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.

The Stereocontrolled Biosynthesis of Mirror-Symmetric 2,4-Diaminobutyric Acid Homopolymers Is Critically Governed by Adenylation Activations.

Among the four bioactive cationic homo-poly(amino acids) discovered in nature, two are mirror-image isomers of poly(2,4-diaminobutyric acid) (poly-Dab) whose biosynthesis has long been unexplained. Their structural analogy plausibly suggested that they could share a common biosynthetic pathway utilizing ε-poly(l-lysine) synthetase-like enzymology but with an unprecedented process for enantiomeric inversion of polymer building blocks. To investigate this possibility, we comparatively explored the biosynthesis of poly-l-Dab and its mirror-image isomer poly-d-Dab in Streptomyces celluloflavus USE31 and Streptoalloteichus hindustanus NBRC15115, respectively, through genome mining, genetic inactivation, and heterologous expression combined with biochemical assays. While they shared the same biosynthetic pathway, the poly-d-Dab biosynthetic gene cluster additionally harbored the racemase gene. The critical finding that poly-d-Dab synthetase, in contrast to the synthetase generating the l-isomer, selectively activated d-Dab through adenylation conclusively demonstrated that free diffusible d-Dab preactivationally generated by the racemase is directly activated to be incorporated into the polymer. Our study thus represents the first demonstration of the stereoselective biosynthesis of a nonribosomal peptide governed by adenylation activity for a d-amino acid other than alanine. In silico sequence comparison between poly-Dab synthetases allowed us to identify amino acid residues potentially responsible for the discrimination of Dab enantiomers. Our results will provide significant insight not only for the future discovery of novel bioactive cationic poly(amino acids) but also for the creation of designer nonribosomal peptides with d-configuration.

The many faces and important roles of protein-protein interactions during non-ribosomal peptide synthesis.

Covering: up to July 2018 Non-ribosomal peptide synthetase (NRPS) machineries are complex, multi-domain proteins that are responsible for the biosynthesis of many important, peptide-derived compounds. By decoupling peptide synthesis from the ribosome, NRPS assembly lines are able to access a significant pool of amino acid monomers for peptide synthesis. This is combined with a modular protein architecture that allows for great variation in stereochemistry, peptide length, cyclisation state and further modifications. The architecture of NRPS assembly lines relies upon a repetitive set of catalytic domains, which are organised into modules responsible for amino acid incorporation. Central to NRPS-mediated biosynthesis is the carrier protein (CP) domain, to which all intermediates following initial monomer activation are bound during peptide synthesis up until the final handover to the thioesterase domain that cleaves the mature peptide from the NRPS. This mechanism makes understanding the protein-protein interactions that occur between different NRPS domains during peptide biosynthesis of crucial importance to understanding overall NRPS function. This endeavour is also highly challenging due to the inherent flexibility and dynamics of NRPS systems. In this review, we present the current state of understanding of the protein-protein interactions that govern NRPS-mediated biosynthesis, with a focus on insights gained from structural studies relating to CP domain interactions within these impressive peptide assembly lines.

Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker's yeast.

Fungi are a valuable source of enzymatic diversity and therapeutic natural products including antibiotics. Here we engineer the baker's yeast Saccharomyces cerevisiae to produce and secrete the antibiotic penicillin, a beta-lactam nonribosomal peptide, by taking genes from a filamentous fungus and directing their efficient expression and subcellular localization. Using synthetic biology tools combined with long-read DNA sequencing, we optimize productivity by 50-fold to produce bioactive yields that allow spent S. cerevisiae growth media to have antibacterial action against Streptococcus bacteria. This work demonstrates that S. cerevisiae can be engineered to perform the complex biosynthesis of multicellular fungi, opening up the possibility of using yeast to accelerate rational engineering of nonribosomal peptide antibiotics.

Natural product discovery from the human microbiome.

Human-associated microorganisms have the potential to biosynthesize numerous secondary metabolites that may mediate important host-microbe and microbe-microbe interactions. However, there is currently a limited understanding of microbiome-derived natural products. A variety of complementary discovery approaches have begun to illuminate this microbial "dark matter," which will in turn allow detailed mechanistic studies of the effects of these molecules on microbiome and host. Herein, we review recent efforts to uncover microbiome-derived natural products, describe the key approaches that were used to identify and characterize these metabolites, discuss potential functional roles of these molecules, and highlight challenges related to this emerging research area.

