Accessing and exploring the unusual chemistry by radical SAM-RiPP enzymes.

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.

The Triceptide Maturase OscB Catalyzes Uniform Cyclophane Topology and Accepts Diverse Gly-Rich Precursor Peptides.

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.

Substrate Promiscuity of the Triceptide Maturase XncB Leads to Incorporation of Various Amino Acids and Detection of Oxygenated Products.

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.

Functional and Promiscuity Studies of Three-Residue Cyclophane Forming Enzymes Show Nonnative C-C Cross-Linked Products and Leader-Dependent Cyclization.

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.

Bacterial cyclophane-containing RiPPs from radical SAM enzymes.

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.

Structural and Biochemical Insights into Post-Translational Arginine-to-Ornithine Peptide Modifications by an Atypical Arginase.

Landornamide A is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product with antiviral activity. Its biosynthetic gene cluster encodes─among other maturases─the peptide arginase OspR, which converts arginine to ornithine units in an unusual post-translational modification. Peptide arginases are a recently discovered RiPP maturase family with few characterized representatives. They show little sequence similarity to conventional arginases, a well-characterized enzyme family catalyzing the hydrolysis of free arginine to ornithine and urea. Peptide arginases are highly promiscuous and accept a variety of substrate sequences. The molecular basis for binding the large peptide substrate and for the high promiscuity of peptide arginases remains unclear. Here, we report the first crystal structure of a peptide arginase at a resolution of 2.6 Å. The three-dimensional structure reveals common features and differences between conventional arginases and the peptide arginase: the binuclear metal cluster and the active-site environment strongly resemble each other, while the quaternary structures diverge. Kinetic analyses of OspR with various substrates provide new insights into the order of biosynthetic reactions during the post-translational maturation of landornamide A. These results provide the basis for pathway engineering to generate derivatives of landornamide A and for the general application of peptide arginases as biosynthetic tools for peptide engineering.

A Prevalent Group of Actinobacterial Radical SAM/SPASM Maturases Involved in Triceptide Biosynthesis.

Triceptides are ribosomally synthesized and post-translationally modified peptides characterized by three-residue cyclophanes. The cyclophanes are installed by radical SAM/SPASM maturases referred to as 3-residue cyclophane forming enzymes (3-CyFEs) which catalyze C(sp2)-Cß(sp3) bond formation on three residue motifs at the C-terminus of precursor peptides. Here, we bioinformatically map uncharacterized rSAM/SPASM enzymes, referred to as Actinobacterial multiple cyclophane maturases. The enzyme FwwB from Actinospira robinae was selected for in vivo functional studies in Escherichia coli, and was found to catalyze formation of multiple Phe- and Trp-derived 3-residue cyclophanes. FwwB was shown to accept a series of engineered substrates but showed specificity for the native 3-residue motif.

The Biosynthetic Landscape of Triceptides Reveals Radical SAM Enzymes That Catalyze Cyclophane Formation on Tyr- and His-Containing Motifs.

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.

Ribosomally derived lipopeptides containing distinct fatty acyl moieties.

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.

Genome-Mining-Based Discovery of the Cyclic Peptide Tolypamide and TolF, a Ser/Thr Forward O-Prenyltransferase.

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.

Post-translational formation of strained cyclophanes in bacteria.

Cyclic peptide natural products have served as important drug molecules, with several examples used clinically. Enzymatic or chemical macrocyclization is the key transformation for constructing these chemotypes. Methods to generate new and diverse cyclic peptide scaffolds enabling the modular and predictable synthesis of peptide libraries are desirable in drug discovery platforms. Here we identify a suite of post-translational modifying enzymes from bacteria that install single or multiple strained cyclophane macrocycles. The crosslinking occurs on three-residue motifs that include tryptophan or phenylalanine to form indole- or phenyl-bridged cyclophanes. The macrocycles display restricted rotation of the aromatic ring and induce planar chirality in the asymmetric indole bridge. The biosynthetic gene clusters originate from a broad range of bacteria derived from marine, terrestrial and human microbiomes. Three-residue cyclophane-forming enzymes define a new and significant natural product family and occupy a distinct region in sequence-function space.

Posttranslationally Acting Arginases Provide a Ribosomal Route to Non-proteinogenic Ornithine Residues in Diverse Peptide Sequences.

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) landornamide 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.

Landornamides: Antiviral Ornithine-Containing Ribosomal Peptides Discovered through Genome Mining.

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 landornamide 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. Landornamide 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.

Bacteria-induced production of the antibacterial sesquiterpene lagopodin B in Coprinopsis cinerea.

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.

Radical S-Adenosylmethionine Peptide Epimerases: Detection of Activity and Characterization of d-Amino Acid Products.

The identification of the polytheonamide (poy) gene cluster led to the discovery of the enzyme PoyD, a member of the radical S-adenosylmethionine superfamily capable of introducing d-amino acids into a ribosomally synthesized peptide. This enzyme was used as a starting point to identify additional radical S-adenosylmethionine peptide epimerases in other cyanobacterial genomes, which show different epimerization patterns compared to PoyD. During the course of studying these enzymes by heterologous expression in Escherichia coli, we developed a two-step strategy to (1) detect epimerase activity and (2) localize where epimerization occurs based on an in vivo deuterium labeling strategy. The procedures for these two methods are described in the following chapter and will set the stage for further study of these enzymes.

Natural noncanonical protein splicing yields products with diverse ß-amino acid residues.

Current textbook knowledge holds that the structural scope of ribosomal biosynthesis is based exclusively on α-amino acid backbone topology. Here we report the genome-guided discovery of bacterial pathways that posttranslationally create ß-amino acid-containing products. The transformation is widespread in bacteria and is catalyzed by an enzyme belonging to a previously uncharacterized radical S-adenosylmethionine family. We show that the ß-amino acids result from an unusual protein splicing process involving backbone carbon-carbon bond cleavage and net excision of tyramine. The reaction can be used to incorporate diverse and multiple ß-amino acids into genetically encoded precursors in Escherichia coli In addition to enlarging the set of basic amino acid components, the excision generates keto functions that are useful as orthogonal reaction sites for chemical diversification.

Toblerols: Cyclopropanol-Containing Polyketide Modulators of Antibiosis in Methylobacteria.

Trans-AT polyketide synthases (PKSs) are a family of biosynthetically versatile modular type 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.

Liposomal Circular Dichroism (L-CD) of Arenoyl Derivatives of Sphingolipids. Amplification of Cotton Effects in Ordered Lipid Bilayers.

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.

Cyanobacterial ent-Sterol-Like Natural Products from a Deviated Ubiquinone Pathway.

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.

Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium.

Uncultivated bacteria represent a massive resource of new enzymes and bioactive metabolites, but such bacteria remain functionally enigmatic. Polytheonamides are potent peptide cytotoxins produced by uncultivated bacteria that exist as symbionts in a marine sponge. Outside glycobiology, polytheonamides represent the most heavily post-translationally modified biomolecules that are derived from amino acids. The biosynthesis of polytheonamides involves up to 50 site-specific modifications to create a membrane-spanning ß-helical structure. Here, we provide functional evidence that only seven enzymes are necessary for this process. They iteratively catalyse epimerization, methylation and hydroxylation of diverse amino acids. To reconstitute C-methylation, we employed the rarely used heterologous host Rhizobium leguminosarum to invoke the activities of two cobalamin-dependent C-methyltransferases. We observed 44 of the modifications to systematically unravel the biosynthesis of one of the most densely modified and metabolically obscure ribosome-derived molecules found in nature.