Nonribosomal Peptide Synthetase Machinery Research Articles

The specificity of intermodular recognition in a prototypical nonribosomal peptide synthetase depends on an adaptor domain.

In the quest for new bioactive substances, nonribosomal peptide synthetases (NRPS) provide biodiversity by synthesizing nonproteinaceous peptides with high cellular activity. NRPS machinery consists of multiple modules, each catalyzing a unique series of chemical reactions. Incomplete understanding of the biophysical principles orchestrating these reaction arrays limits the exploitation of NRPSs in synthetic biology. Here, we use nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to solve the conundrum of how intermodular recognition is coupled with loaded carrier protein specificity in the tomaymycin NRPS. We discover an adaptor domain that directly recruits the loaded carrier protein from the initiation module to the elongation module and reveal its mechanism of action. The adaptor domain of the type found here has specificity rules that could potentially be exploited in the design of engineered NRPS machinery.

Altering glycopeptide antibiotic biosynthesis through mutasynthesis allows incorporation of fluorinated phenylglycine residues.

Glycopeptide antibiotics (GPAs) are peptide natural products used as last resort treatments for antibiotic resistant bacterial infections. They are produced by the sequential activities of a linear nonribosomal peptide synthetase (NRPS), which assembles the heptapeptide core of GPAs, and cytochrome P450 (Oxy) enzymes, which perform a cascade of cyclisation reactions. The GPAs contain proteinogenic and nonproteinogenic amino acids, including phenylglycine residues such as 4-hydroxyphenylglycine (Hpg). The ability to incorporate non-proteinogenic amino acids in such peptides is a distinctive feature of the modular architecture of NRPSs, with each module selecting and incorporating a desired amino acid. Here, we have exploited this ability to produce and characterise GPA derivatives containing fluorinated phenylglycine (F-Phg) residues through a combination of mutasynthesis, biochemical, structural and bioactivity assays. Our data indicate that the incorporation of F-Phg residues is limited by poor acceptance by the NRPS machinery, and that the phenol moiety normally present on Hpg residues is essential to ensure both acceptance by the NRPS and the sequential cyclisation activity of Oxy enzymes. The principles learnt here may prove useful for the future production of GPA derivatives with more favourable properties through mixed feeding mutasynthesis approaches.

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.

Chemical Strategies for Visualizing and Analyzing Endogenous Nonribosomal Peptide Synthetase (NRPS) Megasynthetases.

Nonribosomal peptide (NRP) natural products are among the most promising resources for drug discovery and development, owing to their wide range of biological activities and therapeutic applications. These peptide metabolites are biosynthesized by large multienzyme machinery known as NRP synthetases (NRPSs). The structural complexity of a number of NRPs poses an enormous challenge in their synthesis. A major issue in this field is reprogramming NRPS machineries to allow the biosynthetic production of artificial peptides. NRPS adenylation (A) domains are responsible for the incorporation of a wide variety of amino acids and can be considered as reprogramming sites; therefore, advanced methods to accelerate the functional prediction and assessment of A-domains are required. This Concept article demonstrates that activity-based protein profiling of NRPSs offers a simple, rapid, and robust analytical platform for A-domains and provides insights into enzyme-substrate candidates and active-site microenvironments. It also describes the background associated with the development and application of a method to analyze endogenous NRPS machinery in its natural environment.

Characterization of a Hybrid Nonribosomal Peptide-Carbohydrate Biosynthetic Pathway in Photorhabdus luminescens.

Advances in genome sequencing and analysis have afforded a trove of "orphan" bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the potential for these hypothetical proteins to carry out novel chemistry, orphan pathways serve as a rich reservoir for the discovery of new enzymes responsible for the production of metabolites with both fascinating chemistries and biological functions. We previously identified a rare hybrid nonribosomal peptide synthetase (NRPS)-carbohydrate genomic island in the entomopathogen Photorhabdus luminescens. Heterologous expression of the pathway led to the characterization of oligosaccharides harboring a 1,6-anhydro-β-d- N-acetyl-glucosamine moiety, but these new metabolites lacked modification by the NRPS machinery. Here, through the application of top-down protein mass spectrometry, pathway-targeted molecular networking, stable isotope labeling, and in vitro protein biochemistry, we complete the characterization of this biosynthetic pathway and identify the hybrid product of the pathway, a new "glycoamino acid" metabolite termed photolose. Intriguingly, a hypothetical protein served as a bridge to condense a glycyl unit derived from the NRPS machinery onto the free 1,6-anhydro-β-d- N-acetyl-glucosamine core. We further demonstrate that the gene cluster confers a growth advantage to antimicrobial peptide challenge.

