Mechanistic analysis of nonribosomal peptide synthetases

Considering the ongoing rise of the multidrug-resistant bacterial infections, it is essential to expand the available repertoire of therapeutic agents. Microbial natural products are an indispensable source of novel activities and continue to serve as our main provider of antibiotics and chemotherapeutics. Nonribosomal peptides are among the most widespread natural products in bacteria and fungi. Their importance is best illustrated by their complexity and the amounts of resources dedicated to building the underlying biosynthetic machineries nonribosomal peptide synthetases (NRPS). These gigantic, multidomain enzymes synthesize peptides by linking individual amino acid units in an assembly line fashion. Six decades of NRPS research have resulted in several remarkable tailoring successes. However, the lack of mechanistic understanding of the inner workings of NRPSs has prevented the development of a general workflow which would reliably generate functional enzymes and new drugs. Aspiring to alleviate these obstacles, this thesis offers critical insights into adenylation and the interplay with condensation, two fundamental NRPS reactions

An investigation into the biosynthesis of proximicins

PhD ThesisThe proximicins are a family of three compounds – A-C – produced by two marine Actinomycete Verrucosispora strains – V. maris AB18-032 and V. sp. str. 37 - and are characterised by the presence of 2,4-disubstituted furan rings. Proximicins demonstrate cell-arresting and antimicrobial ability, making them interesting leads for clinical drug development. Proximicin research has been largely overshadowed by other Verrucosispora strain secondary metabolites (SM), and despite the publication of the V. maris AB18-032 draft, the enzymatic machinery responsible for their production has not been established. It has been noted in related research into a pyrrole-containing homolog – congocidine –due to the structural similarity exhibited, proximicins likely utilise a similar biosynthetic route. The initial aim of this research was to confirm the presumed pathway to proximicin biosynthesis. Following the sequencing, assembly and annotation of the second proximicin producer, Verrucosispora sp. str. MG37, and genome mining of V. maris AB18-032, no common clusters mimicked that of congocidine, casting doubt on the previously assumed analogous biosynthetic routes. A putative proximicin biosynthesis (ppb) cluster was identified, containing non-ribosomal peptide synthetase (NRPS) enzymes, exhibiting some homology with congocidine. NRPSsystems represent a network of interacting proteins, which act as a SM assembly line: crucially, adenylation (A)- domain enzymes act as the ‘gate-keeper’, determining which precursors are included into the elongating peptide. To elucidate the route to proximicins, activity characterisation of the four A-domains present in ppb cluster was attempted. The A-domain Ppb120 was shown to possess novel activity, demonstrating a high promiscuity towards heterocycle containing precursors, in addition to the absence of an apparent essential domain. This discovery refutes previous work outlining the core residues which dictate A-domain activity, while also presenting a facile route to novel heterocycle-containing compounds. Despite extensive work, A-domains ppb195 and ppb210, were ineffectively purified in the active form – informing future work into A-domains activity characterisation. Finally, the ppb220 A-domain which lies at the border of ppb, was inactive suggesting over-estimation of the cluster margins. To confirm ppb220 redundancy and confirm ppb boundaries, CRISPR/Cas gene editing studies were done. The gene responsible for the orange pigment of Verrucosispora strains was initially targeted and successfully deleted, and ppb studies commenced. The research here refutes the previously presumed route to proximicin biosynthesis; the ppb cluster instead comprises enzymes exhibiting unique activity and structure. The findings represent the foundations for allowing exploitation of chemistry exhibited within the proximicin family. The novelty exhibited can be utilised in the search for antimicrobial clinical leads, by allowing the production of compounds containing previously inaccessible heterocycle chemistry

Pattern recognition methods for the prediction of chemical structures of fungal secondary metabolites

