RESUMEN
Lipopeptides represent a large group of microbial natural products that include important antibacterial and antifungal drugs and some of the most-powerful known biosurfactants. The vast majority of lipopeptides comprise cyclic peptide backbones N-terminally equipped with various fatty acyl moieties. The known compounds of this type are biosynthesized by nonribosomal peptide synthetases, giant enzyme complexes that assemble their products in a non-gene-encoded manner. Here, we report the genome-guided discovery of ribosomally derived, fatty-acylated lipopeptides, termed selidamides. Heterologous reconstitution of three pathways, two from cyanobacteria and one from an arctic, ocean-derived alphaproteobacterium, allowed structural characterization of the probable natural products and suggest that selidamides are widespread over various bacterial phyla. The identified representatives feature cyclic peptide moieties and fatty acyl units attached to (hydroxy)ornithine or lysine side chains by maturases of the GCN5-related N-acetyltransferase superfamily. In contrast to nonribosomal lipopeptides that are usually produced as congener mixtures, the three selidamides are selectively fatty acylated with C10, C12, or C16 fatty acids, respectively. These results highlight the ability of ribosomal pathways to emulate products with diverse, nonribosomal-like features and add to the biocatalytic toolbox for peptide drug improvement and targeted discovery.
Asunto(s)
Lipopéptidos/biosíntesis , Lipopéptidos/química , Ribosomas/metabolismo , Antibacterianos/metabolismo , Antifúngicos/metabolismo , Vías Biosintéticas , Cianobacterias/metabolismo , Péptido Sintasas/metabolismo , Péptidos CíclicosRESUMEN
Covering: 2016 to 2023Ribosomally synthesized and posttranslationally modified peptides (RiPPs) continue to be a rich source of chemically diverse and bioactive peptide natural products. In recent years, cyclophane-containing RiPP natural products and their biosynthetic pathways have been more frequently encountered. This highlight will focus on bacterial monoaryl cyclophane-containing RiPPs. This class of RiPPs is produced by radical SAM/SPASM enzymes that form a crosslink between the aromatic ring and sidechain of two amino acid residues of the precursor peptide. Selected natural products from these pathways exhibit specific antibacterial activity against gram-negative pathogens. The approaches used to discover these pathways and products will be described and categorized as natural product-first or enzyme-first. The breadth of ring systems formed by the enzymes, enzyme mechanism, and recent reports of synthetic methods for constructing these ring systems will also be presented. Bacterial cyclophane-containing RiPPs and their biosynthetic enzymes represent an untapped source of scaffolds for drug discovery and tools for synthetic biology.
RESUMEN
Peptide-derived cyclophanes inhabit a unique niche in the chemical space of macrocyclic peptides with several examples of pharmaceutical importance. Although both synthetic and biocatalytic methods are available for constructing these macrocycles, versatile (bio)catalysts able to incorporate a variety of amino acids that compose the macrocycle would be useful for the creation of diverse peptide cyclophanes. In this report, we synergized the use of bioinformatic tools to map the biosynthetic landscape of radical SAM enzymes (3-CyFEs) that catalyze three-residue cyclophane formation in the biosynthesis of a new family of RiPP natural products, the triceptides. This analysis revealed 3940 (3113 unique) putative precursor sequences predicted to be modified by 3-CyFEs. Several uncharacterized maturase systems were identified that encode unique precursor types. Functional studies were carried out in vivo in Escherichia coli to identify modified precursors containing His and Tyr residues. NMR analysis of the products revealed that Tyr and His can also be incorporated into cyclophane macrocycles by 3-CyFEs. Collectively, all aromatic amino acids can be incorporated by 3-CyFEs, and the cyclophane formation strictly occurs via a C(sp2)-C(sp3) cross-link between the (hetero)aromatic ring to Cß. In addition to 3-CyFEs, we functionally validated an Fe(II)/α-ketoglutarate-dependent hydroxylase, resulting in ß-hydroxylated residues within the cyclophane rings. This study reveals the potential breadth of triceptide precursors and a systematic approach for studying these enzymes to broaden the diversity of peptide macrocycles.