Genetic and functional analyses of krs, a locus encoding kurstakin, a lipopeptide produced by Bacillus thuringiensis.

Bacteria of the Bacillus genus are able to synthesize several families of lipopeptides. These small molecules are the product of non-ribosomal peptide synthetases. In 2000, it was found that Bacillus thuringiensis, an entomopathogenic bacterium of the Bacillus cereus group, produced a previously unknown lipopeptide: kurstakin. Genomic analyses reveal that the krs locus, encoding the kurstakin synthetases, is specific to the B. cereus group, but is unevenly distributed within this group. Previous work showed that krs transcription requires the necrotrophism quorum-sensor NprR. Here, we demonstrated that the genes of the krs locus form an operon and we defined its transcription start site. Following krs transcription at the population and single-cell levels in multiple culture conditions, we depicted a condition-dependent transcription pattern, indicating that production of kurstakin is subject to environmental regulation. Consistent with this idea, we found krs transcription to be regulated by another master regulator, Spo0A, suggesting that krs expression is fine-tuned by integrating multiple signals. We also reported an unknown DNA palindrome in the krs promoter region that modulates krs expression. Due to their surfactant properties, lipopeptides could play several physiological roles. We showed that the krs locus was required for proper biofilm structuration.

Regions involved in fengycin synthetases enzyme complex formation.

BACKGROUND: Fengycin is a lipopeptide antibiotic synthesized nonribosomally by five fengycin synthetases. These enzymes are linked in a specific order to form the complex. This study investigates how these enzymes interact in the complex and analyzes the regions in the enzymes that are critical to the interactions. METHODS: Deletions were generated in the fengycin synthetases. The interaction of these mutant proteins with their partner enzymes in the complex was analyzed in vitro by a glutathione S-transferase (GST) or nickel pulldown assay. RESULTS: The communication-mediating donor (COM-D) domains of the fengycin synthetases, when fused to GST, specifically pulled down their downstream partner enzymes in the GST-pulldown assays. The communication-mediating acceptor (COM-A) domains were required for binding between two partner enzymes, although the domains alone did not confer specificity of the binding to their upstream partner enzymes. This study found that the COM-A domain, the condensation domain, and a portion of the adenylation domain in fengycin synthetase B (FenB) were required for specific binding to fengycin synthetase A (FenA). CONCLUSION: The interaction between the COM-D and COM-A domains in two partner enzymes is critical for nonribosomal peptide synthesis. The COM-A domain alone is insufficient for interacting with its upstream partner enzyme in the enzyme complex with specificity; a region that contains COM-A, condensation, and a portion of adenylation domains in the downstream partner enzyme is required.

Visualizing A Natural Antibiotic Nanofactory.

Environmental samples are excellent sources of natural products that possess numerous kinds of therapeutic activities. One important family of natural products is the nonribosomal peptides, which includes penicillin, cyclosporin, viomycin and daptomycin (Cubicin). These peptides are made in bacteria or fungi by large enzymes called nonribosomal peptide synthetases (NRPS). NRPSs are true macromolecular machines or nanofactories, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites. Visualization of large fragments of NRPSs at various functional states is required to understand the manner in which NRPSs synthesize their important products. Many excellent structural experiments have been performed to date. Recently, we added to the structural knowledge by visualizing the first module of the NRPS, which makes linear gramicidin, a clinical topical antibiotic, in all its major functional states. These experiments show how the individual domains, including an unusual tailoring domain, function together in assembly-line synthesis. Along with the ever-expanding body of biophysical, biochemical and genetic work, this work brings us closer to a fundamental understanding of these natural antibiotic nanofactories, and perhaps the ability to exploit them to produce novel therapeutics.

Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes.

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.

Biosynthesis of cyclosporins and other natural peptidyl prolyl cis/trans isomerase inhibitors.

