Macrolactam Ring Research Articles

Total Synthesis of the Guangnanmycin A Alcohol

AbstractGuangnanmycin A is a recently discovered congener of the well‐known antitumor drug lead leinamycin; its macrolactam ring, however, is even more strained than that of the parent compound. The first synthetic foray towards this challenging target is reported, which relies on molybdenum‐catalyzed macrocyclization by ring closing alkyne metathesis (RCAM) followed by ruthenium‐catalyzed redox isomerization of the propargyl alcohol thus formed; the resulting enone enabled the introduction of the yet missing exo‐methylene group by a modified Peterson olefination. The signature disulfide moiety of guangnanmycin A was installed by strain‐driven thia‐Michael addition followed by conversion of the thioether thus formed into an unsymmetric disulfide with the aid of (methylthio)dimethylsulfonium tetrafluoroborate and MeSSMe. While this sequence furnished racemic guangnanmycin A alcohol in good overall yield, the final oxidation to the corresponding acid failed, most likely because of the exceptional sensitivity of the strained scaffold.

Total Synthesis of the Guangnanmycin A Alcohol.

Guangnanmycin A is a recently discovered congener of the well-known antitumor drug lead leinamycin; its macrolactam ring, however, is even more strained than that of the parent compound. The first synthetic foray towards this challenging target is reported, which relies on molybdenum-catalyzed macrocyclization by ring closing alkyne metathesis (RCAM) followed by ruthenium-catalyzed redox isomerization of the propargyl alcohol thus formed; the resulting enone enabled the introduction of the yet missing exo-methylene group by a modified Peterson olefination. The signature disulfide moiety of guangnanmycin A was installed by strain-driven thia-Michael addition followed by conversion of the thioether thus formed into an unsymmetric disulfide with the aid of (methylthio)dimethylsulfonium tetrafluoroborate and MeSSMe. While this sequence furnished racemic guangnanmycin A alcohol in good overall yield, the final oxidation to the corresponding acid failed, most likely because of the exceptional sensitivity of the strained scaffold.

Genome mining for macrolactam-encoding gene clusters allowed for the network-guided isolation of β-amino acid-containing cyclic derivatives and heterologous production of ciromicin A

β-Amino acid-containing macrolactams represent a structurally diverse group of bioactive natural products derived from polyketides; however we are currently lacking a comprehensive overview about their abundance across bacterial families and the underlying biosynthetic diversity. In this study, we employed a targeted β-amino acid-specific homology-based multi-query search to identify potential bacterial macrolactam producers. Here we demonstrate that approximately 10% of each of the identified actinobacterial genera harbor a biosynthetic gene cluster (BGC) encoding macrolactam production. Based on our comparative study, we propose that mutations occurring in specific regions of polyketide synthases (PKS) are the primary drivers behind the variation in macrolactam ring sizes. We successfully validated two producers of ciromicin A from the genus Amycolatopsis, revised the composition of the biosynthetic gene cluster region mte of macrotermycins, and confirmed the ciromicin biosynthetic pathway through heterologous expression. Additionally, network-based metabolomic analysis uncovered three previously unreported macrotermycin congeners from Amycolatopsis sp. M39. The combination of targeted mining and network-based analysis serves as a powerful tool for identifying macrolactam producers and our studies will catalyze the future discovery of yet unreported macrolactams.

Genomic Insight into Shimazuella Soli Sp. Nov. Isolated from Soil and Its Putative Novel Class II Lasso Peptide.

