The pearl jubilee of microcin J25: thirty years of research on an exceptional lasso peptide.

Covering: 1992 up to 2023Since their discovery, lasso peptides went from peculiarities to be recognized as a major family of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that were shown to be spread throughout the bacterial kingdom. Microcin J25 was first described in 1992, making it one of the earliest known lasso peptides. No other lasso peptide has since then been studied to such an extent as microcin J25, yet, previous review articles merely skimmed over all the research done on this exceptional lasso peptide. Therefore, to commemorate the 30th anniversary of its first report, we give a comprehensive overview of all literature related to microcin J25. This review article spans the early work towards the discovery of microcin J25, its biosynthetic gene cluster, and the elucidation of its three-dimensional, threaded lasso structure. Furthermore, the current knowledge about the biosynthesis of microcin J25 and lasso peptides in general is summarized and a detailed overview is given on the biological activities associated with microcin J25, including means of self-immunity, uptake into target bacteria, inhibition of the Gram-negative RNA polymerase, and the effects of microcin J25 on mitochondria. The in vitro and in vivo models used to study the potential utility of microcin J25 in a (veterinary) medicine context are discussed and the efforts that went into employing the microcin J25 scaffold in bioengineering contexts are summed up.

ABC-HuMi: the Atlas of Biosynthetic Gene Clusters in the Human Microbiome.

The human microbiome has emerged as a rich source of diverse and bioactive natural products, harboring immense potential for therapeutic applications. To facilitate systematic exploration and analysis of its biosynthetic landscape, we present ABC-HuMi: the Atlas of Biosynthetic Gene Clusters (BGCs) in the Human Microbiome. ABC-HuMi integrates data from major human microbiome sequence databases and provides an expansive repository of BGCs compared to the limited coverage offered by existing resources. Employing state-of-the-art BGC prediction and analysis tools, our database ensures accurate annotation and enhanced prediction capabilities. ABC-HuMi empowers researchers with advanced browsing, filtering, and search functionality, enabling efficient exploration of the resource. At present, ABC-HuMi boasts a catalog of 19 218 representative BGCs derived from the human gut, oral, skin, respiratory and urogenital systems. By capturing the intricate biosynthetic potential across diverse human body sites, our database fosters profound insights into the molecular repertoire encoded within the human microbiome and offers a comprehensive resource for the discovery and characterization of novel bioactive compounds. The database is freely accessible at https://www.ccb.uni-saarland.de/abc_humi/.

Current developments in antibiotic discovery: Global microbial diversity as a source for evolutionary optimized anti-bacterials: Global microbial diversity as a source for evolutionary optimized anti-bacterials.

In light of the pending antibiotic resistance crisis, we need to go back to nature and search for novel anti-bacterial compounds.

Fighting antibiotic resistance-strategies and (pre)clinical developments to find new antibacterials.

Antibacterial resistance is one of the greatest threats to human health. The development of new therapeutics against bacterial pathogens has slowed drastically since the approvals of the first antibiotics in the early and mid-20th century. Most of the currently investigated drug leads are modifications of approved antibacterials, many of which are derived from natural products. In this review, we highlight the challenges, advancements and current standing of the clinical and preclinical antibacterial research pipeline. Additionally, we present novel strategies for rejuvenating the discovery process and advocate for renewed and enthusiastic investment in the antibacterial discovery pipeline.

Discovery of a Unique Structural Motif in Lanthipeptide Synthetases for Substrate Binding and Interdomain Interactions.

Class III lanthipeptide synthetases catalyze the formation of lanthionine/methyllanthionine and labionin crosslinks. We present here the 2.40 Šresolution structure of the kinase domain of a class III lanthipeptide synthetase CurKC from the biosynthesis of curvopeptin. A unique structural subunit for leader binding, named leader recognition domain (LRD), was identified. The LRD of CurKC is responsible for the recognition of the leader peptide and for mediating interactions between the lyase and kinase domains. LRDs are highly conserved among the kinase domains of class III and class IV lanthipeptide synthetases. The discovery of LRDs provides insight into the substrate recognition and domain organization in multidomain lanthipeptide synthetases.