BACKGROUND: Peptidyl-prolyl-cis/trans-isomerases (PPIases) are ubiquitously expressed and have been implicated in a wide range of biological functions. Their inhibition is beneficial in immunosuppression, cancer treatment, treatment of autoimmune diseases, protozoan and viral infections. SCOPE OF REVIEW: Three classes of PPIases are known, each class having their own specific inhibitors. This review will cover the present knowledge on the biosynthesis of the natural PPIase inhibitors. These include for the cyclophilins: the cyclosporins, the analogues of peptolide SDZ 214-103 and the sanglifehrins; for the FKBPs: ascomycin, rapamycin and FK506 and for the parvulins the naphtoquinone juglone. MAJOR CONCLUSIONS: Over the last thirty years much progress has been made in understanding PPIase function and the biosynthesis of natural PPIase inhibitors. Non-immunosuppressive analogues were discovered and served as lead compounds for the development of novel antiviral drugs. There are, however, still unsolved questions which deserve further research into this exciting field. GENERAL SIGNIFICANCE: As all the major natural inhibitors of the cyclophilins and FKBPs are synthesized by complex non-ribosomal peptide synthetases and/or polyketide synthases, total chemical synthesis is not a viable option. Thus, fully understanding the modular enzyme systems involved in their biosynthesis may help engineering enzymes capable of synthesizing novel PPIase inhibitors with improved functions for a wide range of conditions. This article is part of a Special Issue entitled Proline-directed Foldases: Cell signaling catalysts and drug targets.

Classification of the adenylation and acyl-transferase activity of NRPS and PKS systems using ensembles of substrate specific hidden Markov models.

There is a growing interest in the Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) of microbes, fungi and plants because they can produce bioactive peptides such as antibiotics. The ability to identify the substrate specificity of the enzyme's adenylation (A) and acyl-transferase (AT) domains is essential to rationally deduce or engineer new products. We here report on a Hidden Markov Model (HMM)-based ensemble method to predict the substrate specificity at high quality. We collected a new reference set of experimentally validated sequences. An initial classification based on alignment and Neighbor Joining was performed in line with most of the previously published prediction methods. We then created and tested single substrate specific HMMs and found that their use improved the correct identification significantly for A as well as for AT domains. A major advantage of the use of HMMs is that it abolishes the dependency on multiple sequence alignment and residue selection that is hampering the alignment-based clustering methods. Using our models we obtained a high prediction quality for the substrate specificity of the A domains similar to two recently published tools that make use of HMMs or Support Vector Machines (NRPSsp and NRPS predictor2, respectively). Moreover, replacement of the single substrate specific HMMs by ensembles of models caused a clear increase in prediction quality. We argue that the superiority of the ensemble over the single model is caused by the way substrate specificity evolves for the studied systems. It is likely that this also holds true for other protein domains. The ensemble predictor has been implemented in a simple web-based tool that is available at http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/.

The two-step biosynthesis of cyclic peptides from linear precursors in a member of the plant family Caryophyllaceae involves cyclization by a serine protease-like enzyme.

Caryophyllaceae-type cyclic peptides (CPs) of 5-12 proteinogenic amino acids occur in 10 plant families. In Saponaria vaccaria (Caryophyllaceae), they have been shown to be formed from linear peptide precursors derived from ribosomal translation. There is also evidence for such precursors in other members of the Caryophyllaceae, Rutaceae, and Linaceae families. The biosynthesis of CP in the developing seeds of S. vaccaria was investigated with respect to the enzymes involved in precursor processing. Through biochemical assays with seed extracts and synthetic peptides, an enzyme named oligopeptidase 1 (OLP1) was found that catalyzes the cleavage of intermediates at the N terminus of the incipient CP. A second enzyme, peptide cyclase 1 (PCY1), which was separated chromatographically from OLP1, was found to act on the product of OLP1, giving rise to a cyclic peptide and concomitant removal of a C-terminal flanking sequence. PCY1 was partially purified, and using the methods of proteomics, a full-length cDNA clone encoding an enzyme matching the properties of PCY1 was obtained. The substrate specificity of purified recombinant PCY1, believed to be the first cloned plant enzyme whose function is peptide cyclization, was tested with synthetic peptides. The results are discussed in the light of CP biosynthetic systems of other organisms.

Identification of an mRNP complex regulating tumorigenesis at the translational elongation step.