The strain designated as AN120528T was isolated from farmland soil in South Korea. This strain grows well on R2A medium at 28 °C. The cells are an off-white colour and have no hyphae. The phylogenetic analysis indicated that the strain is a member of the genus Shimazuella with a 98.11% similarity to Shimazuella alba KC615T and a 97.05% similarity to S. kribbensis KCTC 9933T, respectively. The strain AN120528T shares common chemotaxonomic features with the other two type strains in the genus. It has MK-9 (H4) and MK-10 (H4) as its predominant menaquinones. The major fatty acids are iso-C14:0, iso-C15:0, anteiso-C15:0 and iso-C16:0. Diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), lipids (L), and aminolipids (AL) were identified as the major cellular polar lipids. Analysis of the peptidoglycan showed the presence of meso-diaminopimelic acid. Whole-genome sequencing revealed that the genome of the strain is approximately 3.3 Mbp in size. The strain showed a 77.5% average nucleotide identity (ANI) with S. alba KC615T. The genomic DNA (gDNA) G + C content is 39.0%. Based on polyphasic taxonomy analysis, it is proposed that this strain, AN120528T, represents a novel species in the genus Shimazuella, designated as Shimazuella soli sp. nov. The type stain is AN120528T (=KCTC 39810T = DSM 103571T). Furthermore, shimazuellin I, a new 15-amino-acid peptide, was discovered in the AN120528T through genome mining; it has the features of a lasso peptide, containing eight amino acids (-G-Q-G-G-S-N-N-D-) that form a macrolactam ring and seven amino acids (-D-G-W-Y-H-S-K-) that form a tail.

C-terminal modification of a de novo designed antimicrobial peptide via capping of macrolactam rings

In this work, by capping a macrolactam ring at the C-terminus of a de novo-designed peptide, namely zp80, we have constructed a small peptide library via the solid phase peptide synthesis for screening. Eight peptides bearing different aspartic acid-rich macrolactam rings but the same linear (IIRR)4 unit exhibited improved antibacterial activities, hemolytic activity, and selectivity index. Mechanistic studies revealed that they could destroy the integrity of bacterial envelope, leading to cytoplasm leakage and rapid dissipation of membrane potential. One of these peptides, zp90 with a macrolactam ring of (KaDGD), demonstrated preferential interaction with calcium ions at a stoichiometric ratio of 1:1, promoting the affinity of designed peptides to bacterial membrane. Overall, this work provides a feasible strategy for medicinal chemists to further develop potent, selective, and multifunctional de novo-designed antimicrobial peptides.

Puzzling Out the Structure of Novofumigatamide: Total Synthesis of Constitutional Isomers. Part II.

The total synthesis of several constitutional isomers showing a different connectivity of the macrolactam ring with the hexahydropyrrolo[2,3-b]indole core, as well as those arising from the positional exchange of the valine and the anthranilate units of the structure originally proposed for (−)-novofumigatamide, has been carried out. The constitutional isomers with 12-membered ring macrolactam connected with the pyrroloindoline framework through the indole nitrogen, and the acetyl group at the pyrrole nitrogen, of endo relative configuration, were prepared through the condensation between the tryptophan and valine edges derived from l- or d-tryptophan and l-valine amino acids. The corresponding exo products are highly unstable structures difficult to isolate and characterize. A second group of isomeric structures synthesized considered the positional exchange between the valine and the anthranilate residues within the macrolactam ring in the originally proposed macrocyclic structure. Comparison of the spectroscopic data allowed us to discard these alternative structures for the natural product.

Inhibiting the Interaction Between von Willebrand Factor and Collagen Using Novel Peptides to Prevent Thrombosis Initiation

