224 research outputs found
Substrate control in stereoselective lanthionine biosynthesis.
Enzymes are typically highly stereoselective catalysts that enforce a reactive conformation on their native substrates. We report here a rare example in which the substrate controls the stereoselectivity of an enzyme-catalysed Michael-type addition during the biosynthesis of lanthipeptides. These natural products contain thioether crosslinks formed by a cysteine attack on dehydrated Ser and Thr residues. We demonstrate that several lanthionine synthetases catalyse highly selective anti-additions in which the substrate (and not the enzyme) determines whether the addition occurs from the re or si face. A single point mutation in the peptide substrate completely inverted the stereochemical outcome of the enzymatic modification. Quantum mechanical calculations reproduced the experimentally observed selectivity and suggest that conformational restraints imposed by the amino-acid sequence on the transition states determine the face selectivity of the Michael-type cyclization
Ribosomally Synthesized and Post-Translationally Modified Peptide Natural Products: New Insights into the Role of Leader and Core Peptides during Biosynthesis
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 review, we discuss the features of the biosynthetic pathways to different RiPP classes, and propose mechanisms regarding recognition of the precursor peptide by the posttranslational 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
Investigation into the mechanism of action of the antimicrobial peptide epilancin 15X
Addressing the current antibiotic-resistance challenge would be aided by the identification of compounds with novel mechanisms of action. Epilancin 15X, a lantibiotic produced by Staphylococcus epidermidis 15 × 154, displays antimicrobial activity in the submicromolar range against a subset of pathogenic Gram-positive bacteria. S. epidermidis is a common member of the human skin or mucosal microbiota. We here investigated the mechanism of action of epilancin 15X. The compound is bactericidal against Staphylococcus carnosus as well as Bacillus subtilis and appears to kill these bacteria by membrane disruption. Structure–activity relationship studies using engineered analogs show that its conserved positively charged residues and dehydroamino acids are important for bioactivity, but the N-terminal lactyl group is tolerant of changes. Epilancin 15X treatment negatively affects fatty acid synthesis, RNA translation, and DNA replication and transcription without affecting cell wall biosynthesis. The compound appears localized to the surface of bacteria and is most potent in disrupting the membranes of liposomes composed of negatively charged membrane lipids in a lipid II independent manner. Epilancin 15X does not elicit a LiaRS response in B. subtilis but did upregulate VraRS in S. carnosus. Treatment of S. carnosus or B. subtilis with epilancin 15X resulted in an aggregation phenotype in microscopy experiments. Collectively these studies provide new information on epilancin 15X activity
Biosynthesis of Rhizocticins, Antifungal Phosphonate Oligopeptides Produced by Bacillus subtilis ATCC6633
SummaryRhizocticins are phosphonate oligopeptide antibiotics containing the C-terminal nonproteinogenic amino acid (Z)-l-2-amino-5-phosphono-3-pentenoic acid (APPA). Here we report the identification and characterization of the rhizocticin biosynthetic gene cluster (rhi) in Bacillus subtilis ATCC6633. Rhizocticin B was heterologously produced in the nonproducer strain Bacillus subtilis 168. A biosynthetic pathway is proposed on the basis of bioinformatics analysis of the rhi genes. One of the steps during the biosynthesis of APPA is an unusual aldol reaction between phosphonoacetaldehyde and oxaloacetate catalyzed by an aldolase homolog RhiG. Recombinant RhiG was prepared, and the product of an in vitro enzymatic conversion was characterized. Access to this intermediate allows for biochemical characterization of subsequent steps in the pathway
Discovery of Unique Lanthionine Synthetases Reveals New Mechanistic and Evolutionary Insights
Identification of a new class of lanthionine synthetases provides insight into the mechanism and evolution of cyclic peptide biosynthesis
Molecular Cloning and Heterologous Expression of the Dehydrophos Biosynthetic Gene Cluster
SummaryDehydrophos is a vinyl phosphonate tripeptide produced by Streptomyces luridus with demonstrated broad-spectrum antibiotic activity. To identify genes necessary for biosynthesis of this unusual compound we screened a fosmid library of S. luridus for the presence of the phosphoenolpyruvate mutase gene, which is required for biosynthesis of most phosphonates. Integration of one such fosmid clone into the chromosome of S. lividans led to heterologous production of dehydrophos. Deletion analysis of this clone allowed identification of the minimal contiguous dehydrophos cluster, which contained 17 open reading frames (ORFs). Bioinformatic analyses of these ORFs are consistent with a proposed biosynthetic pathway that generates dehydrophos from phosphoenolpyruvate. The early steps of this pathway are supported by analysis of intermediates accumulated by blocked mutants and in vitro biochemical experiments
Evolutionary radiation of lanthipeptides in marine cyanobacteria
Lanthipeptides are ribosomally derived peptide secondary metabolites that undergo extensive posttranslational modification. Prochlorosins are a group of lanthipeptides produced by certain strains of the ubiquitous marine picocyanobacteria Prochlorococcus and Synechococcus. Unlike other lanthipeptide-producing bacteria, picocyanobacteria use an unprecedented mechanism of substrate promiscuity for the production of numerous and diverse lanthipeptides using a single lanthionine synthetase. Through a cross-scale analysis of prochlorosin biosynthesis genes-from genomes to oceanic populations-we show that marine picocyanobacteria have the collective capacity to encode thousands of different cyclic peptides, few of which would display similar ring topologies. To understand how this extensive structural diversity arises, we used deep sequencing of wild populations to reveal genetic variation patterns in prochlorosin genes. We present evidence that structural variability among prochlorosins is the result of a diversifying selection process that favors large, rather than small, sequence changes in the precursor peptide genes. This mode of molecular evolution disregards any conservation of the ancestral structure and enables the emergence of extensively different cyclic peptides through short mutational paths based on indels. Contrary to its fast-evolving peptide substrates, the prochlorosin lanthionine synthetase evolves under a strong purifying selection, indicating that the diversification of prochlorosins is not constrained by commensurate changes in the biosynthetic enzyme. This evolutionary interplay between the prochlorosin peptide substrates and the lanthionine synthetase suggests that structure diversification, rather than structure refinement, is the driving force behind the creation of new prochlorosin structures and represents an intriguing mechanism by which natural product diversity arises. Keywords: lanthipeptides; prochlorosin; RiPPs; Prochlorococcus; SynechococcusGordon and Betty Moore Foundation (Grant GBMF495
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