Control of mRNA translation by dynamic ribosome modification.

Control of mRNA translation is a crucial regulatory mechanism used by bacteria to respond to their environment. In the soil bacterium Pseudomonas fluorescens, RimK modifies the C-terminus of ribosomal protein RpsF to influence important aspects of rhizosphere colonisation through proteome remodelling. In this study, we show that RimK activity is itself under complex, multifactorial control by the co-transcribed phosphodiesterase trigger enzyme (RimA) and a polyglutamate-specific protease (RimB). Furthermore, biochemical experimentation and mathematical modelling reveal a role for the nucleotide second messenger cyclic-di-GMP in coordinating these activities. Active ribosome regulation by RimK occurs by two main routes: indirectly, through changes in the abundance of the global translational regulator Hfq and directly, with translation of surface attachment factors, amino acid transporters and key secreted molecules linked specifically to RpsF modification. Our findings show that post-translational ribosomal modification functions as a rapid-response mechanism that tunes global gene translation in response to environmental signals.

Biochemical characterization of the Nocardia lactamdurans ACV synthetase.

The L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS) is a nonribosomal peptide synthetase (NRPS) that fulfills a crucial role in the synthesis of ß-lactams. Although some of the enzymological aspects of this enzyme have been elucidated, its large size, at over 400 kDa, has hampered heterologous expression and stable purification attempts. Here we have successfully overexpressed the Nocardia lactamdurans ACVS in E. coli HM0079. The protein was purified to homogeneity and characterized for tripeptide formation with a focus on the substrate specificity of the three modules. The first L-α-aminoadipic acid-activating module is highly specific, whereas the modules for L-cysteine and L-valine are more promiscuous. Engineering of the first module of ACVS confirmed the strict specificity observed towards its substrate, which can be understood in terms of the non-canonical peptide bond position.

Probing the limits of interrupted adenylation domains by engineering a trifunctional enzyme capable of adenylation, N-, and S-methylation.

The adenylation (A) domains found in nonribosomal peptide synthetases (NRPSs) exhibit tremendous plasticity. Some A domains have been shown to display the ability to contain within them the catalytic portion of an auxiliary domain, most commonly that of a methyltransferase (M) enzyme. This unique feature of A domains interrupted by M domains allows them to possess bifunctionality, where they can both adenylate and methylate an amino acid substrate. Additionally, these types of inserted M domains are able to selectively carry out either backbone or side chain methylation of amino acids. Interruptions with M domains are naturally found to occur either between the a2-a3 or the a8-a9 of the ten conserved motifs of A domains. Herein, we set out to answer the following question: Can one A domain support two different M domain interruptions occurring in two different locations (a2-a3 and a8-a9) of the A domain and possess the ability to adenylate an amino acid and methylate it on both its side chain and backbone? To answer this question we added a backbone methylating M3S domain from TioS(A3aM3SA3b) between the a8-a9 region of a mono-interrupted A domain, TioN(AaMNAb), that already contained a side chain methylating MN domain between its a2-a3 region. We evaluated the di-interrupted A domain TioN(AMNAM3SA) with a series of radiometric and mass spectrometry assays and found that this engineered enzyme was indeed capable of all three activities. These findings show that production of an active trifunctional di-interrupted A domain is possible and represents an exciting new avenue for future nonribosomal peptide (NRP) derivatization.

Stuffed Methyltransferase Catalyzes the Penultimate Step of Pyochelin Biosynthesis.

Nonribosomal peptide synthetases use tailoring domains to incorporate chemical diversity into the final natural product. A structurally unique set of tailoring domains are found to be stuffed within adenylation domains and have only recently begun to be characterized. PchF is the NRPS termination module in pyochelin biosynthesis and includes a stuffed methyltransferase domain responsible for S-adenosylmethionine (AdoMet)-dependent N-methylation. Recent studies of stuffed methyltransferase domains propose a model in which methylation occurs on amino acids after adenylation and thiolation rather than after condensation to the nascent peptide chain. Herein, we characterize the adenylation and stuffed methyltransferase didomain of PchF through the synthesis and use of substrate analogues, steady-state kinetics, and onium chalcogen effects. We provide evidence that methylation occurs through an SN2 reaction after thiolation, condensation, cyclization, and reduction of the module substrate cysteine and is the penultimate step in pyochelin biosynthesis.

