An in vitro assay to explore condensation domain specificity from non-ribosomal peptide synthesis.

Non-ribosomal peptide synthesis produces a wide range of bioactive peptide natural products and is reliant on a modular architecture based on repeating catalytic domains able to generate diverse peptide sequences. In this chapter we detail an in vitro biochemical assay to explore the substrate specificity of condensation domains, which are responsible for peptide elongation, from the biosynthetic machinery that produces from the siderophore fuscachelin. This assay removes the requirement to utilise the specificity of adjacent adenylation domains and allows the acceptance of a wide range of synthetic substrates to be explored.

Biosynthesis and recruitment of reactive amino acids in nonribosomal peptide assembly lines.

Reactive amino acid side chains play important roles in the binding of peptides to specific targets. In addition, their reactivity enables selective peptide conjugation and functionalization for pharmaceutical purposes. Diverse reactive amino acids are incorporated into nonribosomal peptides, which serve as a source for drug candidates. Notable examples include (poly)unsaturated (enamine, alkyne, and furyl) and halogenated residues, strained carbacycles (cyclopropyl and cyclopropanol), small heterocycles (oxirane and aziridine), and reactive N-N functionalities (hydrazones, diazo compounds, and diazeniumdiolates). Their biosynthesis requires diverse biocatalysts for sophisticated reaction mechanisms. Several avenues have been identified for their incorporation into peptides, the recruitment by adenylation domains or ligases, on-line modifications, and enzymatic tailoring reactions. Combined with protein engineering approaches, this knowledge provides new opportunities in synthetic biology and bioorthogonal chemistry.

Recent progress in the reprogramming of nonribosomal peptide synthetases.

Nonribosomal peptide synthetases (NRPSs) biosynthesize nonribosomal peptide (NRP) natural products, which belong to the most promising resources for drug discovery and development because of their wide range of therapeutic applications. The results of genetic, biochemical, and bioinformatics analyses have enhanced our understanding of the mechanisms of the NRPS machinery. A major goal in NRP biosynthesis is to reprogram the NRPS machinery to enable the biosynthetic production of designed peptides. Reprogramming strategies for the NRPS machinery have progressed considerably in recent years, thereby increasing the yields and generating modified peptides. Here, the recent progress in NRPS reprogramming and its application in peptide synthesis are described.

Not always an innocent bystander: the impact of stabilised phosphopantetheine moieties when studying nonribosomal peptide biosynthesis.

Nonribosomal peptide synthetases produce many important peptide natural products and are centred around carrier proteins (CPs) that deliver intermediates to various catalytic domains. We show that the replacement of CP substrate thioesters by stabilised ester analogues leads to active condensation domain complexes, whereas amide stabilisation generates non-functional complexes.

The Assembly-Line Enzymology of Nonribosomal Peptide Biosynthesis.

Peptide natural products constitute a major class of secondary metabolites produced by microorganisms (mostly bacteria and fungi). In the past several decades, researchers have gained extensive knowledge about nonribosomal peptides (NRPs) generated by ribosome-independent systems, namely, NRP synthetases (NRPSs). NRPSs are multifunctional enzymes consisting of semiautonomous domains that form a peptide backbone. Using a thiotemplate mechanism that employs assembly-line logic with multiple modules, NRPSs activate, tether, and modify amino acid building blocks, sequentially elongating the peptide chain before releasing the complete peptide. Adenylation, thiolation, condensation, and thioesterase domains play central roles in these reactions. This chapter focuses on the current understanding of these central domains in NRPS assembly-line enzymology.

Engineering the biosynthesis of fungal nonribosomal peptides.

Covering: 2011 up to the end of 2021.Fungal nonribosomal peptides (NRPs) and the related polyketide-nonribosomal peptide hybrid products (PK-NRPs) are a prolific source of bioactive compounds, some of which have been developed into essential drugs. The synthesis of these complex natural products (NPs) utilizes nonribosomal peptide synthetases (NRPSs), multidomain megaenzymes that assemble specific peptide products by sequential condensation of amino acids and amino acid-like substances, independent of the ribosome. NRPSs, collaborating polyketide synthase modules, and their associated tailoring enzymes involved in product maturation represent promising targets for NP structure diversification and the generation of small molecule unnatural products (uNPs) with improved or novel bioactivities. Indeed, reprogramming of NRPSs and recruiting of novel tailoring enzymes is the strategy by which nature evolves NRP products. The recent years have witnessed a rapid development in the discovery and identification of novel NRPs and PK-NRPs, and significant advances have also been made towards the engineering of fungal NRP assembly lines to generate uNP peptides. However, the intrinsic complexities of fungal NRP and PK-NRP biosynthesis, and the large size of the NRPSs still present formidable conceptual and technical challenges for the rational and efficient reprogramming of these pathways. This review examines key examples for the successful (and for some less-successful) re-engineering of fungal NRPS assembly lines to inform future efforts towards generating novel, biologically active peptides and PK-NRPs.

