RESUMO
Bacillaene, a novel polyene antibiotic, was discovered and isolated from fermentation broths of a strain of Bacillus subtilis. The novel antibiotic has a nominal molecular weight of 580 and an empirical formula of C35H48O7. Bacillaene is active against a broad spectrum of bacteria in agar-plate diffusion assays. Studies in vitro indicate that the antibiotic inhibits prokaryotic protein synthesis but not eukaryotic protein synthesis. Cell survival studies performed with strains of Escherichia coli indicate that the antibiotic is a bacteriostatic agent.
Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/química , Antibacterianos/farmacologia , Bacillus subtilis , Escherichia coli/efeitos dos fármacos , Fermentação , Testes de Sensibilidade Microbiana , Polienos/química , Polienos/isolamento & purificação , Polienos/farmacologiaRESUMO
Eupenifeldin was isolated from cultures of Eupenicillium brefeldianum ATCC 74184 by extraction and crystallization. The compound was identified as a pentacyclic bistropolone on the basis of spectral data and its complete structure was established by single-crystal X-ray analysis. The compound is cytotoxic against the HCT-116 cell line and has in vivo antitumor activity in the P388 leukemia model.
Assuntos
Antibióticos Antineoplásicos , Tropolona/análogos & derivados , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/isolamento & purificação , Antibióticos Antineoplásicos/farmacologia , Ascomicetos/metabolismo , Fenômenos Químicos , Físico-Química , Ensaios de Seleção de Medicamentos Antitumorais , Fermentação , Humanos , Camundongos , Estrutura Molecular , Tropolona/química , Tropolona/isolamento & purificação , Tropolona/farmacologiaRESUMO
The gene encoding Anacystis nidulans 5-deazaflavin-dependent photolyase (phr) was inserted into the Streptomyces vector pIJ385 to form a transcriptional fusion with the neomycin resistance (aph) gene. The resulting plasmid, pANPL, was introduced into Streptomyces coelicolor, a host which exhibits no detectable photolyase activity and provides 5-deazaflavins. Transformants expressed functional photolyase and could be cultured at much higher cell densities than A. nidulans. A two-step affinity protocol was used to purify photolyase to homogeneity. High-pressure liquid chromatographic analysis established the presence of 5-deazaflavin cofactors in the enzyme, showing that this expression system allows heterologous production of 5-deazaflavin-class photolyases.
Assuntos
Cianobactérias/genética , Desoxirribodipirimidina Fotoliase/genética , Genes , Streptomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , Cianobactérias/enzimologia , Desoxirribodipirimidina Fotoliase/isolamento & purificação , Desoxirribodipirimidina Fotoliase/metabolismo , Expressão Gênica , Vetores Genéticos , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Streptomyces/enzimologia , Transcrição GênicaRESUMO
Thiolase proceeds via covalent catalysis involving an acetyl-S-enzyme. The active-site thiol nucleophile is identified as Cys89 by acetylation with [14C]acetyl-CoA, rapid denaturation, tryptic digestion, and sequencing of the labeled peptide. The native acetyl enzyme is labile to hydrolytic decomposition with t 1/2 of 2 min at pH 7, 25 degrees C. Cys89 has been converted to the alternate nucleophile Ser89 by mutagenesis and the C89S enzyme overproduced, purified, and assessed for activity. The Ser89 enzyme retains 1% of the Vmax of the Cys89 enzyme in the direction of acetoacetyl-CoA thiolytic cleavage and 0.05% of the Vmax in the condensation of two acetyl-CoA molecules. A covalent acetyl-O-enzyme intermediate is detected on incubation with [14C]acetyl-CoA and isolation of the labeled Ser89-containing tryptic peptide. Comparisons of the Cys89 and Ser89 enzymes have been made for kinetic and thermodynamic stability of the acetyl enzyme intermediates both by isolation and by analysis of [32P]CoASH/acetyl-CoA partial reactions and for rate-limiting steps in catalysis with trideuterioacetyl-CoA.