RESUMO
The α',ß'-epoxyketone moiety of proteasome inhibitors confers high binding specificity to the N-terminal threonine in catalytic proteasome ß-subunits. We recently identified the epoxomicin and eponemycin biosynthetic gene clusters and have now conducted isotope-enriched precursor feeding studies and comprehensive gene deletion experiments to shed further light on their biosynthetic pathways. Leucine and two methyl groups from S-adenosylmethionine were readily incorporated into the epoxyketone warhead, suggesting decarboxylation of the thioester intermediate. Formation of the α',ß'-epoxyketone is likely mediated by conserved acyl-CoA dehydrogenase-like enzymes, as indicated by complete loss of epoxomicin and eponemycin production in the respective knockout mutants. Our results clarify crucial questions in the formation of epoxyketone compounds and lay the foundation for in vitro biochemical studies on the biosynthesis of this pharmaceutically important class of proteasome inhibitors.
Assuntos
Acil-CoA Desidrogenase/metabolismo , Serina/análogos & derivados , Acil-CoA Desidrogenase/genética , Amidas/química , Cromatografia Líquida de Alta Pressão , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Metionina/metabolismo , Família Multigênica , Oligopeptídeos/biossíntese , Oligopeptídeos/química , Inibidores de Proteassoma/química , Inibidores de Proteassoma/metabolismo , Serina/biossíntese , Serina/química , Streptomyces/genética , Streptomyces/metabolismo , Espectrometria de Massas em TandemRESUMO
Aminocoumarins are potent antibiotics belonging to a relatively small group of secondary metabolites produced by actinomycetes. Genome mining of Catenulispora acidiphila has recently led to the discovery of a gene cluster responsible for biosynthesis of novel aminocoumarins, cacibiocins. However, regulation of the expression of this novel gene cluster has not yet been analyzed. In this study, we identify transcriptional regulators of the cacibiocin gene cluster. Using a heterologous expression system, we show that the CabA and CabR proteins encoded by cabA and cabR genes in the cacibiocin gene cluster control the expression of genes involved in the biosynthesis, modification, regulation, and potentially, efflux/resistance of cacibiocins. CabA positively regulates the expression of cabH (the first gene in the cabHIYJKL operon) and cabhal genes encoding key enzymes responsible for the biosynthesis and halogenation of the aminocoumarin moiety, respectively. We provide evidence that CabA is a direct inducer of cacibiocin production, whereas the second transcriptional factor, CabR, is involved in the negative regulation of its own gene and cabT-the latter of which encodes a putative cacibiocin transporter. We also demonstrate that CabR activity is negatively regulated in vitro by aminocoumarin compounds, suggesting the existence of analogous regulation in vivo. Finally, we propose a model of multilevel regulation of gene transcription in the cacibiocin gene cluster by CabA and CabR.
Assuntos
Actinomycetales/genética , Aminocumarinas/metabolismo , Antibacterianos/biossíntese , Proteínas de Bactérias/genética , Genes Bacterianos , Genoma Bacteriano , Fatores de Transcrição/genética , Actinomycetales/química , Actinomycetales/metabolismo , Sequência de Aminoácidos , Aminocumarinas/química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , Farmacorresistência Bacteriana , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Óperon , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Genome mining led to the discovery of a novel aminocoumarin gene cluster in the rare actinomycete Catenulispora acidiphila DSM 44928. Sequence analysis revealed the presence of genes putatively involved in export/resistance, regulation, and biosynthesis of the aminocoumarin moiety and its halogenation, as well as several genes with so far unknown function. Two new aminocoumarins, cacibiocin A and B, were identified in the culture broth of C. acidiphila. Heterologous expression of the putative gene cluster in Streptomyces coelicolor M1152 confirmed that this cluster is responsible for cacibiocin biosynthesis. Furthermore, total production levels of cacibiocins could be increased by heterologous expression and screening of different culture media from an initial yield of 4.9 mg L(-1) in C. acidiphila to 60 mg L(-1) in S. coelicolor M1152. By HR-MS and NMR analysis, cacibiocin A was found to contain a 3-amino-4,7-dihydroxycoumarin moiety linked by an amide bond to a pyrrole-2,5-dicarboxylic acid. The latter structural motif has not been identified previously in any natural compound. Additionally, cacibiocin B contains two chlorine atoms at positions 6' and 8' of the aminocoumarin moiety.
Assuntos
Actinomycetales/química , Aminocumarinas/química , Antibacterianos/química , Pirróis/química , Actinomycetales/genética , Actinomycetales/metabolismo , Aminocumarinas/metabolismo , Aminocumarinas/farmacologia , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Genoma Bacteriano , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Conformação Molecular , Família Multigênica , Pirróis/metabolismo , Pirróis/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Streptomyces/metabolismoRESUMO
The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique α',ß'-epoxyketone pharmacophore allows binding to the catalytic ß-subunits of the proteasome with extraordinary specificity. Here, we report the characterization of the first gene clusters for the biosynthesis of natural peptidyl-epoxyketones. The clusters for epoxomicin, the lead compound for the anticancer drug Kyprolis, and for eponemycin were identified in the actinobacterial producer strains ATCC 53904 and Streptomyces hygroscopicus ATCC 53709, respectively, using a modified protocol for Ion Torrent PGM genome sequencing. Both gene clusters code for a hybrid nonribosomal peptide synthetase/polyketide synthase multifunctional enzyme complex and homologous redox enzymes. Epoxomicin and eponemycin were heterologously produced in Streptomyces albus J1046 via whole pathway expression. Moreover, we employed mass spectral molecular networking for a new comparative metabolomics approach in a heterologous system and discovered a number of putative epoxyketone derivatives. With this study, we have definitively linked epoxyketone proteasome inhibitors and their biosynthesis genes for the first time in any organism, which will now allow for their detailed biochemical investigation.