The field of peptide pharmaceuticals is gaining important prominence. Peptides have an attractive pharmacological profile and excellent safety, tolerability and efficacy in humans which makes them ideal candidates for new drugs. The macrocyclic arrangements of the amino acids in peptides can provide added stability, therefore the use of cyclic or looped peptides displaying unique secondary structure can aid in stability and efficacy. Heart attacks and strokes, many of which are attributed to arterial blood clots, remain major health concerns in America. Current therapies and preventatives target platelet interactions at the site of a clot and are rife with complications. We have previously targeted the interaction between von Willebrand Factor (vWF) and collagen, which initiates thrombosis, using our exceptionally stable heat‐to‐tail cyclized peptides to moderate potency. Due to the favorability of peptides as drug candidates, as well as the need to target thrombosis in a unique way that precludes platelets, our objective is to develop peptide inhibitors to clot initiation. Our current objective is broken into three key pieces: 1) the development of cyclic peptides with unnatural amino acid substitutions, 2) honing the structures and sequences of our cyclic peptides using rational design and 3) targeting the interaction using substituted lasso peptides displaying an epitope from vWF. Lasso peptides represent a special class of bacterial natural products containing a threaded macrocycle structure with a looped macrolactam ring, an isopeptide bond between the N‐terminus and an acidic side chain, and the C‐terminal tail threaded and trapped by steric hindrance. These peptides can behave as a macromolecular scaffold, retaining their structure when substitutions are made to the loop region. We hypothesize continued success in developing remarkably stable cyclic peptides that target the vWF‐collagen interface, as well as lasso peptides with unique function. A comparison between the cyclic and lasso peptides will provide relationships between stability, structure and efficacy. The potency of each peptide is tested using our novel fluorescently‐linked immunosorbent assay, using anti‐vWF antibodies to detect the presence or absence of vWF bound to collagen‐coated plates when treated with our peptides in a dose‐dependent manner. Additionally, protease stability assays testing our peptides with a panel of peptide‐degrading enzymes in cellular conditions provides a baseline of stability. In summation, we found that unnatural amino acid substitutions were well tolerated by the cyclic peptides and led to some improvements in efficacy and stability. The lasso peptides recombinantly express in abundance and are currently being tested for stability and inhibitor capabilities in comparison to the cyclic peptides. Therefore, all of these designs provide a springboard for future advances in exceptionally stable, active cyclic peptide drugs. Overall, our studies inform future peptidomimetic designs, especially in the development of short, structured peptides with biological function.

Discovery and Characterization of Rubrinodin Provide Clues into the Evolution of Lasso Peptides.

Lasso peptides are unique natural products that comprise a class of ribosomally synthesized and post-translationally modified peptides. Their defining three-dimensional structure is a lariat knot, in which the C-terminal tail is threaded through a macrolactam ring formed between the N-terminal amino group and an Asp or Glu side chain (i.e., an isopeptide bond). Recent genome mining strategies have revealed various types of lasso peptide biosynthetic gene clusters and have thus redefined the known chemical space of lasso peptides. To date, over 20 different types of these gene clusters have been discovered, including several different clades from Proteobacteria. Despite the diverse architectures of these gene clusters, which may or may not encode various tailoring enzymes, most currently known lasso peptides are synthesized by two discrete clades defined by the presence of an ATP-binding cassette transporter or its absence and (sometimes) concurrent appearance of an isopeptidase, raising questions about their evolutionary history. Herein, we discovered and characterized the lasso peptide rubrinodin, which is assembled by a gene cluster encoding both an ATP-binding cassette transporter and an isopeptidase. Our bioinformatics analyses of this and other representative cluster types provided new clues into the evolutionary history of lasso peptides. Furthermore, our structural and biochemical investigations of rubrinodin permitted the conversion of this thermolabile lasso peptide into a more thermostable scaffold.

Recent Advances and Perspectives on Expanding the Chemical Diversity of Lasso Peptides

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of natural products that exhibit a range of structures and bioactivities. Initially assembled from the twenty proteinogenic amino acids in a ribosome-dependent manner, RiPPs assume their peculiar bioactive structures through various post-translational modifications. The essential modifications representative of each subfamily of RiPP are performed on a precursor peptide by the so-called processing enzymes; however, various tailoring enzymes can also embellish the precursor peptide or processed peptide with additional functional groups. Lasso peptides are an interesting subfamily of RiPPs characterized by their unique lariat knot-like structure, wherein the C-terminal tail is inserted through a macrolactam ring fused by an isopeptide bond between the N-terminal amino group and an acidic side chain. Until recently, relatively few lasso peptides were found to be tailored with extra functional groups. Nevertheless, the development of new routes to diversify lasso peptides and thus introduce novel or enhanced biological, medicinally relevant, or catalytic properties is appealing. In this review, we highlight several strategies through which lasso peptides have been successfully modified and provide a brief overview of the latest findings on the tailoring of these peptides. We also propose future directions for lasso peptide tailoring as well as potential applications for these peptides in hybrid catalyst design.

