transferring the genes of S. nogalater polyketide synthetase into a nonproducing S. galilaeus mutant. The resulting anthracycline antibiotics that were produced possessed structural features characteristic of compounds from both of the undoctored Streptomycesstrains.
Asunto(s)
Antibióticos Antineoplásicos/biosíntesis , Streptomyces/genética , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/aislamiento & purificación , Secuencia de Carbohidratos , Fermentación , Ingeniería Genética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutación/genética , Espectrofotometría Ultravioleta , Streptomyces/químicaRESUMEN
We report the isolation and characterization of a cDNA encoding the mitochondrial short chain delta 3, delta 2-enoyl-CoA isomerase from rat liver. Tryptic fragments of the purified protein were generated, purified, and sequenced. A rat liver cDNA library, constructed in the plasmid vector pUEX1 was screened with oligonucleotides synthesized on the basis of peptide sequences. The obtained clone contained 783 bases predicting to code the entire mature protein of 261 amino acids. The molecular weight of 29,300 predicted from cDNA-derived sequences was consistent with the subunit size determined earlier. A high degree of similarity was noted between the amino acid sequence of isomerase and that of the amino-terminal half of peroxisomal multifunctional isomerase-hydratase-dehydrogenase enzyme and mitochondrial 2-enoyl-CoA hydratase in rat liver. These similarities also appeared at the level of predicted secondary structural elements, suggesting that hte rat multifunctional enzyme has both the isomerization and hydration activities in the amino-terminal domain. This idea is further supported by the proposed existence of only one CoA-binding site in the amino-terminal half of the multifunctional enzyme and by previous studies suggesting that the transfer of the substrate from the isomerization site to the hydration site occurs without aqueous bulk phase (Palosaari P.M., and Hiltunen, J. K. (1990) J. Biol. Chem. 265, 2446-2449).
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/genética , Isomerasas de Doble Vínculo Carbono-Carbono , Enoil-CoA Hidratasa/genética , Isomerasas/genética , Hígado/enzimología , Microcuerpos/enzimología , Mitocondrias Hepáticas/enzimología , Complejos Multienzimáticos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Dodecenoil-CoA Isomerasa , Biblioteca de Genes , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Enzima Bifuncional Peroxisomal , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
A method for incorporating into proteins a nonradioactive Eu3+ label, which exhibits fluorescence of a long decay time in the presence of suitable ligands, is described. As an example of the use of this label the method has been developed to work as a sensitive protease assay. By hydrolyzing the Eu3+-labeled casein, bound to an insoluble matrix (Sepharose 4B or Affi-Gel 10), with proteases and measuring the Eu3+ released with a pulsed time-resolved fluorometer it was possible to detect as low as 2.5, 1.0, or 1.0 ng of alpha-chymotrypsin, trypsin, or subtilisin, respectively.
Asunto(s)
Quimotripsina/metabolismo , Europio , Subtilisinas/metabolismo , Tripsina/metabolismo , Animales , Bacillus/enzimología , Bovinos , Cinética , Microquímica , Páncreas/enzimología , Espectrometría de Fluorescencia/métodos , PorcinosRESUMEN
Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed.
Asunto(s)
Bacillus megaterium/enzimología , Glutamato Deshidrogenasa/metabolismo , Glutamato Sintasa/metabolismo , Transaminasas/metabolismo , Aminoácidos/análisis , Amoníaco , Fenómenos Químicos , Química , Dimetil Suberimidato , Electroforesis en Gel de Poliacrilamida , Glutamato Deshidrogenasa/aislamiento & purificación , Glutamato Sintasa/aislamiento & purificación , CinéticaRESUMEN
Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5'-phosphate and this inactivation increased 3--4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5'-phosphate.