Asunto(s)
Biología Computacional , Péptidos , Catálisis , Biología Computacional/métodos , Escherichia coli/metabolismo , Péptidos/químicaRESUMEN
Cyanobactins comprise a widespread group of peptide metabolites produced by cyanobacteria that are often diversified by post-translational prenylation. Several enzymes have been identified in cyanobactin biosynthetic pathways that carry out chemically diverse prenylation reactions, representing a resource for the discovery of post-translational alkylating agents. Here, genome mining was used to identify orphan cyanobactin prenyltransferases, leading to the isolation of tolypamide from the freshwater cyanobacterium Tolypothrix sp. The structure of tolypamide was confirmed by spectroscopic methods, degradation, and enzymatic total synthesis. Tolypamide is forward-prenylated on a threonine residue, representing an unprecedented post-translational modification. Biochemical characterization of the cognate enzyme TolF revealed a prenyltransferase with strict selectivity for forward O-prenylation of serine or threonine but with relaxed substrate selectivity for flanking peptide sequences. Since cyanobactin pathways often exhibit exceptionally broad substrate tolerance, these enzymes represent robust tools for synthetic biology.
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Proteínas Bacterianas/química , Dimetilaliltranstransferasa/química , Péptidos Cíclicos/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Cianobacterias/enzimología , Dimetilaliltranstransferasa/aislamiento & purificación , Dimetilaliltranstransferasa/metabolismo , Estructura Molecular , Péptidos Cíclicos/metabolismo , Treonina/química , Treonina/metabolismoRESUMEN
Fungi defend their ecological niche against antagonists by producing antibiosis molecules. Some of these molecules are only produced upon confrontation with the antagonist. The basidiomycete Coprinopsis cinerea induces the expression of the sesquiterpene synthase-encoding gene cop6 and its two neighboring genes coding for cytochrome P450 monooxygenases in response to bacteria. We further investigated this regulation of cop6 and examined if the gene product is involved in the production of antibacterials. Cell-free supernatants of axenic cultures of the Gram-positive bacterium Bacillus subtilis were sufficient to induce cop6 transcription assessed using a fluorescent reporter strain. Use of this strain in a microfluidic device revealed that the cop6 gene was induced in all hyphae directly exposed to the supernatant and that induction occurred within less than one hour. Targeted replacement of the cop6 gene demonstrated the requirement of the encoded synthase for the biosynthesis of the sesquiterpene lagopodin B, a previously reported antibacterial compound from related species. Accordingly, lagopodin B from C. cinerea inhibited the growth of several Gram-positive bacteria including B. subtilis but not Gram-negative bacteria. Our results demonstrate that the C. cinerea vegetative mycelium responds to soluble compounds of a bacterial culture supernatant by local production of an antibacterial secondary metabolite.
Asunto(s)
Agaricales/metabolismo , Antibacterianos/metabolismo , Bacillus subtilis/fisiología , Sesquiterpenos/metabolismo , Agaricales/enzimología , Agaricales/genética , Antibacterianos/farmacología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/crecimiento & desarrollo , Sesquiterpenos/farmacologíaRESUMEN
Ornithine is a component of many bioactive nonribosomal peptides but is challenging to incorporate into ribosomal products. We recently identified OspR, a cyanobacterial arginase-like enzyme that installs ornithines in the antiviral ribosomally synthesised and posttranslationally modified peptide (RiPP) landornamideâ A. Here we report that OspR belongs to a larger family of peptide arginases from diverse organisms and RiPP types. In E. coli, seven selected enzymes converted arginine into ornithine with little preference for the leader type. A broad range of peptide sequences was modified, including polyarginine repeats. We also generated analogues of ornithine-containing nonribosomal peptides using RiPP technology. Five pseudo-nonribosomal products with ornithines at the correct positions were obtained, including a brevicidine analogue containing ornithine and a d-amino acid installed by the peptide epimerase OspD. These results suggest new opportunities for peptide bioengineering.
