How a Subfamily of Radical S-Adenosylmethionine Enzymes Became a Mainstay of Ribosomally Synthesized and Post-translationally Modified Peptide Discovery (original) (raw)
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Chemistry - A European Journal, 2013
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with a high degree of structural diversity and a wide variety of bioactivities. Understanding the biosynthetic machinery of these RiPPs will benefit the discovery and development of new molecules with potential pharmaceutical applications. In this Concept article, we discuss the features of the biosynthetic pathways to different RiPP classes, and propose mechanisms regarding recognition of the precursor peptide by the post-translational modification enzymes. We propose that the leader peptides function as allosteric regulators that bind the active form of the biosynthetic enzymes in a conformational selection process. We also speculate how enzymes that generate polycyclic products of defined topologies may have been selected for during evolution.
Cell chemical biology, 2016
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large group of structurally diverse natural products. Their biological activities and unique biosynthetic pathways have sparked a growing interest in RiPPs. Furthermore, the relatively low genetic complexity associated with RiPP biosynthesis makes them excellent candidates for synthetic biology applications. This Review highlights recent developments in the understanding of the biosynthesis of several bacterial RiPP family members, the use of the RiPP biosynthetic machinery for generating novel macrocyclic peptides, and the implementation of tools designed to guide the discovery and characterization of novel RiPPs.
Autocatalytic backbone N-methylation in a family of ribosomal peptide natural products
Nature Chemical Biology, 2017
Peptide backbone N-methylations as in cyclosporin A have been an exclusive characteristic of non-ribosomal peptides. We have identified the first posttranslationally modified peptide or protein harboring internal α-N-methylations through discovery of the genetic locus for the omphalotins, cyclic N-methylated peptides produced by the fungus Omphalotus olearius. We show iterative autocatalytic activity of an N-methyltransferase fused to its peptide substrate is the signature of a new family of ribosomally encoded metabolites. The fidelity and complexity of protein-and peptide-mediated interactions is predominantly achieved through the vast chemical space provided by the 20 canonical proteinogenic amino acids. This space is extended by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, acetylation, amidation, and many others 1. An expanded repertoire of PTMs can be found in ribosomally synthesized and posttranslationally modified peptides (RiPPs), a class of natural products that is found in all kingdoms of life and invokes an extensive array of biological activities 2. RiPPs are produced by translation of protein/peptide precursors followed by posttranslational modification of these precursors by additional enzymes. Many of these accessory proteins are presumably recruited by the Nterminal leader sequence of the precursor to amend the C-terminal peptide core 2. Proteolytic release and export of the core affords the active metabolite. In contrast to RiPP biosynthesis, nonribosomal peptide synthetases (NRPSs) create structurally diverse peptides through direct incorporation of non-proteinogenic amino acids 3. This feat is achieved in an assembly line-like fashion where individual modules within giant multimodular NRPS proteins are responsible for the activation, incorporation, and optional further modification of amino acids. Recent insights into RiPP enzymes have begun to challenge the view that certain residues and peptide modifications are only accessible to NRPS pathways 4. Nonetheless, while the methylation of peptide N-termini and side chains have been observed in RiPPs 2 , backbone N-methylation has only been found in NRPS-derived peptides and shown to occur prior to amide bond formation 5. These modifications have been demonstrated to improve therapeutic peptide metabolic stability, membrane permeability, target selectivity, affinity and oral bioavailability 5. Despite these favorable properties, backbone α-N-methylation has to our knowledge not been reported as a PTM in any naturally occurring ribosomal peptide or protein 5 .
Mechanistic elucidation of the mycofactocin-biosynthetic radical-S-adenosylmethionine protein, MftC
The Journal of biological chemistry, 2017
Ribosomally synthesized and posttranslationally modified peptide (RiPP) pathways produce a diverse array of natural products. A subset of these pathways depend on radical S-adenosylmethionine (RS) proteins to modify the RiPP-produced peptide. Mycofactocin biosynthesis is one example of an RS protein-dependent RiPP pathway. Recently, it has been shown that MftC catalyzes the oxidative decarboxylation of the C-terminal tyrosine (Tyr30) on the mycofactocin precursor peptide MftA; however, this product has not been verified by techniques other than MS. Herein, we provide a more detailed study of MftC catalysis and report a revised mechanism for MftC chemistry. We show that MftC catalyzes the formation of two isomeric products. Using a combination of MS, isotope labeling, and 1H and 13C NMR techniques, we established that the major product, MftA*, is a tyramine-valine crosslinked peptide formed by MftC through two S-adenosylmethionine-dependent turnovers. In addition, we show that the hy...
Nature Communications
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are structurally complex natural products with diverse bioactivities. Here we report discovery of a RiPP, kintamdin, for which the structure is determined through spectroscopy, spectrometry and genomic analysis to feature a bis-thioether macrocyclic ring and a β-enamino acid residue. Biosynthetic investigation demonstrated that its pathway relies on four dedicated proteins: phosphotransferase KinD, Lyase KinC, kinase homolog KinH and flavoprotein KinI, which share low homologues to enzymes known in other RiPP biosynthesis. During the posttranslational modifications, KinCD is responsible for the formation of the characteristic dehydroamino acid residues including the β-enamino acid residue, followed by oxidative decarboxylation on the C-terminal Cys and subsequent cyclization to provide the bis-thioether ring moiety mediated by coordinated action of KinH and KinI. Finally, conserved genomic investigation allow...
The Role of 23S Ribosomal RNA Residue A2451 in Peptide Bond Synthesis Revealed by Atomic Mutagenesis
Chemistry & Biology, 2008
Peptide bond formation is a fundamental reaction in biology, catalyzed by the ribosomal peptidyl-transferase ribozyme. Although all active-site 23S ribosomal RNA nucleotides are universally conserved, atomic mutagenesis suggests that these nucleobases do not carry functional groups directly involved in peptide bond formation. Instead, a single ribose 2 0 -hydroxyl group at A2451 was identified to be of pivotal importance. Here, we altered the chemical characteristics by replacing its 2 0 -hydroxyl with selected functional groups and demonstrate that hydrogen donor capability is essential for transpeptidation. We propose that the A2451-2 0 -hydroxyl directly hydrogen bonds to the P-site tRNA-A76 ribose. This promotes an effective A76 ribose C2 0 -endo conformation to support amide synthesis via a proton shuttle mechanism. Simultaneously, the direct interaction of A2451 with A76 renders the intramolecular transesterification of the peptide from the 3 0 -to 2 0 -oxygen unfeasible, thus promoting effective peptide bond synthesis.