RESUMO
The azinomycins are a family of aziridine-containing antitumor antibiotics and represent a treasure trove of biosynthetic reactions. The formation of the azabicyclo[3.1.0]hexane ring and functionalization of this ring system remain the least understood aspects of the pathway. This study reports the incorporation of 18O-labeled molecular oxygen in azinomycin biosynthesis including both oxygens of the diol that ultimately adorn the aziridino[1,2- a]pyrrolidine moiety. Likewise, two other sites of heavy atom incorporation are observed.
Assuntos
Dipeptídeos/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Oxigênio/metabolismo , Streptomyces/metabolismo , Compostos Azabicíclicos , Cromatografia em Camada Fina , Peptídeos e Proteínas de Sinalização Intercelular/isolamento & purificação , Naftalenos/isolamento & purificação , Streptomyces/químicaRESUMO
The biosynthesis of the azabicyclic ring system of the azinomycin family of antitumor agents represents the "crown jewel" of the pathway and is a complex process involving at least 14 enzymatic steps. This study reports on the first biosynthetic step, the inroads, in the construction of the novel aziridino [1,2-a]pyrrolidine, azabicyclic core, allowing us to support a new mechanism for azabicycle formation.
Assuntos
Aldeído Oxirredutases/metabolismo , Aminoácido N-Acetiltransferase/metabolismo , Antineoplásicos Alquilantes/metabolismo , Compostos Azabicíclicos/metabolismo , Proteínas de Bactérias/metabolismo , Desenho de Fármacos , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Pirrolidinas/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Aldeído Oxirredutases/genética , Aminoácido N-Acetiltransferase/genética , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacologia , Compostos Azabicíclicos/química , Compostos Azabicíclicos/farmacologia , Proteínas de Bactérias/genética , Biocatálise , Dipeptídeos/química , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Técnicas de Inativação de Genes , Ácido Glutâmico/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Estrutura Molecular , Mutação , Naftalenos/química , Naftalenos/metabolismo , Naftalenos/farmacologia , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Fosfotransferases (Aceptor do Grupo Carboxila)/genética , Pirrolidinas/química , Pirrolidinas/farmacologia , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces/metabolismo , Especificidade por SubstratoRESUMO
A protein identified from the Streptomyces sahachiroi genome exhibits a protective effect against the DNA alkylator azinomycin B when heterologously expressed in S. lividans and E. coli. The protein, dubbed AziR for azinomycin resistance, is homologous to aminoglycoside phosphotransferases but behaves as an azinomycin binding protein and fails to chemically modify azinomycin. While AziR confers resistance to azinomycin B, it is inactive against aminoglycoside antibiotics and other DNA alkylators. A nucleic acid staining assay indicates that the protein enhances cell survival, and also prevents DNA damage effects normally observed following azinomycin treatment. Knowledge of an azinomycin resistance mechanism aids in setting the stage for future engineered biosynthesis of functionally useful azinomycin analogues.
Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Streptomyces/efeitos dos fármacos , Streptomyces/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Naftalenos/química , Naftalenos/metabolismo , Naftalenos/farmacologia , Peptídeos/química , Streptomyces/genéticaRESUMO
Experiments reveal that the metabolic precursor aminoacetone is a key intermediate in the production of the antitumor agent azinomycin A relative to the structurally and functionally related agent, azinomycin B. Azinomycin A and B arise through bifurcation of the biosynthetic pathway and competition between metabolic substrates. The availability of the biosynthetic precursors in vivo, aminoacetone for azinomycin A and threonine for azinomycin B, controls the overall ratio of azinomycin A to B produced.
Assuntos
Acetona/análogos & derivados , Antineoplásicos/síntese química , Glicopeptídeos/síntese química , Acetona/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos Azabicíclicos , Dipeptídeos , Glicopeptídeos/química , Glicopeptídeos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Estrutura Molecular , Naftalenos , PeptídeosRESUMO
Streptomyces sahachiroi whole cell feeding experiments, utilizing putative precursors labeled with stable isotopes, established that the epoxide unit of the DNA cross-linked agents, azinomycin A and B, proceeds via a valine-dependent pathway and that hydroxylation and dehydration precedes formation of the terminal epoxide. Sodium 3-methyl-2-oxobutenoate, formed through a transimination reaction, was shown to be the penultimate precursor incorporated into the azinomycin epoxide.
Assuntos
Compostos de Epóxi/química , Compostos de Epóxi/metabolismo , Naftalenos/química , Naftalenos/metabolismo , Compostos Azabicíclicos , Dipeptídeos , Glicopeptídeos/química , Peptídeos e Proteínas de Sinalização Intercelular , Estrutura Molecular , Peptídeos/química , Fatores de TempoRESUMO
Azinomycin B is an environmental DNA crosslinking agent produced by the soil microorganism Streptomyces sahachiroi. While the agent displays potent cytotoxic activities against leukemic cell lines and animal mouse models, the lack of a consistent supply of the natural product has hampered detailed biological investigations on the compound, including its mode of action and biosynthesis. We report here a significant methodological improvement in the culturing of the bacterium, which allows reliable and steady production of the natural product in good yields. The key experimental step involves the culturing of the strain on dehydrated plates, followed by the generation of a two-stage starter culture and subsequent fermentation of the strain under nutrient-starved conditions. We illustrate use of this culture system by investigating the formation of the enol fragment of the molecule in isotopic labeling experiments with threonine and several advanced precursors (beta-ketoamino acid 3, beta-hydroxyamino aldehyde 4, and beta-ketoaminoaldehyde 5). The results unequivocally show that threonine is the most advanced precursor accepted by the NRPS (non-ribosomal peptidyl synthetase) machinery for final processing and construction of the enol moiety of the natural product.
Assuntos
Peptídeos/metabolismo , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismo , Técnicas Bacteriológicas/métodos , Meios de Cultura , Fermentação , Peptídeos e Proteínas de Sinalização Intercelular , Espectroscopia de Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/normas , Conformação Molecular , Naftalenos/síntese química , Naftalenos/química , Naftalenos/metabolismo , Peptídeos/síntese química , Peptídeos/química , Padrões de Referência , Streptomyces/isolamento & purificação , Fatores de TempoRESUMO
Studies on the mechanism of action of the antitumor agent azinomycin B in vitro suggest that the drug elicits its lethal effects by the formation of interstrand crosslinks within the major groove of DNA. Here, we demonstrate the biological effects of the drug in vivo. Fluorescence imaging revealed localization of azinomycin B in the nuclear region of yeast. Moreover, experiments with oligonucleotide microarrays examined the effects of the drug across the yeast transcriptome. The results demonstrated a robust DNA damage response that supports the proposed role of the drug as a covalent DNA modifying agent. RT-PCR analysis validated the gene changes, and flow cytometry of azinomycin-treated yeast cells demonstrated a phenotypic S phase shift consistent with transcriptional effects.
Assuntos
Antineoplásicos/farmacologia , Peptídeos/farmacologia , Alquilação , Sequência de Bases , Ciclo Celular/efeitos dos fármacos , Dano ao DNA , Primers do DNA , DNA Fúngico/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular , Naftalenos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
[reaction: see text] Azinomycins have potential therapeutic value as antitumor agents; however, their biosynthesis is poorly understood. Here, we provide the first demonstration of a protein cell-free system capable of supporting complete in vitro biosynthesis of the antitumor agent azinomycin B. The cell-free system is utilized to probe the cofactor dependence and substrate requirements of the pathway en route to azinomycin.