Asunto(s)
Arginasa/metabolismo , Cianobacterias/enzimología , Ornitina/metabolismo , Péptidos/metabolismo , Ribosomas/metabolismo , Secuencia de Aminoácidos , Arginasa/química , Conformación Molecular , Ornitina/química , Péptidos/química , Procesamiento Proteico-Postraduccional , Ribosomas/químicaRESUMEN
Proteusins are a family of bacterial ribosomal peptides that largely remain hypothetical genome-predicted metabolites. The only known members are the polytheonamide-type cytotoxins, which have complex structures due to numerous unusual posttranslational modifications (PTMs). Cyanobacteria contain large numbers of putative proteusin loci. To investigate their chemical and pharmacological potential beyond polytheonamide-type compounds, we characterized landornamideâ A, the product of the silent osp gene cluster from Kamptonema sp. PCC 6506. Pathway reconstruction in E. coli revealed a peptide combining lanthionines, d-residues, and, unusually, two ornithines introduced by the arginase-like enzyme OspR. Landornamideâ A inhibited lymphocytic choriomeningitis virus infection in mouse cells, thus making it one of the few known anti-arenaviral compounds. These data support proteusins as a rich resource of chemical scaffolds, new maturation enzymes, and bioactivities.
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Antivirales/síntesis química , Proteínas Bacterianas/síntesis química , Minería de Datos , Bases de Datos Genéticas , Ornitina/química , Péptidos/química , Proteínas Ribosómicas/síntesis química , Ribosomas/química , Animales , Antivirales/farmacología , Proteínas Bacterianas/farmacología , Línea Celular , Biología Computacional , Cianobacterias/química , Escherichia coli/genética , Coriomeningitis Linfocítica/tratamiento farmacológico , Virus de la Coriomeningitis Linfocítica , Ratones , Familia de Multigenes , Péptidos/síntesis química , Péptidos/farmacología , Procesamiento Proteico-Postraduccional , Proteínas Ribosómicas/farmacologíaRESUMEN
Trans-AT polyketide synthases (PKSs) are a family of biosynthetically versatile modular typeâ I PKSs that generate bioactive polyketides of impressive structural diversity. In this study, we detected, in the genome of several bacteria a cryptic, architecturally unusual trans-AT PKS gene cluster which eluded automated PKS prediction. Genomic mining of one of these strains, the model methylotroph Methylobacterium extorquens AM1, revealed unique epoxide- and cyclopropanol-containing polyketides named toblerols. Relative and absolute stereochemistry were determined by NMR experiments, chemical derivatization, and the comparison of CD data between the derivatized natural product and a synthesized model compound. Biosynthetic data suggest that the cyclopropanol moiety is generated by carbon-carbon shortening of a more extended precursor. Surprisingly, a knock-out strain impaired in polyketide production showed strong inhibitory activity against other methylobacteria in contrast to the wild-type producer. The activity was inhibited by complementation with toblerols, thus suggesting that these compounds modulate an as-yet unknown methylobacterial antibiotic.
Asunto(s)
Éteres Cíclicos/química , Methylobacterium/enzimología , Sintasas Poliquetidas/metabolismo , Policétidos/química , Antibiosis , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Methylobacterium/efectos de los fármacos , Methylobacterium/genética , Familia de Multigenes , Sintasas Poliquetidas/antagonistas & inhibidores , Sintasas Poliquetidas/genética , Policétidos/metabolismo , Policétidos/farmacologíaRESUMEN
Actin-targeting macrolides comprise a large, structurally diverse group of cytotoxins isolated from remarkably dissimilar micro- and macroorganisms. In spite of their disparate origins and structures, many of these compounds bind actin at the same site and exhibit structural relationships reminiscent of modular, combinatorial drug libraries. Here we investigate biosynthesis and evolution of three compound groups: misakinolides, scytophycin-type compounds and luminaolides. For misakinolides from the sponge Theonella swinhoei WA, our data suggest production by an uncultivated 'Entotheonella' symbiont, further supporting the relevance of these bacteria as sources of bioactive polyketides and peptides in sponges. Insights into misakinolide biosynthesis permitted targeted genome mining for other members, providing a cyanobacterial luminaolide producer as the first cultivated source for this dimeric compound family. The data indicate that this polyketide family is bacteria-derived and that the unusual macrolide diversity is the result of combinatorial pathway modularity for some compounds and of convergent evolution for others.