Biosynthesis of 3-thia-α-amino acids on a carrier peptide.

A subset of natural products, such as polyketides and nonribosomal peptides, is biosynthesized while tethered to a carrier peptide via a thioester linkage. Recently, we reported that the biosyntheses of 3-thiaglutamate and ammosamide, single amino acid-derived natural products, employ a very different type of carrier peptide to which the biosynthetic intermediates are bound via an amide linkage. During their biosyntheses, a peptide aminoacyl-transfer ribonucleic acid (tRNA) ligase (PEARL) first loads an amino acid to the C terminus of the carrier peptide for subsequent modification by other enzymes. Proteolytic removal of the modified C-terminal amino acid yields the mature product. We termed natural products that are biosynthesized using such pathways pearlins. To investigate the diversity of pearlins, in this study we experimentally characterized another PEARL-encoding biosynthetic gene cluster (BGC) from Tistrella mobilis (tmo). The enzymes encoded in the tmo BGC transformed cysteine into 3-thiahomoleucine both in vitro and in Escherichia coli. During this process, a cobalamin-dependent radical S-adenosylmethionine (SAM) enzyme catalyzes C-isopropylation. This work illustrates that the biosynthesis of amino acid-derived natural products on a carrier peptide is a widespread strategy in nature and expands the spectrum of thiahemiaminal analogs of amino acids that may serve a broader, currently unknown function.

Structure and Function of a Dehydrating Condensation Domain in Nonribosomal Peptide Biosynthesis.

Dehydroamino acids are important structural motifs and biosynthetic intermediates for natural products. Many bioactive natural products of nonribosomal origin contain dehydroamino acids; however, the biosynthesis of dehydroamino acids in most nonribosomal peptides is not well understood. Here, we provide biochemical and bioinformatic evidence in support of the role of a unique class of condensation domains in dehydration (CmodAA). We also obtain the crystal structure of a CmodAA domain, which is part of the nonribosomal peptide synthetase AmbE in the biosynthesis of the antibiotic methoxyvinylglycine. Biochemical analysis reveals that AmbE-CmodAA modifies a peptide substrate that is attached to the donor carrier protein. Mutational studies of AmbE-CmodAA identify several key residues for activity, including four residues that are mostly conserved in the CmodAA subfamily. Alanine mutation of these conserved residues either significantly increases or decreases AmbE activity. AmbE exhibits a dimeric conformation, which is uncommon and could enable transfer of an intermediate between different protomers. Our discovery highlights a central dehydrating function for CmodAA domains that unifies dehydroamino acid biosynthesis in diverse nonribosomal peptide pathways. Our work also begins to shed light on the mechanism of CmodAA domains. Understanding CmodAA domain function may facilitate identification of new natural products that contain dehydroamino acids and enable engineering of dehydroamino acids into nonribosomal peptides.

A male-derived nonribosomal peptide pheromone controls female schistosome development.

Schistosomes cause morbidity and death throughout the developing world due to the massive numbers of eggs female worms deposit into the blood of their host. Studies dating back to the 1920s show that female schistosomes rely on constant physical contact with a male worm both to become and remain sexually mature; however, the molecular details governing this process remain elusive. Here, we uncover a nonribosomal peptide synthetase that is induced in male worms upon pairing with a female and find that it is essential for the ability of male worms to stimulate female development. We demonstrate that this enzyme generates ß-alanyl-tryptamine that is released by paired male worms. Furthermore, synthetic ß-alanyl-tryptamine can replace male worms to stimulate female sexual development and egg laying. These data reveal that peptide-based pheromone signaling controls female schistosome sexual maturation, suggesting avenues for therapeutic intervention and uncovering a role for nonribosomal peptides as metazoan signaling molecules.

