Tryptophan-Centric Bioinformatics Identifies New Lasso Peptide Modifications.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by a macrolactam linkage between the N-terminus and the side chain of an internal aspartic acid or glutamic acid residue. Instead of adopting a branched-cyclic conformation, lasso peptides are "threaded", with the C-terminal tail passing through the macrocycle to present a kinetically trapped rotaxane conformation. The availability of enhanced bioinformatics methods has led to a significant increase in the number of secondary modifications found on lasso peptides. To uncover new ancillary modifications in a targeted manner, a bioinformatic strategy was developed to discover lasso peptides with modifications to tryptophan. This effort identified numerous putative lasso peptide biosynthetic gene clusters with core regions of the precursor peptides enriched in tryptophan. Parsing of these tryptophan (Trp)-rich biosynthetic gene clusters uncovered several putative ancillary modifying enzymes, including halogenases and dimethylallyltransferases expected to act upon Trp. Characterization of two gene products yielded a lasso peptide with two 5-Cl-Trp modifications (chlorolassin) and another bearing 5-dimethylallyl-Trp and 2,3-didehydro-Tyr modifications (wygwalassin). Bioinformatic analysis of the requisite halogenase and dimethylallyltransferase revealed numerous other putative Trp-modified lasso peptides that remain uncharacterized. We anticipate that the Trp-centric strategy reported herein may be useful in discovering ancillary modifications for other RiPP classes and, more generally, guide the functional prediction of enzymes that act on specific amino acids.

Catalytic Site Proximity Profiling for Functional Unification of Sequence-Diverse Radical S-Adenosylmethionine Enzymes.

The radical S-adenosylmethionine (rSAM) superfamily has become a wellspring for discovering new enzyme chemistry, especially regarding ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we report a compendium of nearly 15,000 rSAM proteins with high-confidence involvement in RiPP biosynthesis. While recent bioinformatics advances have unveiled the broad sequence space covered by rSAM proteins, the significant challenge of functional annotation remains unsolved. Through a combination of sequence analysis and protein structural predictions, we identified a set of catalytic site proximity residues with functional predictive power, especially among the diverse rSAM proteins that form sulfur-to-α carbon thioether (sactionine) linkages. As a case study, we report that an rSAM protein from Streptomyces sparsogenes (StsB) shares higher full-length similarity with MftC (mycofactocin biosynthesis) than any other characterized enzyme. However, a comparative analysis of StsB to known rSAM proteins using "catalytic site proximity" predicted that StsB would be distinct from MftC and instead form sactionine bonds. The prediction was confirmed by mass spectrometry, targeted mutagenesis, and chemical degradation. We further used "catalytic site proximity" analysis to identify six new sactipeptide groups undetectable by traditional genome-mining strategies. Additional catalytic site proximity profiling of cyclophane-forming rSAM proteins suggests that this approach will be more broadly applicable and enhance, if not outright correct, protein functional predictions based on traditional genomic enzymology principles.

A scalable platform to discover antimicrobials of ribosomal origin.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a promising source of new antimicrobials in the face of rising antibiotic resistance. Here, we report a scalable platform that combines high-throughput bioinformatics with automated biosynthetic gene cluster refactoring for rapid evaluation of uncharacterized gene clusters. As a proof of concept, 96 RiPP gene clusters that originate from diverse bacterial phyla involving 383 biosynthetic genes are refactored in a high-throughput manner using a biological foundry with a success rate of 86%. Heterologous expression of all successfully refactored gene clusters in Escherichia coli enables the discovery of 30 compounds covering six RiPP classes: lanthipeptides, lasso peptides, graspetides, glycocins, linear azol(in)e-containing peptides, and thioamitides. A subset of the discovered lanthipeptides exhibit antibiotic activity, with one class II lanthipeptide showing low µM activity against Klebsiella pneumoniae, an ESKAPE pathogen. Overall, this work provides a robust platform for rapidly discovering RiPPs.

Reactivity-based screening for natural product discovery.

Natural products have traditionally been a fruitful source of chemical matter that has been developed into novel therapeutics. Actinomycetes and several other bacterial taxa are especially gifted in biosynthesizing natural products. However, many decades of intense bioactivity-based screening led to a large rediscovery problem, rendering industrial natural product discovery pipelines uneconomical. Numerous methods for circumventing the rediscovery problem have been developed, among them various chemistry-focused strategies, including reactivity-based screening. Emerging from the field of chemical proteomics, reactivity-based screening relies on a reactive probe that chemoselectively modifies a functional group of interest in the context of a complex biological sample. Reactivity-based probes for several distinct functional groups have been deployed to discover new polyketide and peptidic natural products. This chapter describes the protocols to conduct a reactivity-based screening campaign, including bacteria cultivation and screening of cellular extracts with phenylglyoxal-, tetrazine-, thiol-, and aminooxy-functionalized probes, which respectively target primary uriedo, electron-rich olefins, Michael acceptors, and reactive carbonyls. In addition, a recent case study is presented that employs reactivity-based screening as a component of a forward genetics screen to identify a previously unknown peptidyl arginine deiminase. We anticipate that these methods will be useful for those interested in discovering natural products that evade detection by traditional, bioassay-guided methods and others who wish to rapidly connect metabolic chemotype with genotype.