Asunto(s)
Actinas/metabolismo , Evolución Biológica , Cianobacterias/metabolismo , Deltaproteobacteria/metabolismo , Policétidos/metabolismo , Actinas/química , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cianobacterias/genética , Deltaproteobacteria/genética , Expresión Génica , Macrólidos/química , Macrólidos/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Péptidos , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Policétidos/química , Unión Proteica , Piranos/química , Piranos/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Simbiosis , Theonella/microbiologíaRESUMEN
Liposomal circular dichroism (L-CD) of acyclic amino alcohols exhibit amplification of Cotton effects when measured in highly uniform, unilamellar liposomes. The effect is likely due to intermolecular associations-H-aggregates-that self-assemble spontaneously within the lipid bilayer, and persists over long time scales. L-CD spectra of N,O,O'-tri-(6'methoxy-2'-naphthoyl)-d-erythro-sphingosine, or the corresponding dihydro-derivative (sphinganine), shows ~10-fold amplification of magnitudes of Cotton effects over conventional CD spectra recorded in isotropic solution.
Asunto(s)
Dicroismo Circular/métodos , Membrana Dobles de Lípidos/química , Liposomas/química , Esfingolípidos/química , Esfingosina/química , Ácidos Acíclicos/química , Esfingosina/análogos & derivados , EstereoisomerismoRESUMEN
Radical S-adenosyl methionine peptide epimerases (RSPEs) are an enzyme family that accomplishes regiospecific and irreversible introduction of multiple d-configured residues into ribosomally encoded peptides. Collectively, RSPEs can generate diverse epimerization patterns in a wide range of substrates. Previously, the lack of rapid methods to localize epimerized residues has impeded efforts to investigate the function and applicative potential of RSPEs. An efficient mass spectrometry-based assay is introduced that permits characterization of products generated in E. coli. Applying this to a range of non-natural peptide-epimerase combinations, it is shown that the d-amino acid pattern is largely but not exclusively dictated by the core peptide sequence, while the epimerization order is dependent on the enzyme-leader pair. RSPEs were found to be highly promiscuous, which allowed for modular introduction of peptide segments with defined patterns.
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Aminoácidos/biosíntesis , Carbohidrato Epimerasas/metabolismo , Óxido de Deuterio/metabolismo , S-Adenosilmetionina/metabolismo , Aminoácidos/química , Carbohidrato Epimerasas/química , Óxido de Deuterio/química , Radicales Libres/química , Radicales Libres/metabolismo , Conformación Molecular , S-Adenosilmetionina/químicaRESUMEN
Natural products from marine animals show high potential for the development of new medicines, but drug development based on these compounds is commonly hampered by their low natural abundance. Since many of these metabolites are suspected or known to be produced by uncultivated bacterial symbionts, the rapidly growing diversity of sequenced prokaryotic genomes offers the opportunity to identify alternative, culturable sources of natural products computationally. In this work, we investigated the potential of using this sequenced resource to facilitate the production of meroterpenoid-like compounds related to those from marine sources. This genome-mining strategy revealed a biosynthetic gene cluster for highly modified cytotoxic meroterpenoids related to pelorol and other compounds isolated from sponges. Functional characterization of the terpene cyclase MstE showed that it generates an ent-sterol-like skeleton fused to an aryl moiety from an open-chain precursor and is therefore a promising tool for the chemoenzymatic preparation of synthetically challenging chemical scaffolds.