PKS-NRPS Enzymology and Structural Biology: Considerations in Protein Production.

The structural diversity and complexity of marine natural products have made them a rich and productive source of new bioactive molecules for drug development. The identification of these new compounds has led to extensive study of the protein constituents of the biosynthetic pathways from the producing microbes. Essential processes in the dissection of biosynthesis have been the elucidation of catalytic functions and the determination of 3D structures for enzymes of the polyketide synthases and nonribosomal peptide synthetases that carry out individual reactions. The size and complexity of these proteins present numerous difficulties in the process of going from gene to structure. Here, we review the problems that may be encountered at the various steps of this process and discuss some of the solutions devised in our and other labs for the cloning, production, purification, and structure solution of complex proteins using Escherichia coli as a heterologous host.

Characterization and validation of Entamoeba histolytica pantothenate kinase as a novel anti-amebic drug target.

The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells.

Preliminary Results on the Evaluation of the Occurrence of Tetrodotoxin Associated to Marine Vibrio spp. in Bivalves from the Galician Rias (Northwest of Spain).

Tetrodotoxins (TTX) are a potent group of natural neurotoxins putatively produced by symbiotic microorganisms and affecting the aquatic environment. These neurotoxins have been recently found in some species of bivalves and gastropods along the European Coasts (Greece, UK, and The Netherlands) linked to the presence of high concentrations of Vibrio, in particular Vibrio parahaemolyticus. This study is focused on the evaluation of the presence of Vibrio species and TTX in bivalves (mussels, oysters, cockles, clams, scallops, and razor clams) from Galician Rias (northwest of Spain). The detection and isolation of the major Vibrio spp. and other enterobacterial populations have been carried out with the aim of screening for the presence of the pathways genes, poliketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) possibly involved in the biosynthesis of these toxins. Samples containing Vibrio spp. were analyzed by biochemical (API20E-galery) and genetic tests (PCR-RT). These samples were then screened for TTX toxicity by a neuroblastoma cell-based assay (N2a) and the presence of TTX was further confirmed by LC-MS/MS. TTX was detected in two infaunal samples. This is the first confirmation of the presence of TTX in bivalve molluscs from the Galician Rias.

Expanding tryptophan-containing cyclodipeptide synthase spectrum by identification of nine members from Streptomyces strains.

Cyclodipeptide synthases (CDPSs) comprise normally 200-300 amino acid residues and are mainly found in bacteria. They hijack aminoacyl-tRNAs from the ribosomal machinery for cyclodipeptide formation. In this study, nine new CDPS genes from eight Streptomyces strains were cloned into pET28a vector and expressed in Escherichia coli. Structural elucidation of the isolated products led to the identification of one cyclo-L-Trp-L-Leu, two cyclo-L-Trp-L-Pro, and three cyclo-L-Trp-L-Trp synthases. Other three CDPSs produce cyclo-L-Trp-L-Ala or cyclo-L-Trp-L-Tyr as the major cyclodipeptide. Total product yields of 46 to 211 mg/L E. coli culture were obtained. Our findings represent rare examples of CDPS family derived from actinobacteria that form various tryptophan-containing cyclodipeptides. Furthermore, this study highlights the potential of the microbial machinery for tryptophan-containing cyclodipeptide biosynthesis and provides valid experimental basis for further combination of these CDPS genes with other modification genes in synthetic biology.

Estimation of structure and stability of MurE ligase from Salmonella enterica serovar Typhi.