Non-Ribosomal Peptide Synthetases (NRPS) are mega synthetases that are predominantly found in bacteria and fungi. They produce small peptides that serve numerous biological functions and crucial ecological roles. Adenylation (A) domains of NRPSs catalyze ATP dependent activation of substrates harboring carboxy terminus. A-domain substrates include not only natural amino acids (D and L forms) but also non-proteinogenic amino acids. As the substrate repertoire is large and specificity rules for fungi are not established well, there is a difficulty in predicting substrates for fungal A-domains. In bacteria, ten amino acid residues were established as NRPS code, which determine specificity of A-domains. To study relationships between fungal A-domains and their specificity, the cluster analysis of NRPS code residues was done. NRPS code residues were encoded by physicochemical properties essential for binding small molecules and these residues were clustered. Cluster analysis showed similar NRPS codes for α-amino adipic acid, and tryptophan, etc. between bacteria and fungi. Fungal NRPS codes for substrates such as tyrosine, and proline, did not cluster together with bacteria, which indicates an independent evolution of substrate specificity in fungi. This emphasizes the need for the development of a fungus-specific prediction tool. Currently available A-domain substrate specificity prediction tools accurately identify substrates for bacteria but fail to provide correct predictions for fungi. A novel approach for fungal A-domain substrate specificity prediction is presented here. Neural Network based A-domain substrate specificity classifier (NNassc) was developed using Keras with TensorFlow backend. NNassc was trained solely using fungal NRPS codes and combines physicochemical and structural features for specificity predictions. Internal and external validation datasets of experimentally verified NRPS codes were used to assess the performance of NNassc

Identification of Sare0718 As an Alanine-Activating Adenylation Domain in Marine Actinomycete Salinispora arenicola CNS-205

BACKGROUND: Amino acid adenylation domains (A domains) are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs represent a large group of valuable natural products that are widely applied in medicine, agriculture, and biochemical research. Salinispora arenicola CNS-205 is a representative strain of the first discovered obligate marine actinomycete genus, whose genome harbors a large number of cryptic secondary metabolite gene clusters. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate cryptic NRP-related metabolites in S. arenicola CNS-205, we cloned and identified the putative gene sare0718 annotated "amino acid adenylation domain". Firstly, the general features and possible functions of sare0718 were predicted by bioinformatics analysis, which suggested that Sare0718 is a soluble protein with an AMP-binding domain contained in the sequence and its cognate substrate is L-Val. Then, a GST-tagged fusion protein was expressed and purified to further explore the exact adenylation activity of Sare0718 in vitro. By a newly mentioned nonradioactive malachite green colorimetric assay, we found that L-Ala but not L-Val is the actual activated amino acid substrate and the basic kinetic parameters of Sare0718 for it are K(m) = 0.1164±0.0159 (mM), V(max) = 3.1484±0.1278 (µM/min), k(cat) = 12.5936±0.5112 (min(-1)). CONCLUSIONS/SIGNIFICANCE: By revealing the biochemical role of sare0718 gene, we identified an alanine-activating adenylation domain in marine actinomycete Salinispora arenicola CNS-205, which would provide useful information for next isolation and function elucidation of the whole cryptic nonribosomal peptide synthetase (NRPS)-related gene cluster covering Sare0718. And meanwhile, this work also enriched the biochemical data of A domain substrate specificity in newly discovered marine actinomycete NRPS system, which bioinformatics prediction will largely depend on

Structural characterization of Arabidopsis thaliana ethylene signaling molecules and the non-ribosomal peptide synthetase from Planktothrix agardhii