The biosynthetic implications of late-stage condensation domain selectivity during glycopeptide antibiotic biosynthesis.

Non-ribosomal peptide synthesis is a highly important biosynthetic pathway for the formation of many secondary metabolites of medical relevance. Due to the challenges associated with the chemical synthesis of many of the products of these assembly lines, understanding the activity and selectivity of non-ribosomal peptide synthetase (NRPS) machineries is an essential step towards the redesign of such machineries to produce new bioactive peptides. Whilst the selectivity of the adenylation domains responsible for amino acid activation during NRPS synthesis has been widely studied, the selectivity of the essential peptide bond forming domains - known as condensation domains - is not well understood. Here, we present the results of a combination of in vitro and in vivo investigations into the final condensation domain from the NRPS machinery that produces the glycopeptide antibiotics (GPAs). Our results show that this condensation domain is tolerant for a range of peptide substrates and even those with unnatural stereochemistry of the peptide C-terminus, which is in contrast to the widely ascribed role of these domains as a stereochemical gatekeeper during NRPS synthesis. Furthermore, we show that this condensation domain has a significant preference for linear peptide substrates over crosslinked peptides, which indicates that the GPA crosslinking cascade targets the heptapeptide bound to the final module of the NRPS machinery and reinforces the role of the unique GPA X-domain in this process. Finally, we demonstrate that the peptide bond forming activity of this condensation domain is coupled to the rate of amino acid activation performed by the subsequent adenylation domain. This is a significant result with implications for NRPS redesign, as it indicates that the rate of amino acid activation of modified adenylation domains must be maintained to prevent unwanted peptide hydrolysis from the NRPS due to a loss of the productive coupling of amino acid selection and peptide bond formation. Taken together, our results indicate that assessing condensation domain activity is a vital step in not only understanding the biosynthetic logic and timing of NRPS-mediated peptide assembly, but also the rules which redesign efforts must obey in order to successfully produce functional, modified NRPS assembly lines.

Design and Biosynthesis of Dimeric Alboflavusins with Biaryl Linkages via Regiospecific C-C Bond Coupling.

Alboflavusins (AFNs) are a group of cyclohexapeptides with moderate antibacterial and antitumor activities from Streptomyces alboflavus sp. 313. In vivo and in vitro studies proposed that AFNs are biosynthesized by a nonribosomal peptide synthetase machinery, and the 6-Cl-L-Trp precursor is supplied by a tryptophan halogenase gene located outside the afn gene cluster. Guided by the structure-activity relationship knowledge about the AFN-like cyclohexapeptides, two dimeric AFNs (di-AFNs) with regiospecific biaryl linkages were designed and generated biotechnologically by expressing the P450 gene hmtS or clpS in S. alboflavus wild-type and mutant strains. The di-AFNs displayed much better antibacterial and antitumor activities than their monomers as anticipated, exemplifying a rational strategy to generate natural product congeners with improved bioactivities.

Activity-Based Protein Profiling of Non-ribosomal Peptide Synthetases.

Non-ribosomal peptide (NRP) natural products are one of the most promising resources for drug discovery and development because of their wide-ranging of therapeutic potential, and their behavior as virulence factors and signaling molecules. The NRPs are biosynthesized independently of the ribosome by enzyme assembly lines known as the non-ribosomal peptide synthetase (NRPS) machinery. Genetic, biochemical, and bioinformatics analyses have provided a detailed understanding of the mechanism of NRPS catalysis. However, proteomic techniques for natural product biosynthesis remain a developing field. New strategies are needed to investigate the proteomes of diverse producer organisms and directly analyze the endogenous NRPS machinery. Advanced platforms should verify protein expression, protein folding, and activities and also enable the profiling of the NRPS machinery in biological samples from wild-type, heterologous, and engineered bacterial systems. Here, we focus on activity-based protein profiling strategies that have been recently developed for studies aimed at visualizing and monitoring the NRPS machinery and also for rapid labeling, identification, and biochemical analysis of NRPS enzyme family members as required for proteomic chemistry in natural product sciences.

