RESUMO
Ribosomally synthesized post-translationally modified peptides (RiPPs) are ubiquitous and represent a structurally diverse class of natural products. The ribosomally encoded precursor polypeptides are often extensively modified post-translationally by enzymes that are encoded by coclustered genes. Radical S-adenosyl-l-methionine (SAM) enzymes catalyze numerous chemically challenging transformations. In RiPP biosynthetic pathways, these transformations include the formation of C-H, C-C, C-S, and C-O linkages. In this paper, we show that the Geobacter lovleyi sbtM gene encodes a radical SAM protein, SbtM, which catalyzes the cyclization of a Cys/SeCys residue in a minimal peptide substrate. Biochemical studies of this transformation support a mechanism involving H-atom abstraction at the C-3 of the substrate Cys to initiate the chemistry. Several possible cyclization products were considered. The collective biochemical, spectroscopic, mass spectral, and computational observations point to a thiooxazole as the product of the SbtM-catalyzed modification. To our knowledge, this is the first example of a radical SAM enzyme that catalyzes a transformation involving a SeCys-containing peptide and represents a new paradigm for formation of oxazole-containing RiPP natural products.
Assuntos
Peptídeos Antimicrobianos/metabolismo , Geobacter/metabolismo , S-Adenosilmetionina/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/fisiologia , Peptídeos Antimicrobianos/fisiologia , Produtos Biológicos/metabolismo , Catálise , Geobacter/patogenicidade , Espectrometria de Massas/métodos , Oxazóis , Processamento de Proteína Pós-Traducional/fisiologia , Proteômica/métodos , Ribossomos , S-Adenosilmetionina/metabolismoRESUMO
7-Carboxy-7-deazaguanine (CDG) is a common intermediate in the biosynthesis of 7-deazapurine-containing natural products. The biosynthesis of CDG from GTP requires three enzymes: GTP cyclohydrolase I, 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) synthase, and CDG synthase (QueE). QueE is a member of the radical S-adenosyl-l-methionine (SAM) superfamily and catalyzes the SAM-dependent radical-mediated ring contraction of CPH4 to generate CDG. This chapter focuses on methods to reconstitute the activity of QueE in vitro.