Plants employ a complex network of signaling pathways to regulate developmental processes and to mediate the responses to both environmental and biological stress factors. Ethylene is one of the key plant hormones involved in controlling this network, which has made it and its signaling pathway a target of intense research for several decades. In the model plant Arabidopsis thaliana, the plant hormone is detected by a group of five receptors (ETR1, ERS1, ETR2, ERS2 and EIN4) that resemble the sensor histidine kinases of bacterial two-component system. The main aim in this thesis study was the expression and purification of the full-length ETR1 for structural studies to gain insights into the initial steps in ethylene signaling. The FL ETR1 was successfully expressed in baculovirus expression vector system but the isolation of the receptor from the membrane was hampered. In addition to the FL ETR1, the cytosolic portion of the receptor was studied using Small Angle X-ray Scattering. The resulting SAXS model had the expected dimeric arrangement. EDR1 from A. thaliana is a CTR1-like MAPKKK that is involved in regulating disease resistance responses, cell death and also ethylene-induced senescence. It possesses an N-terminal regulatory domain and C-terminal catalytic domain wit Ser/Thr kinase activity. As EDR1 has been shown to autophosphorylate in trans, the mechanism of this was studied using X-ray crystallography. A crystal structure for the catalytically inactive kinase domain of EDR1 (EDR1-D792N) was obtained in the presence of the ATP substrate analog AMP-PNP. The asymmetric unit contained two molecules, one of which surprisingly was in an active-like conformation. Furthermore, the active-like EDR1-D792N molecule was found to form an authentic trans-autophosphorylation complex with the inactive monomer from the adjacent asymmetric unit. In addition to the plant defense signaling proteins, an adenylation (A) domain from cyanobacterial non-ribosomal peptide synthetase (NRPS) was studied. NRPSs are large multidomain enzymes that are found from a number of fungal and bacterial species and catalyze the ribosome-independent assembly of biologically active peptides with diverse composition and function. The A domain plays a central role in the NRPS system as it recognizes and activates the amino acid, which is incorporated into the growing peptide. The A domain ApnA A1 from the Anabaenopeptin synthetase of Planktothrix agardhii is an interesting member of its class as it has an unusual ability to activate two very distinct amino acids (arginine and tyrosine). Structural studies on this enzyme were performed to elucidate its bi-specificity. Based on the solved ApnA A1 structures, two active site residues with a crucial role in the substrate binding were identified. The mutation of these residues led to enzyme variants, which were mono-specific for either tyrosine or arginine, or in some instances were able to activate L-tryptophan. Additionally a number of ApnA A1 mutants were shown to activate unnatural amino acids (4-fluorophenylalanine and 4-azidophenylalanine). A final peptide product with an unnatural amino acid incorporated, could possibly have useful industrial or pharmaceutical applications

NRPSsp: non-ribosomal peptide synthase substrate predictor

ABSTRACT Summary: Non-Ribosomal Peptide Synthetases (NRPSs) are multimodular enzymes which biosynthesize many important peptide compounds produced by bacteria and fungi. Some studies have revealed that an individual domain within the NRPSs shows significant substrate selectivity. The discovery and characterisation of nonribosomal peptides are of great interest for the biotechnological industries. We have applied computational mining methods in order to build a database of NRPSs modules which bind to specific substrates. We have used this database to build an HMM predictor of substrates which bind to a given NRPS. Availability: The database and the predictor are freely available on an easy-to-use website at www.nrpssp.com

Identification of a conserved N-terminal domain in the first module of ACV synthetases

Abstract The l‐δ‐(α‐aminoadipoyl)‐l‐cysteinyl‐d‐valine synthetase (ACVS) is a trimodular nonribosomal peptide synthetase (NRPS) that provides the peptide precursor for the synthesis of β‐lactams. The enzyme has been extensively characterized in terms of tripeptide formation and substrate specificity. The first module is highly specific and is the only NRPS unit known to recruit and activate the substrate l‐α‐aminoadipic acid, which is coupled to the α‐amino group of l‐cysteine through an unusual peptide bond, involving its δ‐carboxyl group. Here we carried out an in‐depth investigation on the architecture of the first module of the ACVS enzymes from the fungus Penicillium rubens and the bacterium Nocardia lactamdurans. Bioinformatic analyses revealed the presence of a previously unidentified domain at the N‐terminus which is structurally related to condensation domains, but smaller in size. Deletion variants of both enzymes were generated to investigate the potential impact on penicillin biosynthesis in vivo and in vitro. The data indicate that the N‐terminal domain is important for catalysis

