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.     

A Self‐Sacrificing N‐Methyltransferase Is the Precursor of the Fungal Natural Product Omphalotin

Research on ribosomally synthesized and posttranslationally modified peptides (RiPPs) has led to an increasing understanding of biosynthetic mechanisms, mostly drawn from bacterial examples. In contrast, reports on RiPPs from fungal producers, apart from the amanitins and phalloidins, are still scarce. The fungal cyclopeptide omphalotin A carries multiple N‐methylations on the peptide backbone, a modification previously known only from nonribosomal peptides. Mining the genome of the omphalotin‐producing fungus for a precursor peptide led to the identification of two biosynthesis genes, one encoding a methyltransferase OphMA that catalyzes the automethylation of its C‐terminus, which is then released and cyclized by the protease OphP. Our findings suggest a novel biosynthesis mechanism for a RiPP in which a modifying enzyme bears its own precursor peptide.     

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.     

Discovery and characterisation of an amidine-containing ribosomally-synthesised peptide that is widely distributed in nature

Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural product with a wide range of bioactivities. Genome mining for RiPP biosynthetic gene clusters (BGCs) is often hampered by poor annotation of the short precursor peptides that are ultimately modified into the final molecule. Here, we utilise a previously described genome mining tool, RiPPER, to identify novel RiPP precursor peptides near YcaO-domain proteins, enzymes that catalyse various RiPP post-translational modifications including heterocyclisation and thioamidation. Using this dataset, we identified a novel and diverse family of RiPP BGCs spanning over 230 species of Actinobacteria and Firmicutes. A representative BGC from Streptomyces albidoflavus J1074 (formerly known as Streptomyces albus) was characterised, leading to the discovery of streptamidine, a novel amidine-containing RiPP. This new BGC family highlights the breadth of unexplored natural products with structurally rare features, even in model organisms.

Genome mining for pathways containing YcaO proteins revealed a widespread novel family of RiPP gene clusters. A model gene cluster was characterised through genetic and chemical analyses, which yielded streptamidine, a novel amidine-containing RiPP.     

A Lanthipeptide‐like N‐Terminal Leader Region Guides Peptide Epimerization by Radical SAM Epimerases: Implications for RiPP Evolution

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 class I lanthipeptide biosynthesis. Beyond this region, similarities were identified between proteusin and further RiPP families, including class 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.     

Ribosomally Synthesized and Post‐Translationally Modified Peptide Natural Products: New Insights into the Role of Leader and Core Peptides during Biosynthesis

Ribosomally synthesized and post‐translationally modified peptides (RiPPs) are a major class of natural products with a high degree of structural diversity and a wide variety of bioactivities. Understanding the biosynthetic machinery of these RiPPs will benefit the discovery and development of new molecules with potential pharmaceutical applications. In this Concept article, we discuss the features of the biosynthetic pathways to different RiPP classes, and propose mechanisms regarding recognition of the precursor peptide by the post‐translational modification enzymes. We propose that the leader peptides function as allosteric regulators that bind the active form of the biosynthetic enzymes in a conformational selection process. We also speculate how enzymes that generate polycyclic products of defined topologies may have been selected for during evolution.     

An Atypical Arginine Dihydrolase Involved in the Biosynthesis of Cyclic Hexapeptide Longicatenamides

The incorporation of non-proteinogenic amino acids (NPAAs) enriches the structural diversity of nonribosomal peptides. Recently, four NPAA-containing cyclic hexapeptides, longicatenamides A−D, were isolated using a combined-culture strategy. Based on in silico analysis, we discovered their putative biosynthetic gene cluster (lon) and proposed a possible biosynthetic mechanism. Surprisingly, the lon22 gene encodes an atypical arginine dihydrolase, which can also catalyze the hydrolysis of citrulline to ornithine. Phylogenetic analysis showed that Lon22-like proteins form a novel clade that is separated from other guanidine-modifying enzymes. After rational design, the catalytic efficiencies of a Lon22 Y80F mutant for arginine and citrulline substrates were 2.31- and 4.70-fold that of the wild-type (WT), respectively. In addition, characterization of the Lon20-A4 adenylation domain suggested that it can incorporate both ornithine and lysine into the final products.     