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.

Attempting to synthesize lasso peptides using high pressure.

Lasso peptides are unique in that the tail of the lasso peptide threads through its macrolactam ring. The unusual structure and biological activity of lasso peptides have generated increased interest from the scientific community in recent years. Because of this, many new types of lasso peptides have been discovered. These peptides can be synthesized by microorganisms efficiently, and yet, their chemical assembly is challenging. Herein, we investigated the possibility of high pressure inducing the cyclization of linear precursors of lasso peptides. Unlike other molecules like rotaxanes which mechanically interlock at high pressure, the threaded lasso peptides did not form, even at pressures the high pressure up to 14 000 kbar.

New Sipanmycin Analogues Generated by Combinatorial Biosynthesis and Mutasynthesis Approaches Relying on the Substrate Flexibility of Key Enzymes in the Biosynthetic Pathway.

The appearance of new infectious diseases, the increase in multidrug-resistant bacteria, and the need for more effective chemotherapeutic agents have oriented the interests of researchers toward the search for metabolites with novel or improved bioactivities. Sipanmycins are disaccharyl glycosylated macrolactams that exert antibiotic and cytotoxic activities. By applying combinatorial biosynthesis and mutasynthesis approaches, we have generated eight new members of the sipanmycin family. The introduction of plasmids harboring genes responsible for the biosynthesis of several deoxysugars into sipanmycin-producing Streptomyces sp. strain CS149 led to the production of six derivatives with altered glycosylation patterns. After structural elucidation of these new metabolites, we conclude that some of these sugars are the result of the combination of the enzymatic machinery encoded by the introduced plasmids and the native enzymes of the d-sipanose biosynthetic pathway of the wild-type CS149 strain. In addition, two analogues of the parental compounds with a modified polyketide backbone were generated by a mutasynthesis approach, feeding cultures of a mutant strain defective in sipanmycin biosynthesis with 3-aminopentanoic acid. The generation of new sipanmycin analogues shown in this work relied on the substrate flexibility of key enzymes involved in sipanmycin biosynthesis, particularly the glycosyltransferase pair SipS9/SipS14 and enzymes SipL3, SipL1, SipL7, and SipL2, which are involved in the incorporation of the polyketide synthase starting unit.IMPORTANCE Combinatorial biosynthesis has proved its usefulness in generating derivatives of already known compounds with novel or improved pharmacological properties. Sipanmycins are a family of glycosylated macrolactams produced by Streptomyces sp. strain CS149, whose antiproliferative activity is dependent on the sugar moieties attached to the aglycone. In this work, we report the generation of several sipanmycin analogues with different deoxysugars, showing the high degree of flexibility exerted by the glycosyltransferase machinery with respect to the recognition of diverse nucleotide-activated sugars. In addition, modifications in the macrolactam ring were introduced by mutasynthesis approaches, indicating that the enzymes involved in incorporating the starter unit have a moderate ability to introduce different types of β-amino acids. In conclusion, we have proved the substrate flexibility of key enzymes involved in sipanmycin biosynthesis, especially the glycosyltransferases, which can be exploited in future experiments.

Downsizing Class II Lasso Peptides: Genome Mining-Guided Isolation of Huascopeptin Containing the First Gly1-Asp7 Macrocycle.

A new lasso peptide, huascopeptin, was isolated following genome-mined discovery of a new biosynthetic gene cluster in extremotolerant Streptomyces huasconensis HST28T from Salar de Huasco, Atacama Desert, Chile. Compound 1 is a 13-residue class II lasso peptide containing a novel Gly1-Asp7 macrolactam ring, a three-residue loop, and a three-residue tail, making it the smallest lasso peptide isolated to date. The lasso structure was confirmed using NOE restraint-based molecular dynamics simulations.