Study of the complete genome sequence of Streptomyces scabies (or scabiei) 87.22

A study of the complete genome sequence of Streptomyces scabies 87.22, a common causative agent of scab disease of tubers including potato (Solanum tuberosum), is described. This work includes annotation of the genome and in-depth description of gene clusters likely to encode biosynthetic pathways for complex natural products and not also found in either “Streptomyces coelicolor” A3(2) or Streptomyces avermitilis MA-4680. Twenty-eight gene clusters were identified as likely to encode enzymes for the biosynthesis of complex natural products. Substances predicted by this work, not previously known to be made by S. scabies 87.22, were confirmed by collaborators as products - desferrioxamines, germicidins, and hopene. Of the clusters identified, fourteen gene clusters are not conserved in the other two streptomycete genome sequences for which comparisons have been undertaken. The Streptomyces genus is a reservoir of producer organisms from which many complex natural products of therapeutic importance have been isolated. These findings suggest that the cargo of cryptic and silent gene clusters amongst other members of this genus may add significantly to previous estimates of undiscovered bioactive natural products. Methods developed in this work could enable other researchers to rapidly identify gene clusters likely to encode enzymes involved in biosynthesis of complex natural products from complete genome sequences. De-replication is a problem for approaches to drug discovery based on activity screening and isolation of wild producer organisms. Computational methods in this work allow rapid de-replication of gene clusters following sequencing which may lead to discovery of many new natural products with therapeutic benefit. Sequences predicted to be involved in scab disease pathogenicity are not found in only one ‘pathogenicity island’ location as expected, but at several loci. Two possible mechanisms were identified from sequence data which it is suggested could be involved in regulation of pathogenicity traits: an MbtH-like protein family and an iron box sequence likely to be triggered response to low iron conditions

The surfactin biosynthetic complex of Bacillus subtilis: COM domain-mediated biocombinatorial synthesis, and single step purification of native multi-modular NRPSs and multi-enzyme complexes

Most biosynthetic templates for the assembly of peptide natural products are composed of two or more nonribosomal peptide synthetases (NRPSs). For example, the surfactin biosynthetic complex consists of three NRPSs (SrfA-A, SrfA-B, and SrfA-C), which are encoded by the polycistronic srfA operon within the chromosome of the producer strain Bacillus subtilis. According to the molecular logic employed by NRPS assembly lines, the biosynthesis of a defined product relies on the proper, well-orchestrated interaction between partner-enzymes (i.e. SrfA-A/SrfA-B, and SrfA-B/SrfA-C), and the prevention of futile interactions between non-partner enzymes (i.e. SrfA-A/SrfA-C). Based on most recent in vitro studies, these selective interactions between NRPSs are controlled by the interplay of communication-mediating (COM) domains, located at the C- and N-termini of the corresponding donor and acceptor enzymes. In the first part of this study, the potential of COM domains was exploited for the directed reprogramming of the surfactin biosynthetic complex, and the setting up of an in vivo system for the true biocombinatorial synthesis of lipopeptides. To this end, the first COM domain pair, facilitating the selective interaction between SrfA-A and SrfA-B, was substituted against various cognate, mis-cognate and non-cognate COM domain pairs. The consequences of these manipulations were then analyzed by means of HPLC and high-resolution MS. These experiments verified that COM domain pairs of the tyrocidine biosynthetic complex retain their functionality and selectivity even in the context of a heterologous host and NRPS system. Furthermore, utilization of a designated non-cognate COM domain pair allowed for an intended skipping of the second NRPS SrfA-B, the enforcement of a productive interaction between the natural non-partner enzymes SrfA-A and SrfA-C, and thus the directed synthesis of a shortened lipotetrapeptide product. In another experiment, all donor and acceptor enzymes of the biosynthetic complex were equipped with the same set of cognate COM domain pair. The resulting abrogation of the selectivity-barrier led to the establishment of an so-called universal communication system, and afforded the envisioned biocombinatorial synthesis of two lipopeptide products. All these experiments verified – for the first time in vivo, and within the context of a natural NRP assembly line – the decisive role of COM domains for the control of protein-protein communication between NRPSs. The second objective of this work was the establishment of a gentle method for the purification of NRPSs and multi-enzymatic NRPS complexes. The approach taken was based on the utilization of polyol-responsive monoclonal antibodies (PR-mAb), which are able to release their bound antigen under gentle, non-denaturating conditions, in the presence of polyols. PR-mAbs were originally developed and used for the purification of the E. coli RNA polymerase holo-enzyme complex, including low-affinity bound -factors. Among others, these studies led to the identification of a antigen/antibody pair epitope/NT73. Within the scope of this work, the coding sequence of this epitope tag was fused to the 3’-end of the srfA-A gene within the chromosome of B. subtilis. Subsequently, the encoded SrfA-A-epi protein could be purified from cleared crude extracts of the resulting mutant using immunoaffininty chromatography. SDS-PAGE and MS/MS analyses, as well as biochemical characterizations unequivocally verified the purification of the epitop-tagged SrfA-A protein in active holo-form, as well as co-purification of SrfA-B (molecular weight of the dimeric complex: approx. 803 kDa). Under the conditions tested, the third NRPS, SrfA-C, as well as additional proteins, associated with the surfactin complex, could not be detected