The Thioesterase Domain in Glycopeptide Antibiotic Biosynthesis Is Selective for Cross-Linked Aglycones.

The biosynthesis of the glycopeptide antibiotics (GPAs)-which include teicoplanin and vancomycin-is a complex enzymatic process relying on the interplay of nonribosomal peptide synthesis and a cytochrome P450-mediated cyclization cascade. This unique cyclization cascade generates the highly cross-linked state of these nonribosomal peptides, which is crucial for their antimicrobial activity. Given that these essential oxidative transformations occur while the peptide remains bound to the terminal module of the nonribosomal peptide synthetase (NRPS) machinery, it is important to assess the selectivity of the terminal thioesterase (TE) domain and how this domain contributes to the maintenance of an efficient biosynthetic pathway while at the same time ensuring GPA maturation is completed. In this study, we report the in vitro characterization of the thioesterase domain from teicoplanin biosynthesis, the first GPA thioesterase to be characterized. Our results show that the activity of this TE domain relies on the presence of an unusual extended N-terminal linker region that appears to be unique to the NRPS machineries found in GPA biosynthesis. In addition, we show that the activity of this domain against carrier protein bound substrates is dramatically enhanced for mature GPA aglycones as opposed to linear peptides and partially cyclized intermediates. These results demonstrate how the interplay between NRPS and P450s during late stage GPA biosynthesis is not only maintained but also leads to the efficient production of mature GPA aglycones. Thus, GPA TE domains represent another impressive example of the ability of TE domains to act as logic gates during NRPS biosynthesis, ensuring that essential late-stage peptide modifications are completed before catalyzing the release of the mature, bioactive peptide product.

Capturing the Structure of the Substrate Bound Condensation Domain

Condensation domains of nonribosomal peptide synthetase machineries have so far escaped detailed structural analysis. In this issue of Cell Chemical Biology, Bloudoff etal. (2016) describe a protein tethering technique that allowed the authors to obtain structural information on the substrate bound state of the first condensation domain from calcium-dependent antibiotic biosynthesis, thus opening a new window into how these important biosynthetic machineries function.

Facile Synthetic Access to Glycopeptide Antibiotic Precursor Peptides for the Investigation of Cytochrome P450 Action in Glycopeptide Antibiotic Biosynthesis.

The glycopeptide antibiotics are an important class of complex, medically relevant peptide natural products. Given that the production of such compounds all stems from in vivo biosynthesis, understanding the mechanisms of the natural assembly system--consisting of a nonribosomal-peptide synthetase machinery (NRPS) and further modifying enzymes--is vital. In order to address the later steps of peptide biosynthesis, which are catalyzed by Cytochrome P450s that interact with the peptide-producing nonribosomal peptide synthetase, peptide substrates are required: these peptides must also be in a form that can be conjugated to carrier protein domains of the nonribosomal peptide synthetase machinery. Here, we describe a practical and effective route for the solid phase synthesis of glycopeptide antibiotic precursor peptides as their Coenzyme A (CoA) conjugates to allow enzymatic conjugation to carrier protein domains. This route utilizes Fmoc-chemistry suppressing epimerization of racemization-prone aryl glycine derivatives and affords high yields and excellent purities, requiring only a single step of simple solid phase extraction for chromatographic purification. With this, comprehensive investigations of interactions between various NRPS-bound substrates and Cytochrome P450s are enabled.

X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis.

Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450-X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro--in particular the installation of the second crosslink in GPA biosynthesis--occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.

Structure of the terminal PCP domain of the non-ribosomal peptide synthetase in teicoplanin biosynthesis.