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.

Structural Basis of Leader Peptide Recognition in Lasso Peptide Biosynthesis Pathway

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique 3D-interlocked structure, in which an N-terminal macrolactam ring is threaded by a linear C-terminal part. The unique structure of lasso peptides is introduced into ribosomally translated precursor peptides by lasso peptide synthetase encompassing proteins B and C or B1, B2, and C when the B enzyme is split into two distinct proteins. The B1 protein recognizes the leader sequence of the precursor peptide, and then the B2 protein cleaves it. The C protein catalyzes the formation of the macrolactam ring. However, the detailed mechanism of lasso peptide maturation has remained elusive, due to the lack of structural information about the responsible proteins. Here we report the crystal structure of the B1 protein from the thermophilic actinobacteria, Thermobifida fusca (TfuB1), complexed with the leader peptide (TfuA-Leader), which revealed the detailed mechanism of leader peptide recognition. The structure of TfuB1 consists of an N-terminal β-sheet and three C-terminal helices. The leader peptide is docked on one edge of the N-terminal β-sheet of TfuB1, as an additional β strand. Three conserved amino acid residues of the leader peptide (TfuA Tyr-17, Pro-14, and Leu-12) fit well on the hydrophobic cleft between the β-sheet and adjacent helices. Biochemical analysis demonstrated that these conserved residues are essential for affinity between TfuB1 and the TfuA-Leader. Furthermore, we found that TfuB1 and the leader peptide jointly form a hydrophobic patch on the β-sheet, which includes the highly conserved TfuA Phe-6 and TfuB1 Tyr33. Homology modeling and mutational analysis of the B1 protein from a firmicute, Bacillus pseudomycoides (PsmB1), revealed that the hydrophobic patch is conserved in a wide range of species and involved in the cleavage activity of the B2 protein, indicating it forms the interaction surface for the B2 protein or the core part of the precursor peptide.

Structural signatures of the class III lasso peptide BI-32169 and the branched-cyclic topoisomers using trapped ion mobility spectrometry-mass spectrometry and tandem mass spectrometry.

Lasso peptides are a class of bioactive 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. BI-32169 is a class III lasso peptide containing one disulfide bond that further stabilizes the lasso structure. In contrast to its branched-cyclic analog, BI-32169 has higher stability and is known to exert a potent inhibitory activity against the human glucagon 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) experiments were carried out to evidence specific structural signatures of the two topologies. CID experiments showed similar fragmentation patterns for the two topoisomers, where a part of the C-terminal tail remains covalently linked to the macrolactam ring by the disulfide bond, which cannot clearly constitute a signature of the lasso topology. ECD experiments of BI-32169 showed an increase of hydrogen migration events in the loop region when compared with those of its branched-cyclic topoisomer evidencing specific structural signatures for the lasso topology. The high mobility resolving power of TIMS resulted in the identification of multiple conformations for the protonated species but did not allow the clear differentiation of the two topologies in mixture. When in complex with cesium metal ions, a reduced number of conformations led to a clear identification of the two structures. Experiments reducing and alkylating the disulfide bond of BI-32169 showed that the lasso structure is preserved and heat stable and the associated conformational changes provide new insights about the role of the disulfide bond in the inhibitory activity against the human glucagon receptor. Graphical abstract ᅟ.

General rules of fragmentation evidencing lasso structures in CID and ETD.