RESUMEN
Bacteria are a major source of natural products that provide rich opportunities for both chemical and biological investigation. Although the vast majority of known bacterial metabolites derive from free-living organisms, increasing evidence supports the widespread existence of chemically prolific bacteria living in symbioses. A strategy based on bioinformatic prediction, symbiont cultivation, isotopic enrichment, and advanced analytics was used to characterize a unique polyketide, nosperin, from a lichen-associated Nostoc sp. cyanobacterium. The biosynthetic gene cluster and the structure of nosperin, determined from 30 µg of compound, are related to those of the pederin group previously known only from nonphotosynthetic bacteria associated with beetles and marine sponges. The presence of this natural product family in such highly dissimilar associations suggests that some bacterial metabolites may be specific to symbioses with eukaryotes and encourages exploration of other symbioses for drug discovery and better understanding of ecological interactions mediated by complex bacterial metabolites.
Asunto(s)
Vías Biosintéticas/genética , Líquenes/genética , Metagenoma/genética , Sintasas Poliquetidas/genética , Simbiosis/genética , Secuencia de Bases , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Análisis por Conglomerados , Biología Computacional , Minería de Datos , Componentes del Gen , Islandia , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Metagenómica/métodos , Datos de Secuencia Molecular , Estructura Molecular , Familia de Multigenes/genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) exhibit diverse structures and bioactivities and are classified into distinct biosynthetic families. A recently reported family is the proteusins, with the prototype members polytheonamides being generated by almost 50 maturation steps, including introduction of d-residues at multiple positions by an unusual radical SAM epimerase. A region in the protein-like N-terminal leader of proteusin precursors is identified that is crucial for epimerization. It resembles a precursor motif previously shown to mediate interaction in thioether bridge-formation in classâ I lanthipeptide biosynthesis. Beyond this region, similarities were identified between proteusin and further RiPP families, including classâ I lanthipeptides. The data suggest that common leader features guide distinct maturation types and that nitrile hydratase-like enzymes are ancestors of several RiPP classes.
RESUMEN
Two new peptides, stylissamides G and H, were isolated from extracts of a sample of Stylissa caribica collected in deep waters of the Caribbean Sea. A single sample of S. caribica among a collection of 10 samples that were examined by LC-MS appeared to be a different chemotype from the others in that it lacked the familiar pyrrole-2-aminoimidazole alkaloids, stevensine and oroidin, and contained peptides of the stylissamide class. The structures of the title compounds were solved by integrated analysis of the MS and NMR spectra and chemical degradation. The solution conformation of stylissamide G was briefly examined by electronic circular dichroism and temperature-dependent (1)H NMR chemical shifts of amide NH signals, which supported a conformationally rigid macrocycle.
Asunto(s)
Péptidos Cíclicos/química , Péptidos Cíclicos/aislamiento & purificación , Poríferos/química , Alcaloides/química , Animales , Región del Caribe , Imidazoles/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Pirroles/químicaRESUMEN
PoyD is a radical S-adenosyl methionine epimerase that introduces multiple D-configured amino acids at alternating positions into the highly complex marine peptides polytheonamide A and B. This novel post-translational modification contributes to the ability of the polytheonamides to form unimolecular minimalistic ion channels and its cytotoxic activity at picomolar levels. Using a genome mining approach we have identified additional PoyD homologues in various bacteria. Three enzymes were expressed in E. coli with their cognate as well as engineered peptide precursors and shown to introduce diverse D-amino acid patterns into all-L peptides. The data reveal a family of architecturally and functionally distinct enzymes that exhibit high regioselectivity, substrate promiscuity, and irreversible action and thus provide attractive opportunities for peptide engineering.
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Aminoácidos/química , Racemasas y Epimerasas/química , S-Adenosilmetionina/química , Productos Biológicos/metabolismo , EstereoisomerismoRESUMEN
Radical SAM enzymes involved in the biosynthesis of ribosomally synthesized and post-translationally modified peptides catalyze unusual transformations that lead to unique peptide scaffolds and building blocks. Several natural products from these pathways show encouraging antimicrobial activities and represent next-generation therapeutics for infectious diseases. These systems are uniquely configured to benefit from genome-mining approaches because minimal substrate and cognate modifying enzyme expression can reveal unique, chemically complex transformations that outperform late-stage chemical reactions. This report highlights the main strategies used to reveal these enzymatic transformations, which have relied mainly on genome mining using enzyme-first approaches. We describe the general biosynthetic components for rSAM enzymes and highlight emerging approaches that may broaden the discovery and study of rSAM-RiPP enzymes. The large number of uncharacterized rSAM proteins, coupled with their unpredictable transformations, will continue to be an essential and exciting resource for enzyme discovery.