Phylogenomics reveals subfamilies of fungal nonribosomal peptide synthetases and their evolutionary relationships

<p><b>Abstract</b></p> <p>Background</p> <p>Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes. Although their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. We identified genes (<it>NPS</it>) encoding NRPS and NRPS-like proteins in 38 fungal genomes and undertook phylogenomic analyses in order to identify fungal NRPS subfamilies, assess taxonomic distribution, evaluate levels of conservation across subfamilies, and address mechanisms of evolution of multimodular NRPSs. We also characterized relationships of fungal NRPSs, a representative sampling of bacterial NRPSs, and related adenylating enzymes, including α-aminoadipate reductases (AARs) involved in lysine biosynthesis in fungi.</p> <p>Results</p> <p>Phylogenomic analysis identified nine major subfamilies of fungal NRPSs which fell into two main groups: one corresponds to <it>NPS </it>genes encoding primarily mono/bi-modular enzymes which grouped with bacterial NRPSs and the other includes genes encoding primarily multimodular and exclusively fungal NRPSs. AARs shared a closer phylogenetic relationship to NRPSs than to other acyl-adenylating enzymes. Phylogenetic analyses and taxonomic distribution suggest that several mono/bi-modular subfamilies arose either prior to, or early in, the evolution of fungi, while two multimodular groups appear restricted to and expanded in fungi. The older mono/bi-modular subfamilies show conserved domain architectures suggestive of functional conservation, while multimodular NRPSs, particularly those unique to euascomycetes, show a diversity of architectures and of genetic mechanisms generating this diversity.</p> <p>Conclusions</p> <p>This work is the first to characterize subfamilies of fungal NRPSs. Our analyses suggest that mono/bi-modular NRPSs have more ancient origins and more conserved domain architectures than most multimodular NRPSs. It also demonstrates that the α-aminoadipate reductases involved in lysine biosynthesis in fungi are closely related to mono/bi-modular NRPSs. Several groups of mono/bi-modular NRPS metabolites are predicted to play more pivotal roles in cellular metabolism than products of multimodular NRPSs. In contrast, multimodular subfamilies of NRPSs are of more recent origin, are restricted to fungi, show less stable domain architectures, and biosynthesize metabolites which perform more niche-specific functions than mono/bi-modular NRPS products. The euascomycete-only NRPS subfamily, in particular, shows evidence for extensive gain and loss of domains suggestive of the contribution of domain duplication and loss in responding to niche-specific pressures.</p