Ribosome-independent peptide biosynthesis: the challenge of a unifying nomenclature.

The first machineries for non-ribosomal peptide (NRP) biosynthesis were uncovered over 50 years ago, and the dissection of these megasynthetases set the stage for the nomenclature system that has been used ever since. Although the number of exceptions to the canonical biosynthetic pathways has surged in the intervening years, the NRP synthetase (NRPS) classification system has remained relatively unchanged. This has led to the exclusion of many biosynthetic pathways whose biosynthetic machineries violate the classical rules for NRP assembly, and ultimately to a rupture in the field of NRP biosynthesis. In an attempt to unify the classification of NRP pathways and to facilitate the communication within the research field, we propose a revised framework for grouping ribosome-independent peptide biosynthetic pathways based on recognizable commonalities in their biosynthetic logic. Importantly, the framework can be further refined as needed.

Nonribosomal peptide synthetases and their biotechnological potential in Penicillium rubens.

Nonribosomal peptide synthetases (NRPS) are large multimodular enzymes that synthesize a diverse variety of peptides. Many of these are currently used as pharmaceuticals, thanks to their activity as antimicrobials (penicillin, vancomycin, daptomycin, echinocandin), immunosuppressant (cyclosporin) and anticancer compounds (bleomycin). Because of their biotechnological potential, NRPSs have been extensively studied in the past decades. In this review, we provide an overview of the main structural and functional features of these enzymes, and we consider the challenges and prospects of engineering NRPSs for the synthesis of novel compounds. Furthermore, we discuss secondary metabolism and NRP synthesis in the filamentous fungus Penicillium rubens and examine its potential for the production of novel and modified ß-lactam antibiotics.

Intracellular artificial supramolecules based on de novo designed Y15 peptides.

De novo designed self-assembling peptides (SAPs) are promising building blocks of supramolecular biomaterials, which can fulfill a wide range of applications, such as scaffolds for tissue culture, three-dimensional cell culture, and vaccine adjuvants. Nevertheless, the use of SAPs in intracellular spaces has mostly been unexplored. Here, we report a self-assembling peptide, Y15 (YEYKYEYKYEYKYEY), which readily forms ß-sheet structures to facilitate bottom-up synthesis of functional protein assemblies in living cells. Superfolder green fluorescent protein (sfGFP) fused to Y15 assembles into fibrils and is observed as fluorescent puncta in mammalian cells. Y15 self-assembly is validated by fluorescence anisotropy and pull-down assays. By using the Y15 platform, we demonstrate intracellular reconstitution of Nck assembly, a Src-homology 2 and 3 domain-containing adaptor protein. The artificial clusters of Nck induce N-WASP (neural Wiskott-Aldrich syndrome protein)-mediated actin polymerization, and the functional importance of Nck domain valency and density is evaluated.

Competitive interactions as a mechanism for chemical diversity maintenance in Nodularia spumigena.

Nodularia spumigena is a bloom-forming diazotrophic cyanobacterium inhabiting brackish waters worldwide. This species produces non-ribosomal peptides (NRPs), including the hepatotoxin nodularin, often referred to as cyanotoxin. Several known classes of NRPs have various biological activities, although their modes of action are poorly understood. In the Baltic N. spumigena, there is a high NRP chemodiversity among strains, allowing their grouping in specific chemotypes and subgroups. Therefore, it is relevant to ask whether the NRP production is affected by intraspecific interactions between the co-existing strains. Using a novel approach that combines culture technique and liquid chromatography-tandem mass spectrometry for the NRP analysis, we examined N. spumigena strains under mono- and co-culture conditions. The test strains were selected to represent N. spumigena belonging to the same or different chemotype subgroups. In this setup, we observed physiological and metabolic responses in the test strains grown without cell contact. The changes in NRP levels to co-culture conditions were conserved within a chemotype subgroup but different between the subgroups. Our results suggest that intraspecific interactions may promote a chemical diversity in N. spumigena population, with higher NRP production compared to a single-strain population. Studying allelochemical signalling in this cyanobacterium is crucial for understanding toxicity mechanisms and plankton community interactions in the Baltic Sea and other aquatic systems experiencing regular blooms.

Biosynthetic Functionalization of Nonribosomal Peptides.