Unexpected Methyllanthionine Stereochemistry in the Morphogenetic Lanthipeptide SapT.

Lanthipeptides are polycyclic peptides characterized by the presence of lanthionine (Lan) and/or methyllanthionine (MeLan). They are members of the ribosomally synthesized and post-translationally modified peptides (RiPPs). The stereochemical configuration of (Me)Lan cross-links is important for the bioactivity of lanthipeptides. To date, MeLan residues in characterized lanthipeptides have either the 2S,3S or 2R,3R stereochemistry. Herein, we reconstituted in Escherichia coli the biosynthetic pathway toward SapT, a class I lanthipeptide that exhibits morphogenetic activity. Through the synthesis of standards, the heterologously produced peptide was shown to possess three MeLan residues with the 2S,3R stereochemistry (d-allo-l-MeLan), the first time such stereochemistry has been observed in a lanthipeptide. Bioinformatic analysis of the biosynthetic enzymes suggests this stereochemistry may also be present in other lanthipeptides. Analysis of another gene cluster in Streptomyces coelicolor that is widespread in actinobacteria confirmed another example of d-allo-l-MeLan and verified the bioinformatic prediction. We propose a mechanism for the origin of the unexpected stereochemistry and provide support using site-directed mutagenesis.

Disrupting Transcription and Folate Biosynthesis Leads to Synergistic Suppression of Escherichia coli Growth.

The use of synergistic antibiotic combinations has emerged as a viable approach to contain the rapid spread of antibiotic-resistant pathogens. Here we report the discovery of a new strongly synergistic pair - microcin J25 and sulfamonomethoxine. The former is a lasso peptide that inhibits the function of RNA polymerase and the latter is a sulfonamide antibacterial agent that disrupts the folate pathway. Key to our discovery was a screening strategy that focuses on an antibiotic (microcin J25) that targets a hub (transcription) in the densely interconnected network of cellular pathways. The rationale was that disrupting such a hub likely weakens the entire network, generating weak links that potentiate the growth inhibitory effect of other antibiotics. We found that MccJ25 potentiates five other antibiotics as well. These results showcase the merit of taking a more targeted approach in the search and study of synergistic antibiotic pairs.

Combined thermal and carboxypeptidase Y stability assays for probing the threaded fold of lasso peptides.

Lasso peptides are natural products belonging to the superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). The defining characteristic of lasso peptides is their threaded structure, which is reminiscent of a lariat knot. When working with lasso peptides, it is therefore of major importance to understand and evidence their threaded folds. While the full elucidation of their three-dimensional structures via NMR spectroscopy or crystallization remains the gold standard, these methods are time-consuming, require large quantities of highly pure lasso peptides, and therefore might not always be applicable. Instead, the unique properties of lasso peptides in context of their behavior at elevated temperatures and toward carboxypeptidase Y treatment can be leveraged as a tool to investigate and evidence the threaded lasso fold using only minute amounts of compound that does not need to be purified first. This chapter will provide insights into the thermal stability properties of lasso peptides and their behavior when treated with carboxypeptidase Y in comparison to a branched-cyclic peptide with the same amino acid sequence. Furthermore, it will be described in detail how to set up a combined thermal and carboxypeptidase Y stability assay and how to analyze its outcomes.

A Bifunctional Leader Peptidase/ABC Transporter Protein Is Involved in the Maturation of the Lasso Peptide Cochonodin I from Streptococcus suis.

Lasso peptides are members of the natural product superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we describe the first lasso peptide originating from a biosynthetic gene cluster belonging to a unique lasso peptide subclade defined by the presence of a bifunctional protein harboring both a leader peptidase (B2) and an ABC transporter (D) domain. Bioinformatic analysis revealed that these clusters also encode homologues of the NisR/NisK regulatory system and the NisF/NisE/NisG immunity factors, which are usually associated with the clusters of antimicrobial class I lanthipeptides, such as nisin, another distinct RiPP subfamily. The cluster enabling the heterologous production of the lasso peptide cochonodin I in E. coli originated from Streptococcus suis LSS65, and the threaded structure of cochonodin I was evidenced through extensive MS/MS analysis and stability assays. It was shown that the ABC transporter domain from SsuB2/D is not essential for lasso peptide maturation. By extensive genome mining dedicated exclusively to other lasso peptide biosynthetic gene clusters featuring bifunctional B2/D proteins, it was furthermore revealed that many bacteria associated with human or animal microbiota hold the biosynthetic potential to produce cochonodin-like lasso peptides, implying that these natural products might play roles in human and animal health.

