eponemycin and epoxomicin

The α',β'-epoxyketone moiety of proteasome inhibitors confers high binding specificity to the N-terminal threonine in catalytic proteasome β-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',β'-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.

Topics: Acyl-CoA Dehydrogenase; Amides; Chromatography, High Pressure Liquid; Isotope Labeling; Magnetic Resonance Spectroscopy; Methionine; Multigene Family; Oligopeptides; Proteasome Inhibitors; Serine; Streptomyces; Tandem Mass Spectrometry

The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique α',β'-epoxyketone pharmacophore allows binding to the catalytic β-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.

Topics: Amides; Base Sequence; Genes, Bacterial; Humans; Models, Molecular; Molecular Sequence Data; Multigene Family; Oligopeptides; Proteasome Inhibitors; Serine; Streptomyces

An actinomycete strain No. Q996-17 produced a novel compound, epoxomicin, which exhibited in vivo antitumor activity against B16 melanoma. Structural studies indicated that it is a new member of the epoxy-beta-aminoketone group, and is closely related to eponemycin.

Topics: Actinomycetales; Amides; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Chemical Phenomena; Chemistry, Physical; Fermentation; Magnetic Resonance Spectroscopy; Melanoma, Experimental; Molecular Structure; Oligopeptides; Serine; Spectrophotometry