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Ribosomal synthesis of dehydroalanine-containing peptides.

ABSTRACT: Dehydroalanine is a nonproteinogenic amino acid, but it is a component of a wide variety of natural products with therapeutic activities. Indeed, this alpha,beta-unsaturated residue is a highly versatile building block due to its rigidifying effect on peptide backbones and its electrophilicity which allows site-specific thiol ligations of peptides with small molecules or proteins. To harness such versatility in genetically encoded, combinatorial peptide libraries, we report a simple and robust method for the ribosomal synthesis of dehydroalanine-containing peptides. Selenalysine, a selenium-containing lysine analogue, was recruited as a masked dehydroalanine equivalent. This residue is efficiently incorporated by a reconstituted Escherichia coli translation system at high fidelity and efficiency despite the presence of low levels of lysine. Mild oxidative conditions were used to convert selenalysine into dehydroalanine post-translationally. Using this method, we demonstrate the preparation of polyunsaturated and highly decorated peptides. This report is an important step toward the preparation and selection of large libraries of protein-reactive compounds with potential use as novel drugs or as analytical tools.

SUBMITTER: Seebeck FP

PROVIDER: S-EPMC2572134 | biostudies-other | 2006 Jun

REPOSITORIES: biostudies-other

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Ribosomal synthesis of dehydroalanine-containing peptides.

Seebeck Florian P FP Szostak Jack W JW

Journal of the American Chemical Society 20060601 22

Dehydroalanine is a nonproteinogenic amino acid, but it is a component of a wide variety of natural products with therapeutic activities. Indeed, this alpha,beta-unsaturated residue is a highly versatile building block due to its rigidifying effect on peptide backbones and its electrophilicity which allows site-specific thiol ligations of peptides with small molecules or proteins. To harness such versatility in genetically encoded, combinatorial peptide libraries, we report a simple and robust m ...[more]

PMID: 16734454

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Project description:BackgroundRibosomally synthesized and post-translationally modified peptides (RiPPs) are a burgeoning class of natural products with diverse activity that share a similar origin and common features in their biosynthetic pathways. The precursor peptides of these natural products are ribosomally produced, upon which a combination of modification enzymes installs diverse functional groups. This genetically encoded peptide-based strategy allows for rapid diversification of these natural products by mutation in the precursor genes merged with unique combinations of modification enzymes. Thiazole/oxazole-modified microcins (TOMMs) are a class of RiPPs defined by the presence of heterocycles derived from cysteine, serine, and threonine residues in the precursor peptide. TOMMs encompass a number of different families, including but not limited to the linear azol(in)e-containing peptides (streptolysin S, microcin B17, and plantazolicin), cyanobactins, thiopeptides, and bottromycins. Although many TOMMs have been explored, the increased availability of genome sequences has illuminated several unexplored TOMM producers.MethodsAll YcaO domain-containing proteins (D protein) and the surrounding genomic regions were were obtained from the European Molecular Biology Laboratory (EMBL) and the European Bioinformatics Institute (EBI). MultiGeneBlast was used to group gene clusters contain a D protein. A number of techniques were used to identify TOMM biosynthetic gene clusters from the D protein containing gene clusters. Precursor peptides from these gene clusters were also identified. Both sequence similarity and phylogenetic analysis were used to classify the 20 diverse TOMM clusters identified.ResultsGiven the remarkable structural and functional diversity displayed by known TOMMs, a comprehensive bioinformatic study to catalog and classify the entire RiPP class was undertaken. Here we report the bioinformatic characterization of nearly 1,500 TOMM gene clusters from genomes in the European Molecular Biology Laboratory (EMBL) and the European Bioinformatics Institute (EBI) sequence repository. Genome mining suggests a complex diversification of modification enzymes and precursor peptides to create more than 20 distinct families of TOMMs, nine of which have not heretofore been described. Many of the identified TOMM families have an abundance of diverse precursor peptide sequences as well as unfamiliar combinations of modification enzymes, signifying a potential wealth of novel natural products on known and unknown biosynthetic scaffolds. Phylogenetic analysis suggests a widespread distribution of TOMMs across multiple phyla; however, producers of similar TOMMs are generally found in the same phylum with few exceptions.ConclusionsThe comprehensive genome mining study described herein has uncovered a myriad of unique TOMM biosynthetic clusters and provides an atlas to guide future discovery efforts. These biosynthetic gene clusters are predicted to produce diverse final products, and the identification of additional combinations of modification enzymes could expand the potential of combinatorial natural product biosynthesis.

Dataset Information

Ribosomal synthesis of dehydroalanine-containing peptides.

Publications

Ribosomal synthesis of dehydroalanine-containing peptides.

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