Identification of the Catalytic Residues in the Cyclase Domain of the Class IV Lanthipeptide Synthetase SgbL.

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are subdivided into different classes based on their processing enzymes. The three-domain class IV lanthipeptide synthetases (LanL enzymes) consist of N-terminal lyase, central kinase, and C-terminal cyclase domains. While the catalytic residues of the kinase domains (mediating ATP-dependent Ser/Thr phosphorylations) and the lyase domains (carrying out subsequent phosphoserine/phosphothreonine (pSer/pThr) eliminations to yield dehydroalanine/dehydrobutyrine (Dha/Dhb) residues) have been characterized previously, such studies are missing for LanL cyclase domains. To close this gap of knowledge, this study reports on the identification and validation of the catalytic residues in the cyclase domain of the class IV lanthipeptide synthetase SgbL, which facilitate the nucleophilic attacks by Cys thiols on Dha/Dhb residues for the formation of ß-thioether crosslinks.

Exploring structural signatures of the lanthipeptide prochlorosin 2.8 using tandem mass spectrometry and trapped ion mobility-mass spectrometry.

Lanthipeptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by intramolecular thioether cross-links formed between a dehydrated serine/threonine (dSer/dThr) and a cysteine residue. Prochlorosin 2.8 (Pcn2.8) is a class II lanthipeptide that exhibits a non-overlapping thioether ring pattern, for which no biological activity has been reported yet. The variant Pcn2.8[16RGD] has been shown to bind tightly to the αvß3 integrin receptor. In the present work, tandem mass spectrometry, using collision-induced dissociation (CID) and electron capture dissociation (ECD), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) were used to investigate structural signatures for the non-overlapping thioether ring pattern of Pcn2.8. CID experiments on Pcn2.8 yielded bi and yj fragments between the thioether cross-links, evidencing the presence of a non-overlapping thioether ring pattern. ECD experiments of Pcn2.8 showed a significant increase of hydrogen migration events near the residues involved in the thioether rings with a more pronounced effect at the dehydrated residues as compared to the cysteine residues. The high-resolution mobility analysis, aided by site-directed mutagenesis ([P8A], [P11A], [P12A], [P8A/P11A], [P8A/P12A], [P11A/P12A], and [P8A/P11A/P12A] variants), demonstrated that Pcn2.8 adopts cis/trans-conformations at Pro8, Pro11, and Pro12 residues. These observations were complementary to recent NMR findings, for which only the Pro8 residue was evidenced to adopt cis/trans-orientations. This study highlights the analytical power of the TIMS-MS/MS workflow for the structural characterization of lanthipeptides and could be a useful tool in our understanding of the biologically important structural elements that drive the thioether cyclization process.

Light-Controlled Tyrosine Nitration of Proteins.

Tyrosine nitration of proteins is one of the most important oxidative post-translational modifications in vivo. A major obstacle for its biochemical and physiological studies is the lack of efficient and chemoselective protein tyrosine nitration reagents. Herein, we report a generalizable strategy for light-controlled protein tyrosine nitration by employing biocompatible dinitroimidazole reagents. Upon 390 nm irradiation, dinitroimidazoles efficiently convert tyrosine residues into 3-nitrotyrosine residues in peptides and proteins with fast kinetics and high chemoselectivity under neutral aqueous buffer conditions. The incorporation of 3-nitrotyrosine residues enhances the thermostability of lasso peptide natural products and endows murine tumor necrosis factor-α with strong immunogenicity to break self-tolerance. The light-controlled time resolution of this method allows the investigation of the impact of tyrosine nitration on the self-assembly behavior of α-synuclein.

Structural Insights from Tandem Mass Spectrometry, Ion Mobility-Mass Spectrometry, and Infrared/Ultraviolet Spectroscopy on Sphingonodin I: Lasso vs Branched-Cyclic Topoisomers.