Bioinformatics-Guided Expansion and Discovery of Graspetides.

Graspetides are a class of ribosomally synthesized and post-translationally modified peptide natural products featuring ATP-grasp ligase-dependent formation of macrolactones/macrolactams. These modifications arise from serine, threonine, or lysine donor residues linked to aspartate or glutamate acceptor residues. Characterized graspetides include serine protease inhibitors such as the microviridins and plesiocin. Here, we report an update to Rapid ORF Description and Evaluation Online (RODEO) for the automated detection of graspetides, which identified 3,923 high-confidence graspetide biosynthetic gene clusters. Sequence and co-occurrence analyses doubled the number of graspetide groups from 12 to 24, defined based on core consensus sequence and putative secondary modification. Bioinformatic analyses of the ATP-grasp ligase superfamily suggest that extant graspetide synthetases diverged once from an ancestral ATP-grasp ligase and later evolved to introduce a variety of ring connectivities. Furthermore, we characterized thatisin and iso-thatisin, two graspetides related by conformational stereoisomerism from Lysobacter antibioticus. Derived from a newly identified graspetide group, thatisin and iso-thatisin feature two interlocking macrolactones with identical ring connectivity, as determined by a combination of tandem mass spectrometry (MS/MS), methanolytic, and mutational analyses. NMR spectroscopy of thatisin revealed a cis conformation for a key proline residue, while molecular dynamics simulations, solvent-accessible surface area calculations, and partial methanolytic analysis coupled with MS/MS support a trans conformation for iso-thatisin at the same position. Overall, this work provides a comprehensive overview of the graspetide landscape, and the improved RODEO algorithm will accelerate future graspetide discoveries by enabling open-access analysis of existing and emerging genomes.

Reactivity-Based Screening for Citrulline-Containing Natural Products Reveals a Family of Bacterial Peptidyl Arginine Deiminases.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products defined by a genetically encoded precursor peptide that is processed by associated biosynthetic enzymes to form the mature product. Lasso peptides are a class of RiPP defined by an isopeptide linkage between the N-terminal amine and an internal Asp/Glu residue with the C-terminal sequence threaded through the macrocycle. This unique lariat topology, which typically provides considerable stability toward heat and proteases, has stimulated interest in lasso peptides as potential therapeutics. Post-translational modifications beyond the class-defining, threaded macrolactam have been reported, including one example of Arg deimination to yield citrulline (Cit). Although a Cit-containing lasso peptide (i.e., citrulassin) was serendipitously discovered during a genome-guided campaign, the gene(s) responsible for Arg deimination has remained unknown. Herein, we describe the use of reactivity-based screening to discriminate bacterial strains that produce Arg- versus Cit-bearing citrulassins, yielding 13 new lasso peptide variants. Partial phylogenetic profiling identified a distally encoded peptidyl arginine deiminase (PAD) gene ubiquitous to the Cit-containing variants. Absence of this gene correlated strongly with lasso peptide variants only containing Arg (i.e., des-citrulassin). Heterologous expression of the PAD gene in a des-citrulassin producer resulted in the production of the deiminated analog, confirming PAD involvement in Arg deimination. The PADs were then bioinformatically surveyed to provide a deeper understanding of their taxonomic distribution and genomic contexts and to facilitate future studies that will evaluate any additional biochemical roles for the superfamily.

Microviridin 1777: A Toxic Chymotrypsin Inhibitor Discovered by a Metabologenomic Approach.

The toxicity of the cyanobacterium Microcystis aeruginosa EAWAG 127a was evaluated against the sensitive grazer Thamnocephalus platyurus, and the extract possessed strong activity. To investigate the compounds responsible for cytotoxicity, a series of peptides from this cyanobacterium were studied using a combined genomic and molecular networking approach. The results led to the isolation, structure elucidation, and biological evaluation of microviridin 1777, which represents the most potent chymotrypsin inhibitor characterized from this family of peptides to date. Furthermore, the biosynthetic gene clusters of microviridin, anabaenopeptin, aeruginosin, and piricyclamide were located in the producing organism, and six additional natural products were identified by tandem mass spectrometry analyses. These results highlight the potential of modern techniques for the identification of natural products, demonstrate the ecological role of protease inhibitors produced by cyanobacteria, and raise ramifications concerning the presence of novel, yet uncharacterized, toxin families in cyanobacteria beyond microcystin.