Transcript-selective translational regulation of epithelial-mesenchymal transition (EMT) by transforming growth factor-ß (TGF-ß) is directed by the hnRNP E1-containing TGF-ß-activated-translational (BAT) mRNP complex. Herein, eukaryotic elongation factor-1 A1 (eEF1A1) is identified as an integral component of the BAT complex. Translational silencing of Dab2 and ILEI, two EMT transcripts, is mediated by the binding of hnRNP E1 and eEF1A1 to their 3'UTR BAT element, whereby hnRNP E1 stalls translational elongation by inhibiting the release of eEF1A1 from the ribosomal A site. TGF-ß-mediated hnRNP E1 phosphorylation, through Akt2, disrupts the BAT complex, thereby restoring translation of target EMT transcripts. Attenuation of hnRNP E1 expression in two noninvasive breast epithelial cells (NMuMG and MCF-7) not only induced EMT but also enabled cells to form metastatic lesions in vivo. Thus, translational regulation by TGF-ß at the elongation stage represents a critical checkpoint coordinating the expression of EMT transcripts required during development and in tumorigenesis and metastatic progression.

Molecular identification of N-acetylaspartylglutamate synthase and beta-citrylglutamate synthase.

The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and ß-citrylglutamate, a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and l-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of ß-citrylglutamate. The nature of the reaction products was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress in the understanding of the function of NAAG and ß-citrylglutamate.

Nonribosomal peptide synthetases: structures and dynamics.

Nonribosomal peptide synthetases (NRPSs) are large multimodular biocatalysts that utilize complex regiospecific and stereospecific reactions to assemble structurally and functionally diverse peptides that have important medicinal applications. During this ribosome-independent peptide synthesis, catalytic domains of NRPS select, activate or modify the covalently tethered reaction intermediates to control the iterative chain elongation process and product release. Recent advances in structural elucidation of domains, didomains, and an entire termination module revealed valuable insights into the mechanism of nonribosomal synthesis and are highlighted herein.

Characterization and localization of a hybrid non-ribosomal peptide synthetase and polyketide synthase gene from the toxic dinoflagellate Karenia brevis.

The toxic dinoflagellate Karenia brevis, a causative agent of the red tides in Florida, produces a series of toxic compounds known as brevetoxins and their derivatives. Recently, several putative genes encoding polyketide synthase (PKS) were identified from K. brevis in an effort to elucidate the genetic systems involved in brevetoxin production. In this study, novel PKS sequences were isolated from three clones of K. brevis. Eighteen unique sequences were obtained for the PKS ketosynthase (KS) domain of K. brevis. Phylogenetic comparison with closely related PKS genes revealed that 16 grouped with cyanobacteria sequences, while the remaining two grouped with Apicomplexa and previously reported sequences for K. brevis. A fosmid library was also constructed to further characterize PKS genes detected in K. brevis Wilson clone. Several fosmid clones were positive for the presence of PKS genes, and one was fully sequenced to determine the full structure of the PKS cluster. A hybrid non ribosomal peptide synthetase and PKS (NRPS-PKS) gene cluster of 16,061 bp was isolated. In addition, we assessed whether the isolated gene was being actively expressed using reverse transcription polymerase chain reaction (RT-PCR) and determined its localization at the cellular level by chloroplast isolation. RT-PCR analyses revealed that this gene was actively expressed in K. brevis cultures. The hybrid NRPS-PKS gene cluster was located in the chloroplast, suggesting that K. brevis acquired the ability to produce some of its secondary metabolites through endosymbiosis with ancestral cyanobacteria. Further work is needed to determine the compound produced by the NRPS-PKS hybrid, to find other PKS gene sequences, and to assess their role in K. brevis toxin biosynthetic pathway.

Heterologous production of non-ribosomal peptide LLD-ACV in Saccharomyces cerevisiae.