The biosynthesis of the glycopeptide antibiotics, of which teicoplanin and vancomycin are representative members, relies on the combination of non-ribosomal peptide synthesis and modification of the peptide by cytochrome P450 (Oxy) enzymes while the peptide remains bound to the peptide synthesis machinery. We have structurally characterized the final peptidyl carrier protein domain of the teicoplanin non-ribosomal peptide synthetase machinery: this domain is believed to mediate the interactions with tailoring Oxy enzymes in addition to its function as a shuttle for intermediates between multiple non-ribosomal peptide synthetase domains. Using solution state NMR, we have determined structures of this PCP domain in two states, the apo and the post-translationally modified holo state, both of which conform to a four-helix bundle assembly. The structures exhibit the same general fold as the majority of known carrier protein structures, in spite of the complex biosynthetic role that PCP domains from the final non-ribosomal peptide synthetase module must play in glycopeptide antibiotic biosynthesis. These structures thus support the hypothesis that it is subtle rearrangements, rather than dramatic conformational changes, which govern carrier protein interactions and selectivity during non-ribosomal peptide synthesis.

NMR Characterization of an Unusual 37 kDa Epimerization Domain of Yersinabactin Synthetase

Nonribosomal Peptide Synthetases (NRPSs) are endogenous microbial supramolecular (1-4 MDa) assemblies that are responsible for the biosynthesis of important natural products, including antibiotics. Genetic reprogramming of NRPS machinery for the generation of novel-or perhaps “designer”-antibiotics (or other therapeutics) represents the apogee of NRPS research. Our model NRPS system is yersiniabactin synthetase (YS), which produces the virulence factor yersiniabactin. Heterocycles (e.g. thiazoline rings) in natural products, such as yersiniabactin, exhibit unique medicinal properties (antibiotic, anticancer, immunosuppressant). An unusual epimerization domain (EA) in YS catalyzes the stereoconversion of a chiral heterocyclic center. Interestingly, EA itself is embedded within the primary sequence of a so-called adenylation domain (A, recognizes and activates substrates). To understand the overall choreography of catalysis, we require structural models of each catalytic domain in its monomeric state, bound to its substrate(s), and in complex with other relevant domains. NRPS domains can be difficult to crystallize in the biologically relevant conformations and complexes, so we take advantage of NMR spectroscopy, which can offer structural, kinetic, thermodynamic, and dynamical data. However, conventional bimolecular NMR experiments will fail on domains such as EA (37 kDa) largely because of relaxation losses but also spectral crowding. This work discusses how nonuniform sampling allowed us to rescue sensitivity and collect preliminary data on a partially deuterated sample. We also exploit covariance NMR spectroscopy to ease the challenging backbone assignment procedure. Using such data we report ∼70% backbone assignment. We also present a chemical biology strategy that will allow us to synthesize otherwise challenging substrates, which will facilitate measurement of steady state kinetic parameters and inform our structural models.

A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis

The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-β-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-β-lysine by ORF 5, followed by transfer to the T domain of ORF 18. In contrast, L-β-lysine molecules adenylated by ORF 19 are used to elongate an L-β-lysine peptide chain on ORF 18, a reaction unexpectedly catalyzed by ORF 19 itself. Finally, the C domain of ORF 18 catalyzes the condensation of L-β-lysine oligopeptides covalently bound to ORF 18 with a freely diffusible intermediate to release the ST products. These results highlight an unusual activity for an A domain and unique mechanisms of crosstalk within NRPS machinery.

Hijacking a hydroxyethyl unit from a central metabolic ketose into a nonribosomal peptide assembly line

Nonribosomal peptide synthetases (NRPSs) usually catalyze the biosynthesis of peptide natural products by sequential selection, activation, and condensation of amino acid precursors. It was reported that some fatty acids, α-ketoacids, and α-hydroxyacids originating from amino acid metabolism as well as polyketide-derived units can also be used by NRPS assembly lines as an alternative to amino acids. Ecteinascidin 743 (ET-743), naphthyridinomycin (NDM), and quinocarcin (QNC) are three important antitumor natural products belonging to the tetrahydroisoquinoline family. Although ET-743 has been approved as an anticancer drug, the origin of an identical two-carbon (C(2)) fragment among these three antibiotics has not been elucidated despite much effort in the biosynthetic research in the past 30 y. Here we report that two unexpected two-component transketolases (TKases), NapB/NapD in the NDM biosynthetic pathway and QncN/QncL in QNC biosynthesis, catalyze the transfer of a glycolaldehyde unit from ketose to the lipoyl group to yield the glycolicacyl lipoic acid intermediate and then transfer the C(2) unit to an acyl carrier protein (ACP) to form glycolicacyl-S-ACP as an extender unit for NRPS. Our results demonstrate a unique NRPS extender unit directly derived from ketose phosphates through (α,β-dihydroxyethyl)-thiamin diphosphate and a lipoyl group-tethered ester intermediate catalyzed by the TKase-ACP platform in the context of NDM and QNC biosynthesis, all of which also highlights the biosynthesis of ET-743. This hybrid system and precursor are distinct from the previously described universal modes involving the NRPS machinery. They exemplify an alternate strategy in hybrid NRPS biochemistry and enrich the diversity of precursors for NRPS combinatorial biosynthesis.