Lasso peptides form a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked topology, where the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Sphingonodin I is a lasso peptide that has not yet been structurally characterized using the traditional structural biology tools (e.g., NMR and X-ray crystallography), and its biological function has not yet been elucidated. In the present work, we describe structural signatures characteristic of the class II lasso peptide sphingonodin I and its branched-cyclic analogue using a combination of gas-phase ion tools (e.g., tandem mass spectrometry, MS/MS, trapped ion mobility spectrometry, TIMS, and infrared, IR, and ultraviolet, UV, spectroscopies). Tandem MS/MS CID experiments on sphingonodin I yielded mechanically interlocked species with associated bi and yj fragments demonstrating the presence of a lasso topology, while tandem MS/MS ECD experiments on sphingonodin I showed a significant increase in hydrogen migration in the loop region when compared to the branched-cyclic analogue. The high-mobility resolving power of TIMS permitted the separation of both topoisomers, where sphingonodin I adopted a more compact structure than its branched-cyclic analogue. Cryogenic and room-temperature IR spectroscopy experiments evidenced a different hydrogen bond network between the two topologies, while cryogenic UV spectroscopy experiments clearly demonstrated a distinct phenylalanine environment for the lasso peptide.

Generation of Lasso Peptide-Based ClpP Binders.

The Clp protease system fulfills a plethora of important functions in bacteria. It consists of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25 (MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly, synergistic interactions between 12IGF and ADEP were observed.

Matters of class: coming of age of class III and IV lanthipeptides.

Lanthipeptides belong to the superfamily of ribosomally-synthesized and posttranslationally-modified peptides (RiPPs). Despite the fact that they represent one of the longest known RiPP subfamilies, their youngest members, classes III and IV, have only been described more recently. Since then, a plethora of studies furthered the understanding of their biosynthesis. While there are commonalities between classes III and IV due to the similar domain architectures of their processing enzymes, there are also striking differences that allow their discrimination. In this concise review article, we summarize what is known about the underlying biosynthetic principles of these lanthipeptides and discuss open questions for future research.

Factors Governing the Thermal Stability of Lasso Peptides.

Lasso peptides belong to the natural product superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). They are defined by an N-terminal macrolactam ring that is threaded by the C-terminal tail. In class II lasso peptides, this fold is maintained only through steric hindrance. Nonetheless, this fold can often withstand prolonged incubation at highly elevated temperatures. However, some lasso peptides will irreversibly unthread into their branched-cyclic counterparts upon heating. In recent years, an increasing number of research studies have focused on studying the factors that govern the thermal stability (or the lack thereof) of lasso peptides by using in vitro stability assays, mutational analysis, and molecular dynamics simulations. In this review, the current state of understanding the physicochemical parameters deciding the fate of a lasso peptide at elevated temperatures is discussed, and an overview is given of the techniques developed to streamline the separation and discrimination of lasso peptides from their branched-cyclic topoisomers.

Genome Mining and Heterologous Expression Reveal Two Distinct Families of Lasso Peptides Highly Conserved in Endofungal Bacteria.

Genome mining identified the fungal-bacterial endosymbiosis Rhizopus microsporus-Mycetohabitans (previously Burkholderia) rhizoxinica as a rich source of novel natural products. However, most of the predicted compounds have remained cryptic. In this study, we employed heterologous expression to isolate and characterize three ribosomally synthesized and post-translationally modified peptides with lariat topology (lasso peptides) from the endosymbiont M. rhizoxinica: burhizin-23, mycetohabin-16, and mycetohabin-15. Through coexpression experiments, it was shown that an orphan gene product results in mature mycetohabin-15, albeit encoded remotely from the core biosynthetic gene cluster. Comparative genomics revealed that mycetohabins are highly conserved among M. rhizoxinica and related endosymbiotic bacteria. Gene knockout and reinfection experiments indicated that the lasso peptides are not crucial for establishing symbiosis; instead, the peptides are exported into the environment during endosymbiosis. This is the first report on lasso peptides from endosymbiotic bacteria.