Nonribosomal peptides (NRPs) are a therapeutically important class of secondary metabolites that are produced by modular synthetases in assembly-line fashion. We previously showed that a single Trp-to-Ser mutation in the initial Phe-loading adenylation domain of tyrocidine synthetase completely switches the specificity toward clickable analogues. Here we report that this minimally invasive strategy enables efficient functionalization of the bioactive NRP on the pathway level. In a reconstituted tyrocidine synthetase, the W227S point mutation permitted selective incorporation of Phe analogues with alkyne, halogen, and benzoyl substituents by the initiation module. The respective W2742S mutation in module 4 similarly permits efficient incorporation of these functionalized substrate analogues at position 4, expanding this strategy to elongation modules. Efficient incorporation of an alkyne handle at position 1 or 4 of tyrocidine A allowed site-selective one-step fluorescent labeling of the corresponding tyrocidine analogues by Cu(I)-catalyzed alkyne-azide cycloaddition. By combining synthetic biology with bioorthogonal chemistry, this approach holds great potential for NRP isolation and molecular target elucidation as well as combinatorial optimization of NRP therapeutics.

Structural characterization of a PCP-R didomain from an archaeal nonribosomal peptide synthetase reveals novel interdomain interactions.

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes that produce a wide range of bioactive peptides, such as siderophores, toxins, and antibacterial and insecticidal agents. NRPSs are dynamic proteins characterized by extensive interdomain communications as a consequence of their assembly-line mode of synthesis. Hence, crystal structures of multidomain fragments of NRPSs have aided in elucidating crucial interdomain interactions that occur during different steps of the NRPS catalytic cycle. One crucial yet unexplored interaction is that between the reductase (R) domain and the peptide carrier protein (PCP) domain. R domains are members of the short-chain dehydrogenase/reductase family and function as termination domains that catalyze the reductive release of the final peptide product from the terminal PCP domain of the NRPS. Here, we report the crystal structure of an archaeal NRPS PCP-R didomain construct. This is the first NRPS R domain structure to be determined together with the upstream PCP domain and is also the first structure of an archaeal NRPS to be reported. The structure reveals that a novel helix-turn-helix motif, found in NRPS R domains but not in other short-chain dehydrogenase/reductase family members, plays a major role in the interface between the PCP and R domains. The information derived from the described PCP-R interface will aid in gaining further mechanistic insights into the peptide termination reaction catalyzed by the R domain and may have implications in engineering NRPSs to synthesize novel peptide products.

Monocyclic Peptides: Types, Synthesis and Applications.

Peptides are considered to be appropriate tools in various biological fields. They can be primarily used for the rational design of bioactive molecules. They can act as ligands in the development of targeted therapeutics as well as diagnostics, can be used in the design of vaccines or can be employed in agriculture. Peptides can be classified in two broad structural classes: linear and cyclic peptides. Monocyclic peptides are a class of polypeptides with one macrocyclic ring that bears advantages, such as more selective binding and uptake by the target receptor, as well as higher potency and stability compared to linear types. This paper provides an overview of the categories, synthesis methods and various applications of cyclic peptides. The various applications of cyclic peptides include their use as pro-apoptotic and anti-microbial agents, their application as targeting ligands in drug delivery and diagnostic agents, as well as agricultural and therapeutics applications that are elaborated and discussed in this paper.

Unimodular Methylation by Adenylation-Thiolation Domains Containing an Embedded Methyltransferase.

Nonribosomal peptides (NRPs) are natural products that are biosynthesized by large multi-enzyme assembly lines called nonribosomal peptide synthetases (NRPSs). We have previously discovered that backbone or side chain methylation of NRP residues is carried out by an interrupted adenylation (A) domain that contains an internal methyltransferase (M) domain, while maintaining a monolithic AMA fold of the bifunctional enzyme. A key question that has remained unanswered is at which step of the assembly line mechanism the methylation by these embedded M domains takes place. Does the M domain methylate an amino acid residue tethered to a thiolation (T) domain on same NRPS module (in cis), or does it methylate this residue on a nascent peptide tethered to a T domain on another module (in trans)? In this study, we investigated the kinetics of methylation by wild-type AMAT tridomains from two NRPSs involved in biosynthesis of anticancer depsipeptides thiocoraline and echinomycin, and by mutants of these domains, for which methylation can occur only in trans. The analysis of the methylation kinetics unequivocally demonstrated that the wild-type AMATs methylate overwhelmingly in cis, strongly suggesting that this is also the case in the context of the entire NRPS assembly line process. The mechanistic insight gained in this study will facilitate rational genetic engineering of NRPS to generate unnaturally methylated NRPs.