MurE ligase catalyzes the assembly of peptide moiety, an essential component of bacterial cell wall. We have explored the conformational stability and unfolding equilibrium behaviour of the protein MurE ligase by determining the conformational free energy, entropy and enthalpy parameters under stress conditions. MurE from Salmonella enterica Serovar Typhi was cloned, expressed and purified. Conformational changes associated with increasing concentration of GdmCl- and urea-induced denaturation of MurE were monitored using Circular Dichroism (CD) and fluorescence spectroscopies. The secondary structural content of protein estimated by CD experiment is in close agreement with the predicted MurE ligase structure by homology modeling. Denaturant-induced transition curve was analyzed for thermodynamic parameters. Average values for MurE ligase of ΔGD0 = 3.13 kcal mol-1, m = 1.52 kcal mol-1 M-1 and Cm (=ΔGD0/m) = 2.05 M were calculated in the presence of GdmCl whereas in the case of urea these were ΔGD0 = 3.04 kcal mol-1, m = 1.20 kcal mol-1 M-1 and Cm (=ΔGD0/m) = 2.53 M. The observed superposition of normalized transition curve of two independent optical properties suggested that GdmCl- and urea-induced denaturation follow a two-state process.

Ralstonins A and B, Lipopeptides with Chlamydospore-Inducing and Phytotoxic Activities from the Plant Pathogen Ralstonia solanacearum.

Ralstonia solanacearum has an orphan hybrid polyketide synthase-nonribosomal peptide synthetase gene cluster. We herein isolate its products (named ralstonins A and B) from R. solanacearum and elucidate their structures and biological activities. Ralstonins are unusual lipodepsipeptides composed of 11 amino acids (containing unique amino acids such as ß-hydroxytyrosine and dehydroalanine) and a 3-amino-2-hydroxyoctadecanoic acid, and their production is controlled by quorum sensing, a mechanism of bacterial cell-cell communication. Ralstonins exhibited chlamydospore-inducing activity and phytotoxicity.

Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens.

Covering: up to 2017.Natural products are important secondary metabolites produced by bacterial and fungal species that play important roles in cellular growth and signaling, nutrient acquisition, intra- and interspecies communication, and virulence. A subset of natural products is produced by nonribosomal peptide synthetases (NRPSs), a family of large, modular enzymes that function in an assembly line fashion. Because of the pharmaceutical activity of many NRPS products, much effort has gone into the exploration of their biosynthetic pathways and the diverse products they make. Many interesting NRPS pathways have been identified and characterized from both terrestrial and marine bacterial sources. Recently, several NRPS pathways in human commensal bacterial species have been identified that produce molecules with antibiotic activity, suggesting another source of interesting NRPS pathways may be the commensal and pathogenic bacteria that live on the human body. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) have been identified as a significant cause of human bacterial infections that are frequently multidrug resistant. The emerging resistance profile of these organisms has prompted calls from multiple international agencies to identify novel antibacterial targets and develop new approaches to treat infections from ESKAPE pathogens. Each of these species contains several NRPS biosynthetic gene clusters. While some have been well characterized and produce known natural products with important biological roles in microbial physiology, others have yet to be investigated. This review catalogs the NRPS pathways of ESKAPE pathogens. The exploration of novel NRPS products may lead to a better understanding of the chemical communication used by human pathogens and potentially to the discovery of novel therapeutic approaches.

A sensitive single-enzyme assay system using the non-ribosomal peptide synthetase BpsA for measurement of L-glutamine in biological samples.

The ability to rapidly, economically and accurately measure L-glutamine concentrations in biological samples is important for many areas of research, medicine or industry, however there is room for improvement on existing methods. We describe here how the enzyme BpsA, a single-module non-ribosomal peptide synthetase able to convert L-glutamine into the blue pigment indigoidine, can be used to accurately measure L-glutamine in biological samples. Although indigoidine has low solubility in aqueous solutions, meaning direct measurements of indigoidine synthesis do not reliably yield linear standard curves, we demonstrate that resolubilisation of the reaction end-products in DMSO overcomes this issue and that spontaneous reduction to colourless leuco-indigoidine occurs too slowly to interfere with assay accuracy. Our protocol is amenable to a 96-well microtitre format and can be used to measure L-glutamine in common bacterial and mammalian culture media, urine, and deproteinated plasma. We show that active BpsA can be prepared in high yield by expressing it in the apo-form to avoid the toxicity of indigoidine to Escherichia coli host cells, then activating it to the holo-form in cell lysates prior to purification; and that BpsA has a lengthy shelf-life, retaining >95% activity when stored at either -20 °C or 4 °C for 24 weeks.