Peptide macrocyclization: the reductase of the nostocyclopeptide synthetase triggers the self-assembly of a macrocyclic imine

Many biologically active natural products have macrocyclic structures. In nonribosomal peptides macrocyclization is commonly achieved via the formation of intramolecular ester or amide bond catalyzed by thioesterase domains during biosynthesis. A unique and so far unknown type of peptide cyclization occurs in the nostocyclopeptide, a macrocyclic imine produced by the terrestrial cyanobacterium Nostoc sp. ATCC53789. In this work we show that a C-terminal reductase domain of the nostocyclopeptide nonribosomal peptide synthetase catalyzes the reductive release of a linear peptide aldehyde and thereby triggers the spontaneous formation of a stable imino head-to-tail linkage. This type of molecular self-assembly induced by the reductive release of reactive aldehydes may be more commonplace in other complex nonribosomal peptides than originally thought.     

Thuricin Z: A Narrow‐Spectrum Sactibiotic that Targets the Cell Membrane

Sactionine‐containing antibiotics (sactibiotics) are a growing class of peptide antibiotics belonging to the ribosomally synthesized and post‐translationally modified peptide (RiPP) superfamily. We report the characterization of thuricin Z, a novel sactibiotic from Bacillus thuringiensis. Unusually, the biosynthesis of thuricin Z involves two radical S‐adenosylmethionine (SAM) enzymes, ThzC and ThzD. Although ThzC and ThzD are highly divergent from each other, these two enzymes produced the same sactionine ring in the precursor peptide ThzA in vitro. Thuricin Z exhibits narrow‐spectrum antibacterial activity against Bacillus cereus. A series of analyses, including confocal laser scanning microscopy, ultrathin‐sectioning transmission electron microscopy, scanning electron microscopy, and large‐unilamellar‐vesicle‐based fluorescence analysis, suggested that thuricin Z binds to the bacterial cell membrane and leads to membrane permeabilization.     

Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

Antifungal HSAF (heat‐stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single‐module polyketide synthase–nonribosomal peptide synthetase (PKS‐NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two “clean” strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl‐coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene–PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS‐NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide–peptide natural products.     

Dissecting Bottromycin Biosynthesis Using Comparative Untargeted Metabolomics

Bottromycin A₂ is a structurally unique ribosomally synthesized and post‐translationally modified peptide (RiPP) that possesses potent antibacterial activity towards multidrug‐resistant bacteria. The structural novelty of bottromycin stems from its unprecedented macrocyclic amidine and rare β‐methylated amino acid residues. The N‐terminus of a precursor peptide (BtmD) is converted into bottromycin A₂ by tailoring enzymes encoded in the btm gene cluster. However, little was known about key transformations in this pathway, including the unprecedented macrocyclization. To understand the pathway in detail, an untargeted metabolomic approach that harnesses mass spectral networking was used to assess the metabolomes of a series of pathway mutants. This analysis has yielded key information on the function of a variety of previously uncharacterized biosynthetic enzymes, including a YcaO domain protein and a partner protein that together catalyze the macrocyclization.     

Understanding thioamitide biosynthesis using pathway engineering and untargeted metabolomics

Thiostreptamide S4 is a thioamitide, a family of promising antitumour ribosomally synthesised and post-translationally modified peptides (RiPPs). The thioamitides are one of the most structurally complex RiPP families, yet very few thioamitide biosynthetic steps have been elucidated, even though the biosynthetic gene clusters (BGCs) of multiple thioamitides have been identified. We hypothesised that engineering the thiostreptamide S4 BGC in a heterologous host could provide insights into its biosynthesis when coupled with untargeted metabolomics and targeted mutations of the precursor peptide. Modified BGCs were constructed, and in-depth metabolomics enabled a detailed understanding of the biosynthetic pathway to thiostreptamide S4, including the identification of a protein critical for amino acid dehydration that has homology to HopA1, an effector protein used by a plant pathogen to aid infection. We use this biosynthetic understanding to bioinformatically identify diverse RiPP-like BGCs, paving the way for future RiPP discovery and engineering.

Heterologous expression, pathway mutations and detailed metabolomic analysis were used to deduce a model for the biosynthesis of thiostreptamide S4, which belongs the thioamitide family of antitumour RiPPs.     

Unveiling the Biosynthetic Pathway of the Ribosomally Synthesized and Post‐translationally Modified Peptide Ustiloxin B in Filamentous Fungi

The biosynthetic machinery of the first fungal ribosomally synthesized and post‐translationally modified peptide (RiPP) ustiloxin B was elucidated through a series of gene inactivation and heterologous expression studies. The results confirmed an essential requirement for novel oxidases possessing the DUF3328 motif for macrocyclization, and highly unique side‐chain modifications by three oxidases (UstCF1F2) and a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme (UstD). These findings provide new insight into the expression of the RiPP gene clusters found in various fungi.     

Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology

Covering up to the end of 2011Many pharmaceuticals on the market today belong to a large class of natural products called nonribosomal peptides (NRPs). Originating from bacteria and fungi, these peptide-based natural products consist not only of the 20 canonical l-amino acids, but also non-proteinogenic amino acids, heterocyclic rings, sugars, and fatty acids, generating tremendous chemical diversity. As a result, these secondary metabolites exhibit a broad array of bioactivity, ranging from antimicrobial to anticancer. The biosynthesis of these complex compounds is carried out by large multimodular megaenzymes called nonribosomal peptide synthetases (NRPSs). Each module is responsible for incorporation of a monomeric unit into the natural product peptide and is composed of individual domains that perform different catalytic reactions. Biochemical and bioinformatic investigations of these enzymes have uncovered the key principles of NRP synthesis, expanding the pharmaceutical potential of their enzymatic processes. Progress has been made in the manipulation of this biosynthetic machinery to develop new chemoenzymatic approaches for synthesizing novel pharmaceutical agents with increased potency. This review focuses on the recent discoveries and breakthroughs in the structural elucidation, molecular mechanism, and chemical biology underlying the discrete domains within NRPSs.     

Circular logic: nonribosomal peptide-like macrocyclization with a ribosomal peptide catalyst

A protease from ribosomal peptide biosynthesis macrocyclizes diverse substrates, including those resembling nonribosomal peptide and hybrid polyketide-peptide products. The proposed mechanism is analogous to thioesterase-catalyzed chemistry, but the substrates are amide bonds rather than thioesters.     

Nonribosomal Peptide Synthesis—Principles and Prospects

Nonribosomal peptide synthetases (NRPSs) are large multienzyme machineries that assemble numerous peptides with large structural and functional diversity. These peptides include more than 20 marketed drugs, such as antibacterials (penicillin, vancomycin), antitumor compounds (bleomycin), and immunosuppressants (cyclosporine). Over the past few decades biochemical and structural biology studies have gained mechanistic insights into the highly complex assembly line of nonribosomal peptides. This Review provides state‐of‐the‐art knowledge on the underlying mechanisms of NRPSs and the variety of their products along with detailed analysis of the challenges for future reprogrammed biosynthesis. Such a reprogramming of NRPSs would immediately spur chances to generate analogues of existing drugs or new compound libraries of otherwise nearly inaccessible compound structures.     

Introduction of a non-natural amino acid into a nonribosomal peptide antibiotic by modification of adenylation domain specificity

Calcium-dependent antibiotics (CDA) are cyclic lipopeptides assembled by nonribosomal peptide synthetase (NRPS) enzymes. Active site modification of the 3-methyl glutamate activating adenylation (A) domain of the CDA NRPS enables the incorporation of synthetic 3-methyl glutamine into CDA. This provides the first example of how A-domains can be engineered to introduce synthetic "non-natural" amino acids into nonribosomal peptides.     

Solid phase synthesis of mono- or disubstituted arginine containing peptides from an isothiocitrulline precursor

A method for the solid phase synthesis of substituted arginine containing peptides starting from an isothiocitrulline precursor is described. In this procedure, a peptide containing one or more protected ornithine residue(s) was assembled on a solid support. Following selective deprotection, ornithine residue(s) was (were) converted into S-methyl-isothiocitrulline in three steps. Subsequent reaction with primary or secondary amines afforded mono and disubstituted arginine-containing derivatives, respectively. Using lysine instead of ornithine afforded substituted homoarginine-containing derivatives.     

Cacaoidin,First Member of the New Lanthidin RiPP Family

Lantibiotics are ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by the presence of lanthionine or methyllanthionine rings and their antimicrobial activity. Cacaoidin, a novel glycosylated lantibiotic, was isolated from a Streptomyces cacaoi strain and fully characterized by NMR, mass spectrometry, chemical derivatization approaches and genome analysis. The new molecule combines outstanding structural features, such as a high number of d -amino acids, an uncommon glycosylated tyrosine residue and an unprecedented N,N-dimethyl lanthionine. This latter feature places cacaoidin within a new RiPP family located between lanthipeptides and linaridins, here termed lanthidins. Cacaoidin displayed potent antibacterial activity against Gram-positive pathogens including Clostridium difficile. The biosynthetic gene cluster showed low homology with those of other known lanthipeptides or linaridins, suggesting a new RiPP biosynthetic pathway.