Mechanistic Studies of the Kinase Domains of Class IV Lanthipeptide Synthetases.

Lanthipeptides, which belong to the superfamily of ribosomally synthesized and posttranslationally modified peptides (RiPPs), are associated with various interesting biological activities. Lanthipeptides can be subdivided into four classes that are defined by the characteristics of the corresponding posttranslational modification enzymes. Class IV lanthipeptide synthetases consist of an N-terminal lyase, a central kinase, and a C-terminal cyclase domain. Here, we present the first in-depth characterization of such a kinase domain from the globisporin maturation enzyme SgbL that originates from Streptomyces globisporus sp. NRRL B-2293. Catalytic residues were identified by alignments with homologues and structural modeling. Their roles were confirmed by employing proteins with Ala substitutions in in vitro modification and fluorescence polarization binding assays. Furthermore, the protein region that is binding the leader peptide was identified by hydrogen-deuterium exchange-mass spectrometry experiments. By fusion of this protein region to the maltose binding protein, a protein was generated that can specifically bind the SgbA leader peptide, albeit with reduced binding affinity compared to that of full length SgbL. Combined, the results of this study provide a firmer grasp of how lanthipeptide biosynthesis is accomplished by class IV synthetases and suggest by homology analysis that biosynthetic mechanisms are similar in class III lanthipeptide processing enzymes.

Assessing the Flexibility of the Prochlorosin 2.8 Scaffold for Bioengineering Applications.

Cyclization is a common strategy to confer proteolytic resistance to peptide scaffolds. Thus, cyclic peptides have been the focus of extensive bioengineering efforts. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a superfamily of peptidic natural products that often contain macrocycles. In the RiPP family of lanthipeptides, macrocyclization is accomplished through formation of thioether cross-links between cysteines and dehydrated serines/threonines. The recent production of lanthipeptide libraries and development of methods to display lanthipeptides on yeast or phage highlights their potential for bioengineering and synthetic biology. In this regard, the prochlorosins are especially promising as the corresponding class II lanthipeptide synthetase ProcM matures numerous precursor peptides with diverse core peptide sequences. To facilitate future bioengineering projects, one of its native substrates, ProcA2.8, was subjected in this study to in-depth mutational analysis to test the limitations of ProcM-mediated cyclization. Alanine scan mutagenesis was performed on all residues within the two rings, and multiple prolines were introduced at various positions. Moreover, mutation, deletion, and insertion of residues in the region linking the two lanthionine rings was tested. Additional residues were also introduced or deleted from either ring, and inversion of ring forming residues was attempted to generate diastereomers. The findings were used for epitope grafting of the RGD integrin binding epitope within prochlorosin 2.8, resulting in a low nanomolar affinity binder of the αvß3 integrin that was more stable toward proteolysis and displayed higher affinity than the linear counterpart.

Evidence of Cis/Trans-Isomerization at Pro7/Pro16 in the Lasso Peptide Microcin J25.

Microcin J25 is a ribosomal synthesized and post-translationally modified peptide (RiPP) characterized by a mechanically interlocked topology called the lasso fold. This structure provides microcin J25 a potent antimicrobial activity resulting from internalization via the siderophore receptor FhuA and further inhibition of the RNA polymerase. In the present work, nuclear magnetic resonance (NMR) and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) were used to investigate the lasso structure of microcin J25. NMR experiments showed that the lasso peptide microcin J25 can adopt conformational states where Pro16 can be found in the cis- and trans-orientations. The high-resolution mobility analysis, aided by site-directed mutagenesis ([P7A], [P16A], and [P7A/P16A] variants), demonstrated that microcin J25 can adopt cis/cis-, cis/trans-, trans/cis-, and trans/trans-conformations at the Pro7 and Pro16 peptide bonds. It was also shown that interconversion between the conformers can occur as a function of the starting solvent conditions and ion heating (collision-induced activation, CIA) despite the lasso topology. Complementary to NMR findings, the cis-conformations at Pro7 were assigned using TIMS-MS. This study highlights the analytical power of TIMS-MS and site-directed mutagenesis for the study of biological systems with large micro-heterogeneity as a way to further increase our understanding of the receptor-binding dynamics and biological activity.