Genomic and functional features of the biosurfactant producing Bacillus sp. AM13.

Genomic studies provide deeper insights into secondary metabolites produced by diverse bacterial communities, residing in various environmental niches. This study aims to understand the potential of a biosurfactant producing Bacillus sp. AM13, isolated from soil. An integrated approach of genomic and chemical analysis was employed to characterize the antibacterial lipopeptide produced by the strain AM13. Genome analysis revealed that strain AM13 harbors a nonribosomal peptide synthetase (NRPS) cluster; highly similar with known biosynthetic gene clusters from surfactin family: lichenysin (85 %) and surfactin (78 %). These findings were substantiated with supplementary experiments of oil displacement assay and surface tension measurements, confirming the biosurfactant production. Further investigation using LCMS approach exhibited similarity of the biomolecule with biosurfactants of the surfactin family. Our consolidated effort of functional genomics provided chemical as well as genetic leads for understanding the biochemical characteristics of the bioactive compound.

Exploring Peptide Ligase Orthologs in Actinobacteria-Discovery of Pseudopeptide Natural Products, Ketomemicins.

We recently identified a novel peptide ligase (PGM1), an ATP-grasp-ligase, that catalyzes amide bond formation between (S)-2-(3,5-dihydroxy-4-methoxyphenyl)-2-guanidinoacetic acid and ribosomally supplied oligopeptides in pheganomycin biosynthesis. This was the first example of an ATP-grasp-ligase utilizing peptides as nucleophiles. To explore the potential of this type of enzyme, we performed a BLAST search and identified many orthologs. The orthologs of Streptomyces mobaraensis, Salinispora tropica, and Micromonospora sp. were found in similar gene clusters consisting of six genes. To probe the functions of these genes, we heterologously expressed each of the clusters in Streptomyces lividans and detected novel and structurally similar pseudotripeptides in the broth of all transformants. Moreover, a recombinant PGM1 ortholog of Micromonospora sp. was demonstrated to be a novel dipeptide ligase catalyzing amide bond formation between amidino-arginine and dipeptides to yield tripeptides; this is the first report of a peptide ligase utilizing dipeptides as nucleophiles.

Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains.

A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe.

Reconstitution of Fungal Nonribosomal Peptide Synthetases in Yeast and In Vitro.

The emergence of next-generation sequencing has provided new opportunities in the discovery of new nonribosomal peptides (NRPs) and NRP synthethases (NRPSs). However, there remain challenges for the characterization of these megasynthases. While genetic methods in native hosts are critical in elucidation of the function of fungal NRPS, in vitro assays of intact heterologously expressed proteins provide deeper mechanistic insights in NRPS enzymology. Our previous work in the study of NRPS takes advantage of Saccharomyces cerevisiae strain BJ5464-npgA as a robust and versatile platform for characterization of fungal NRPSs. Here we describe the use of yeast recombination strategies in S. cerevisiae for cloning of the NRPS coding sequence in 2µ-based expression vector; the use of affinity chromatography for purification of NRPS from the total S. cerevisiae soluble protein fraction; and strategies for reconstitution of NRPSs activities in vitro.

Characterization of cereulide synthetase, a toxin-producing macromolecular machine.