Asunto(s)
S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , Procesamiento Proteico-Postraduccional , Productos Biológicos/metabolismo , Productos Biológicos/química , Humanos , Enzimas/metabolismo , Enzimas/químicaRESUMEN
Triceptides are a class of ribosomally synthesized and post-translationally modified peptides defined by an aromatic C(sp2) to Cß(sp3) bond. The Gly-rich repeat family of triceptide maturases (TIGR04261) are paired with precursor peptides (TIGR04260) containing a Gly-rich core peptide. These maturases are prevalent in cyanobacteria and catalyze cyclophane formation on multiple Ω1-X2-X3 motifs (Ω1 = Trp and Phe) of the Gly-rich precursor peptide. The topology of the individual rings has not been completely elucidated, and the promiscuity of these enzymes is not known. In this study, we characterized all the cyclophane rings formed by the triceptide maturase OscB and show the ring topology is uniform with respect to the substitution at Trp-C7 and the atropisomerism (planar chirality). Additionally, the enzyme OscB demonstrated substrate promiscuity on Gly-rich precursors and can accommodate a diverse array of engineered sequences. These findings highlight the versatility and implications for using OscB as a biocatalyst for producing polycyclophane-containing peptides for biotechnological applications.
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Glicina , Especificidad por Sustrato , Glicina/química , Glicina/metabolismo , Péptidos/química , Péptidos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cianobacterias/enzimología , Cianobacterias/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Biocatálisis , CiclofanosRESUMEN
Triceptides are cyclophane-containing ribosomally synthesized and post-translationally modified peptides. The characteristic cross-links are formed between an aromatic ring to Cß on three-residue Ω1X2X3 motifs (Ω1 = aromatic). Here, we explored the promiscuity of the XYE family triceptide maturase, XncB from Xenorhabdus nematophila DSM 3370. Single amino acid variants were coexpressed with XncB in vivo in Escherichia coli, and we show that a variety of amino acids can be incorporated into the Phe-Gly-Asn cyclophane. Aromatic amino acids at the X3 position were accepted by the enzyme but yielded hydroxylated, rather than the typical cyclophane, products. These studies show that oxygen can be inserted but diverges in the final product formed relative to daropeptide maturases. Finally, truncations of the leader peptide showed that it is necessary for complete modification by XncB.
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Aminoácidos , Péptidos , Xenorhabdus , Aminoácidos/metabolismo , Péptidos/química , Señales de Clasificación de Proteína , Xenorhabdus/química , Xenorhabdus/enzimología , Xenorhabdus/genética , Xenorhabdus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Especificidad por SustratoRESUMEN
Enzymes catalyzing peptide macrocyclization are important biochemical tools in drug discovery. The three-residue cyclophane-forming enzymes (3-CyFEs) are an emerging family of post-translational modifying enzymes that catalyze the formation of three-residue peptide cyclophanes. In this report, we introduce three additional 3-CyFEs, including ChlB, WnsB, and FnnB, that catalyze cyclophane formation on Tyr, Trp, and Phe, respectively. To understand the promiscuity of these enzymes and those previously reported (MscB, HaaB, and YxdB), we tested single amino acid substitutions at the three-residue motif of modification (Ω1X2X3, Ω1 = aromatic). Collectively, we observe that substrate promiscuity is observed at the Ω1 and X2 positions, but a greater specificity is observed for the X3 residue. Two nonnative cyclophane products were characterized showing a Phe-C3 to Arg-Cß and His-C2 to Pro-Cß cross-links, respectively. We also tested the leader dependence of selected 3-CyFEs and show that a predicted helix region is important for cyclophane formation. These results demonstrate the biocatalytic potential of these maturases and allow rational design of substrates to obtain a diverse array of genetically encoded 3-residue cyclophanes.