Non-ribosomal peptides (NRPs) are a diverse family of secondary metabolites with a broad range of biological activities. We started to develop an eukaryotic microbial platform based on the yeast Saccharomyces cerevisiae for heterologous production of NRPs using delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine (ACV) as a model NRP. The Penicillium chrysogenum gene pcbAB encoding ACV synthetase was expressed in S. cerevisiae from a high-copy plasmid together with phosphopantetheinyl transferase (PPTase) encoding genes from Aspergillus nidulans, P. chrysogenum and Bacillus subtilis, and in all the three cases production of ACV was observed. To improve ACV synthesis, several factors were investigated. Codon optimization of the 5' end of pcbAB did not significantly increase ACV production. However, a 30-fold enhancement was achieved by lowering the cultivation temperature from 30 to 20 degrees C. When ACVS and PPTase encoding genes were integrated into the yeast genome, a 6-fold decrease in ACV production was observed indicating that gene copy number was one of the rate-limiting factors for ACV production in yeast.

Directionality and coordination of dehydration and ring formation during biosynthesis of the lantibiotic nisin.

The lantibiotic nisin is a potent antimicrobial substance, which contains unusual lanthionine rings and dehydrated amino acid residues and is produced by Lactococcus lactis. Recently, the nisin biosynthetic machinery has been applied to introduce lanthionine rings in peptides other than nisin with potential therapeutic use. Due to difficulties in the isolation of the proposed synthetase complex (NisBTC), mechanistic information concerning the enzymatic biosynthesis of nisin is scarce. Here, we present the molecular characterization of a number of nisin mutants that affect ring formation. We have investigated in a systematic manner how these mutations influence dehydration events, which are performed enzymatically by the dehydratase NisB. Specific mutations that hampered ring formation allowed for the dehydration of serine residues that directly follow the rings and are normally unmodified. The combined information leads to the conclusion that 1) nisin biosynthesis is an organized directional process that starts at the N terminus of the molecule and continues toward the C terminus, and 2) NisB and NisC are alternating enzymes, whose activities follow one after another in a repetitive way. Thus, the dehydration and cyclization processes are not separated in time and space. On the basis of these results and previous knowledge, a working model for the sequence of events in the maturation of nisin is proposed.

Chapter 13. Nonribosomal peptide synthetases mechanistic and structural aspects of essential domains.

A widespread class of therapeutically important natural products is of peptidic origin. They are produced nonribosomally by large "assembly line"-like multienzyme complexes, the nonribosomal peptide synthetases (NRPS). In contrast to ribosomal peptide synthesis, nonribosomally assembled peptides contain unique structural features such as D-amino acids, N-terminally attached fatty acid chains, N- and C-methylated amino acids, N-formylated residues, heterocyclic elements, glycosylated amino acids, and phosphorylated residues. In recent research using genetic, biochemical, and structural methods, experiments have revealed profound insights into the molecular mechanism of nonribosomal peptide synthesis. Based on this, it was possible to alter existing nonribosomally produced peptides either by changing their biosynthetic templates or by the combined action of chemical peptide synthesis and subsequent enzyme catalysis. An overview of the structural aspects of the NRPS machinery with a focus on mechanistic and structural aspects of essential domains is presented.

Chapter 15. Plasmid-borne gene cluster assemblage and heterologous biosynthesis of nonribosomal peptides in Escherichia coli.

Nonribosomal peptides (NRPs) are synthesized by modular mega-enzymes called NRP synthetases (NRPSs) that catalyze a peptide bond-forming reaction using natural amino acids as substrates. Most members of this class of natural products exhibit remarkable biological activities, but many of these valuable compounds are often difficult to obtain in sufficient quantities from their natural sources due to low production levels in the producing organisms or difficulty in culturing them. Harnessing recent progress in our genetic and biochemical understanding of the biosynthesis of these nonprimary metabolites, our laboratory has successfully developed an alternative, straightforward approach for obtaining desired natural products by placing the entire biosynthetic gene cluster in our heterologous host of choice, Escherichia coli. This effort led to the first successful de novo production of heterologous bioactive complex NRPs in E. coli. Through developing our heterologous biosynthetic system, we were able to construct a novel platform suitable for generating an NRP library through rational engineering of the natural modular assembly-line array composed of NRPSs and the auxiliary enzymes. This chapter describes the basic concept in establishing an E. coli-based plasmid-borne heterologous NRP biosynthetic system, and gives selected protocols that have been used successfully for engineering NRP biosynthesis.