Cereulide synthetase is a two-protein nonribosomal peptide synthetase system that produces a potent emetic toxin in virulent strains of Bacillus cereus. The toxin cereulide is a depsipeptide, as it consists of alternating aminoacyl and hydroxyacyl residues. The hydroxyacyl residues are derived from keto acid substrates, which cereulide synthetase selects and stereospecifically reduces with imbedded ketoreductase domains before incorporating them into the growing depsipeptide chain. We present an in vitro biochemical characterization of cereulide synthetase. We investigate the kinetics and side chain specificity of α-keto acid selection, evaluate the requirement of an MbtH-like protein for adenylation domain activity, assay the effectiveness of vinylsulfonamide inhibitors on ester-adding modules, perform NADPH turnover experiments and evaluate in vitro depsipeptide biosynthesis. This work also provides biochemical insight into depsipeptide-synthesizing nonribosomal peptide synthetases responsible for other bioactive molecules such as valinomycin, antimycin and kutzneride.

Differences in the purification and solution properties of PurC gene products from Streptococcus pneumoniae and Bacillus anthracis.

4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide synthetase (PurC) is a key enzyme in the de novo purine biosynthetic pathway of bacteria and an ideal target pathway for the discovery of antimicrobials. Bacillus anthracis (Ba) and Streptococcus pneumoniae (Sp) are two of the bacteria shown to be severe detriments to public health. To be able to carry out the experimentation that leads to drug discovery, high yields of pure soluble recombinant protein must first be obtained. We studied two recombinant PurC proteins from B. anthracis and S. pneumoniae, using Escherichia coli as the host cells. These two proteins, with very similar amino acid sequences, exhibit very different solution properties, leading to a large difference in yields during protein purification under the same conditions. The yield for SpPurC (>50mG per gram of cells) is ten times greater than that for BaPurC (<5mG per gram of cells). The BaPurC samples in solution consisted of oligomers and dimers, with dimers as its functional form. Comparing the yields of dimers, SpPurC is 25 times greater than that for BaPurC (∼2mG per gram of cell). Our studies suggest that the difference in exposed hydrophobic surface area is responsible for the difference in yields under the same conditions.

Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis.

Proteases are ubiquitous in nature, whereas naturally occurring peptide ligases, enzymes catalyzing the reverse reactions of proteases, are rare occurrences. Here we describe the discovery of butelase 1, to our knowledge the first asparagine/aspartate (Asx) peptide ligase to be reported. This highly efficient enzyme was isolated from Clitoria ternatea, a cyclic peptide-producing medicinal plant. Butelase 1 shares 71% sequence identity and the same catalytic triad with legumain proteases but does not hydrolyze the protease substrate of legumain. Instead, butelase 1 cyclizes various peptides of plant and animal origin with yields greater than 95%. With Kcat values of up to 17 s(-1) and catalytic efficiencies as high as 542,000 M(-1) s(-1), butelase 1 is the fastest peptide ligase known. Notably, butelase 1 also displays broad specificity for the N-terminal amino acids of the peptide substrate, thus providing a new tool for C terminus-specific intermolecular peptide ligations.

Crystallization and preliminary X-ray crystallographic analysis of a putative nonribosomal peptide synthase AmbB from Pseudomonas aeruginosa.

AmbB is a putative nonribosomal peptide synthase from Pseudomonas aeruginosa, which is involved in the production of IQS, a potent cell-cell communication signal molecule that integrates the quorum-sensing mechanism and stress response. It consists of 1249 amino acids and contains an AMP-binding domain, a phosphopantetheine-binding (PB) domain and a condensation (C) domain. In this report, a truncated form of AmbB that contains the PB domain and the condensation domain was overexpressed with an N-terminal GST tag in Escherichia coli and purified as a monomer using affinity and size-exclusion chromatography. The recombinant AmbBc (comprising residues 727-1249 of full-length AmbB) was crystallized using the hanging-drop vapour-diffusion method and a full data set was collected to 2.45 Šresolution using a synchrotron-radiation source. The crystals belonged to space group P6122 or P6522, with unit-cell parameters a = b = 87.81, c = 286.8 Å, α = 90, ß = 90, γ = 120°, and contained one molecule per asymmetric unit.