RESUMEN
Research on ribosomally synthesized and posttranslationally modified peptides (RiPPs) has led to an increasing understanding of biosynthetic mechanisms, mostly drawn from bacterial examples. In contrast, reports on RiPPs from fungal producers, apart from the amanitins and phalloidins, are still scarce. The fungal cyclopeptide omphalotinâ A carries multiple N-methylations on the peptide backbone, a modification previously known only from nonribosomal peptides. Mining the genome of the omphalotin-producing fungus for a precursor peptide led to the identification of two biosynthesis genes, one encoding a methyltransferase OphMA that catalyzes the automethylation of its C-terminus, which is then released and cyclized by the protease OphP. Our findings suggest a novel biosynthesis mechanism for a RiPP in which a modifying enzyme bears its own precursor peptide.
Asunto(s)
Basidiomycota/enzimología , Productos Biológicos/metabolismo , Metiltransferasas/metabolismo , Péptidos Cíclicos/biosíntesis , Péptidos/metabolismo , Basidiomycota/genética , Catálisis , Cromatografía Líquida de Alta Presión , Escherichia coli/genética , Genes Fúngicos , Metilación , Metiltransferasas/genética , Peso Molecular , Péptidos/química , Péptidos Cíclicos/química , Péptidos Cíclicos/genética , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The bengamides, sponge-derived natural products that have been characterized as inhibitors of methionine aminopeptidases (MetAPs), have been intensively investigated as anticancer compounds. We embarked on a multidisciplinary project to supply bengamides by fermentation of the terrestrial myxobacterium M.â virescens, decipher their biosynthesis, and optimize their properties as drug leads. The characterization of the biosynthetic pathway revealed that bacterial resistance to bengamides is conferred by Leu 154 of the myxobacterial MetAP protein, and enabled transfer of the entire gene cluster into the more suitable production host M.â xanthus DK1622. A combination of semisynthesis of microbially derived bengamides and total synthesis resulted in an optimized derivative that combined high cellular potency in the nanomolar range with high metabolic stability, which translated to an improved half-life in mice and antitumor efficacy in a melanoma mouse model.
Asunto(s)
Azepinas/metabolismo , Productos Biológicos/metabolismo , Biología Marina , Myxococcales/metabolismo , Poríferos/metabolismo , Animales , Área Bajo la Curva , Azepinas/farmacocinética , Azepinas/farmacología , Productos Biológicos/farmacocinética , Productos Biológicos/farmacología , Femenino , Semivida , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Relación Estructura-ActividadRESUMEN
Feglymycin, a peptide antibiotic produced by Streptomyces sp. DSM 11171, consists mostly of nonproteinogenic phenylglycine-type amino acids. It possesses antibacterial activity against methicillin-resistant Staphylococcus aureus strains and antiviral activity against HIV. Inhibition of the early steps of bacterial peptidoglycan synthesis indicated a mode of action different from those of other peptide antibiotics. Here we describe the identification and assignment of the feglymycin (feg) biosynthesis gene cluster, which codes for a 13-module nonribosomal peptide synthetase (NRPS) system. Inactivation of an NRPS gene and supplementation of a hydroxymandelate oxidase mutant with the amino acid l-Hpg proved the identity of the feg cluster. Feeding of Hpg-related unnatural amino acids was not successful. This characterization of the feg cluster is an important step to understanding the biosynthesis of this potent antibacterial peptide.