Lasso peptides constitute a structurally unique class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by a mechanically interlocked structure in which the C-terminal tail of the peptide is threaded and trapped within an N-terminal macrolactam ring. Tandem mass spectrometry using collision induced dissociation (CID) and electron capture dissociation (ECD) have shown previously different fragmentation patterns for capistruin, microcin J25 and their corresponding branched-cyclic forms in which the C-terminal tail is unthreaded. In order to develop general rules that unambiguously discriminate the lasso and branched-cyclic topologies, this report presents experimental evidence for a set of twenty-one lasso peptides analyzed by CID and electron transfer dissociation (ETD). CID experiments on lasso peptides specifically yielded mechanically interlocked species with associated bi and yj fragments. For class II lasso peptides, these lasso-specific fragments were observed only for peptides in which the loop, located above the macrolactam ring, was strictly longer than four amino acid residues. For class I and III lasso peptides, part of the C-terminal tail remains covalently linked to the macrolactam ring by disulfide bonds; associated bi and yj fragments therefore do not clearly constitute a signature of the lasso topology. ETD experiments of lasso peptides showed a significant increase of hydrogen migration events in the loop region when compared to their branched-cyclic topoisomers, leading to the formation of specific ci˙/z'j fragments for all lasso peptides, regardless of their class and loop size. Our experiments enabled us to establish general rules for obtaining structural details from CID and ETD fragmentation patterns, obviating the need for structure determination by NMR or X-ray crystallography.

Isolation and structure determination of a new lasso peptide subterisin from Sphingomonas subterranea

A new lasso peptide named subterisin was isolated from the culture broth of Sphingomonas subterranea NBRC 16086T. The molecular formula of subterisin was established as C78H121O22N21 based on accurate mass analysis. The chemical structure of subterisin was determined by 2D NMR experiments. The presence of macrolactam ring of Gly1–Glu8 was indicated by NOESY experiment and MS/MS analysis. The three-dimensional structure of subterisin in solution was established by calculation based on NMR data. The proposed biosynthetic gene cluster of subterisin was found on the genome of S. subterranea.

Isolation and identification of a new lasso peptide cattlecin from Streptomyces cattleya based on genome mining

Lasso peptides are ribosomally synthesized and posttranslationally modified peptides with diverse biological functions. Recent genome mining has revealed that many species of actinomycetes possibly contain biosynthetic gene clusters of lasso peptides. With genome mining for lasso peptide biosynthesis, we screened several actinomycetes for lasso peptide production using high-performance liquid chromatography and electrospray ionization–mass spectrometry. Consequently, Streptomyces cattleya was identified as a producer of a new lasso peptide named cattlecin. Analysis of amino acid content on cattlecin indicated the presence of four moles each of Asp and His, three moles each of Gly and Tyr, and one mole of Ser. Tandem mass spectrometry (MS/MS) analysis of cattlecin revealed C-terminal sequence of WHHGWYGWWDD. The peptide sequence (SYHWGDYHDWHHGWYGWWDD) was the expected amino acid sequence of cattlecin based on genome mining. As a result of MS/MS analysis, the amine residue of the first Ser was proposed to form a macrolactam ring with the β-carboxyl residue of the ninth Asp. The biosynthetic gene cluster of cattlecin comprised four genes: catA, catC, catB1, and catB2, which is typical of a lasso peptide biosynthetic gene cluster in actinomycetes.

Sphaericin, a Lasso Peptide from the Rare Actinomycete Planomonospora sphaerica

A lasso peptide named sphaericin was isolated from the ISP2 agar medium culture of the rare actinomycete species Planomonospora sphaerica. The analysis of the amino acid composition of sphaericin gave a total of 15 amino acid units (three units each of Gly, Ile, and Pro; one unit each of Arg, Glu, Leu, Phe, Ser, and Tyr). In addition to these amino acids, NMR spectroscopy experiments indicated the presence of three Trp units. The structure of sphaericin was determined to be a peptide comprising 18 amino acids, and an additional peptide bond between the amino group of Gly‐1 and the γ‐carboxyl group of Glu‐9 forms a macrolactam ring, as demonstrated by analyses of the TOF MS/MS and NMR spectra. The analysis of the solution three‐dimensional structural through NOE experiments revealed that sphaericin possesses a typical lasso structure. From the genome data, the biosynthetic gene cluster of sphaericin was determined to consist of four genes, namely sphA, sphB1, sphB2, and sphC. Sphaericin showed a specific antibacterial activity against Micrococcus luteus at a dosage of 50 µg/disk.