Beyond Toxin Transport: Novel Role of ABC Transporter for Enzymatic Machinery of Cereulide NRPS Assembly Line.

Nonribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs) play a pivotal role in the production of bioactive natural products, such as antibiotics and cytotoxins. Despite biomedical and pharmaceutical importance, the molecular mechanisms and architectures of these multimodular enzyme complexes are not fully understood. Here, we report on an ABC transporter that forms a vital part of the nonribosomal peptide biosynthetic machinery. Emetic Bacillus cereus produces the highly potent, mitochondrial active nonribosomal depsipeptide cereulide, synthesized by the NRPS Ces. The ces gene locus includes, next to the structural cesAB genes, a putative ABC transporter, designated cesCD Our study demonstrates that tethering of CesAB synthetase to the cell membrane by CesCD is critical for peptide assembly. In vivo studies revealed that CesAB colocalizes with CesCD on the cell membrane, suggesting direct involvement of this ABC transporter in the biosynthesis of a nonribosomal peptide. Mutation of cesCD, disrupting the assembly of the CesCD complex, resulted in decreased interaction with CesAB and, as a consequence, negatively affected cereulide biosynthesis. Specific domains within CesAB synthetase interacting with CesC were identified. Furthermore, we demonstrated that the structurally similar BerAB transporter from Bacillus thuringiensis complements CesCD function in cereulide biosynthesis, suggesting that the direct involvement of ABC transporter in secondary metabolite biosynthesis could be a widespread mechanism. In summary, our study revealed a novel, noncanonical function for ABC transporter, which is essential for megaenzyme functionality of NRPS. The new insights into natural product biosynthesis gained may facilitate the discovery of new metabolites with bioactive potential.IMPORTANCE This study revealed a novel, potentially conserved mechanism involved in the biosynthesis of microbial natural products, exemplified by the mitochondrial active depsipeptide cereulide. Similar to other bioactive substances, such as the last-resort antibiotics vancomycin and daptomycin, the antitumor drug cryptophycin or the cholesterol-lowering agent lovastatin, cereulide is synthesized nonribosomally by multienzyme machinery, requiring the concerted actions of multiple proteins to ensure correct product assembly. Given the importance of microbial secondary metabolites in human and veterinary medicine, it is critical to understand how these processes are orchestrated within the host cells. By revealing that tethering of a biosynthetic enzyme to the cell membrane by an ABC transporter is essential for nonribosomal peptide production, our study provides novel insights into synthesis of microbial secondary metabolites, which could contribute to isolation of novel compounds from cryptic secondary metabolite clusters or improve the yield of produced pharmaceuticals.

Nonribosomal Peptides Produced by Minimal and Engineered Synthetases with Terminal Reductase Domains.

Nonribosomal peptide synthetases (NRPSs) use terminal reductase domains for 2-electron reduction of the enzyme-bound thioester releasing the generated peptides as C-terminal aldehydes. Herein, we reveal the biosynthesis of a pyrazine that originates from an aldehyde-generating minimal NRPS termed ATRed in entomopathogenic Xenorhabdus indica. Reductase domains were also investigated in terms of NRPS engineering and, although no general applicable approach was deduced, we show that they can indeed be used for the production of similar natural and unnatural pyrazinones.

Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy.

Closthioamide (CTA) is a rare example of a thioamide-containing nonribosomal peptide and is one of only a handful of secondary metabolites described from obligately anaerobic bacteria. Although the biosynthetic gene cluster responsible for CTA production and the thioamide synthetase that catalyzes sulfur incorporation were recently discovered, the logic for peptide backbone assembly has remained a mystery. Here, through the use of in vitro biochemical assays, we demonstrate that the amide backbone of CTA is assembled in an unusual thiotemplated pathway involving the cooperation of a transacylating member of the papain-like cysteine protease family and an iteratively acting ATP-grasp protein. Using the ATP-grasp protein as a bioinformatic handle, we identified hundreds of such thiotemplated yet nonribosomal peptide synthetase (NRPS)-independent biosynthetic gene clusters across diverse bacterial phyla. The data presented herein not only clarify the pathway for the biosynthesis of CTA, but also provide a foundation for the discovery of additional secondary metabolites produced by noncanonical biosynthetic pathways.