Investigations of the MceIJ-Catalyzed Posttranslational Modification of the Microcin E492 C-Terminus: Linkage of Ribosomal and Nonribosomal Peptides To Form “Trojan Horse” Antibiotics

MceIJ is a two protein complex responsible for attachment of a C-glycosylated and linearized derivative of enterobactin, an iron scavenger (siderophore) and product of nonribosomal peptide synthetase machinery, to the C-terminal serine residue of microcin E492 (MccE492), an 84 aa ribosomal antibiotic peptide produced by Klebsiella pneumoniae RYC492. The MceIJ-catalyzed formation of the glycosyl ester linkage between MccE492 and the siderophore requires ATP and Mg(II) as cofactors. This work addresses the ATP utilization, mechanism of C-terminal carboxylate activation, and substrate scope of MceIJ. Formation of the ribosomal peptide-nonribosomal peptide linkage between the MccE492 C-terminal decapeptide and monoglycosylated enterobactin (MGE) requires cleavage of the alpha,beta bond of ATP and formation of a putative peptidyl-CO-AMP intermediate. Attack of the peptidyl-CO-AMP carbonyl by the deprotonated C4' hydroxyl of the glucose moiety forms a glycosyl ester linkage with release of AMP. Site-directed mutagenesis of the three cysteines and five histidines in MceI to alanines reveals that these residues are not required structurally or catalytically. MceIJ recognizes all glycosylated enterobactin derivatives formed by the MccE492 gene cluster members MceC ( C-glycosyltransferase) and MceD (esterase) in vitro and a MGE derivative lacking the C6' hydroxyl moiety. The protein complex also accepts and modifies the C-terminal decapeptide substrate fragments of the structurally related microcins H47, I47, and M. MccE492 C-terminal decapeptides bearing fluorescein and biotin moieties on the N-terminus are also substrates for MceIJ, which provides a route for the chemoenzymatic synthesis of enterobactin conjugates with peptide linkages.

Structural basis for the selectivity of the external thioesterase of the surfactin synthetase

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are found in bacteria, fungi, and plants, in the main, producing antibiotics. They are macromolecular machines that rely on the activity of thioesterases to produce biologically active small molecules. They are of particular interest as an assembly system that might be adapted for the production of novel bioactive compounds with possible therapeutic activity. Frueh et al. have solved the structure of a carrier protein — part of the EntF NRPS subunit of enterobactin synthetase — bound to a type I thioesterase. (Type I thioesterases catalyse the final 'release' step of the small molecule from the NRPS or PKS machinery.) The structure reveals that part of the thioesterase can flip open to reveal the carrier-protein binding site of the enzyme; this movement allows the tether of the carrier protein to access the active site. Koglin et al. determined the structure of conformational sub-states of a thioesterase II enzyme. Type II thioesterases are required to regenerate a functional 4'-phosphopantetheine cofactor when it gets mis-primed by reacting with acetyl- and short chain acyl-residues. Comparison with the structures of type I thioesterases reveals the basis for substrate selectivity and the different modes of interaction of the two types of thioesterases with thiolation domains. The three-dimensional structure of a type II thioesterase, free and in complex with a thiolation domain, is determined. Comparison with type I thioesterases reveal the basis for substrate selectivity and the different modes of interaction of the two types of thioesterases with thiolation domains. Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds1,2. Type I thioesterases (TEI) catalyse the release step from the assembly line3 of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4′-phosphopantetheine (4′-PP) cofactor that is covalently attached to thiolation (T) domains4,5,6,7,8,9. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo-T domains of NRPS and PKS systems10,11,12. Mispriming of 4′-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4′-PP cofactor, is acetylated in bacteria13. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes3,14 shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.