Structure of a Eukaryotic Nonribosomal Peptide Synthetase Adenylation Domain That Activates a Large Hydroxamate Amino Acid in Siderophore Biosynthesis

Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N^δ-cis-anhydromevalonyl-N^δ-hydroxy-l-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.     

Genetic manipulation revealing an unusual N-terminal region in a stand-alone non-ribosomal peptide synthetase involved in the biosynthesis of ramoplanins

Ramoplanins produced by Actinoplanes are new structural class of lipopeptide and are currently in phase III clinical trials for the prevention of vancomycin-resistant enterococcal infections. The depsipeptide structures of ramoplanins are synthesized by non-ribosomal peptide synthetases (NRPS). Romo-orf17, a stand-alone NRPS, is responsible for the recruitment of Thr into the linear NRPS pathways for which the corresponding adenylation domain is absent. Here, systematical gene inactivation and complementation have been carried out in a Actinoplanes sp. using homologous recombination and site-specific integration methods. A hybrid gene coding for the N-terminal region of the stand-alone NRPS and the A-PCP domains of a heterologous NRPS restored production of ramoplanins. The results elucidate the unusual N-terminal region which is essential for the biosynthesis of ramoplanins.     

Substrate selection of adenylation domains for nonribosomal peptide synthetase (NRPS) in bacillamide C biosynthesis by marine <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis> C89

Nonribosomal peptide synthetases (NRPSs) are multi-modular enzymes involved in the biosynthesis of natural products. Bacillamide C was synthesized by Bacillus atrophaeus C89. A nonribosomal peptide synthetase (NRPS) cluster found in the genome of B. atrophaeus C89 was hypothesized to be responsible for the biosynthesis of bacillamide C using alanine and cysteine as substrates. Here, the structure analysis of adenylation domains based on homologous proteins with known crystal structures indicated locations of the substrate-binding pockets. Molecular docking suggested alanine and cysteine as the potential substrates for the two adenylation domains in the NRPS cluster. Furthermore, biochemical characterization of the purified recombinant adenylation domains proved that alanine and cysteine were the optimum substrates for the two adenylation domains. The results provided the in vitro evidence for the hypothesis that the two adenylation domains in the NRPS of B. atrophaeus C89 preferentially select alanine and cysteine, respectively, as a substrate to synthesize bacillamide C. Furthermore, this study on substrates selectivity of adenylation domains provided basis for rational design of bacillamide analogs.     

ClustScan: an integrated program package for the semi-automatic annotation of modular biosynthetic gene clusters and in silico prediction of novel chemical structures

The program package ‘ClustScan’ (Cluster Scanner) is designed for rapid, semi-automatic, annotation of DNA sequences encoding modular biosynthetic enzymes including polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS) and hybrid (PKS/NRPS) enzymes. The program displays the predicted chemical structures of products as well as allowing export of the structures in a standard format for analyses with other programs. Recent advances in understanding of enzyme function are incorporated to make knowledge-based predictions about the stereochemistry of products. The program structure allows easy incorporation of additional knowledge about domain specificities and function. The results of analyses are presented to the user in a graphical interface, which also allows easy editing of the predictions to incorporate user experience. The versatility of this program package has been demonstrated by annotating biochemical pathways in microbial, invertebrate animal and metagenomic datasets. The speed and convenience of the package allows the annotation of all PKS and NRPS clusters in a complete Actinobacteria genome in 2–3 man hours. The open architecture of ClustScan allows easy integration with other programs, facilitating further analyses of results, which is useful for a broad range of researchers in the chemical and biological sciences.     

Substrate Specificity-Conferring Regions of the Nonribosomal Peptide Synthetase Adenylation Domains Involved in Albicidin Pathotoxin Biosynthesis Are Highly Conserved within the Species Xanthomonas albilineans

Albicidin is a pathotoxin produced by Xanthomonas albilineans, a xylem-invading pathogen that causes leaf scald disease of sugarcane. Albicidin is synthesized by a nonribosomal pathway via modular polyketide synthase and nonribosomal peptide synthetase (NRPS) megasynthases, and NRPS adenylation (A) domains are responsible for the recognition and activation of specific amino acid substrates. DNA fragments (0.5 kb) encoding the regions responsible for the substrate specificities of six albicidin NRPS A domains from 16 strains of X. albilineans representing the known diversity of this pathogen were amplified and sequenced. Polymorphism analysis of these DNA fragments at different levels (DNA, protein, and NRPS signature) showed that these pathogenicity loci were highly conserved. The conservation of these loci most likely reflects purifying selective pressure, as revealed by a comparison with the variability of nucleotide and amino acid sequences of two housekeeping genes (atpD and efp) of X. albilineans. Nevertheless, the 16 strains of X. albilineans were differentiated into several groups by a phylogenetic analysis of the nucleotide sequences corresponding to the NRPS A domains. One of these groups was representative of the genetic diversity previously found within the pathogen by random fragment length polymorphism and amplified fragment length polymorphism analyses. This group, which differed by three single synonymous nucleotide mutations, contained only four strains of X. albilineans that were all involved in outbreaks of sugarcane leaf scald. The amount of albicidin produced in vitro in agar and liquid media varied among the 16 strains of X. albilineans. However, no relationship among the amount of albicidin produced in vitro and the pathotypes and genetic diversity of the pathogen was found. The NRPS loci contributing to the synthesis of the primary structure of albicidin apparently are not involved in the observed pathogenicity differences among strains of X. albilineans.     

The Production of Multiple Small Peptaibol Families by Single 14‐Module Peptide Synthetases in Trichoderma/Hypocrea

The most common sequences of peptaibiotics are 11‐residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14‐residue peptaibols sharing partial sequence identity. Genome sequencing projects of three Trichoderma strains of the major clades reveal the presence of up to three types of nonribosomal peptide synthetases with 7, 14, or 18–20 amino acid‐adding modules. Here, we provide evidence that the 14‐module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina), and T. atroviride produces both 11‐ and 14‐residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis of their amino acid‐activating domains and modules. The sequences of these peptides may be predicted from the gene sequences and have been confirmed by analysis of families of 11‐ and 14‐residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 sequences determined, 2 new), and the recently established trichovirins A from T. virens. The distribution of 11‐ and 14‐residue products is strain‐specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.     

Phylogenetic analysis of type I polyketide synthase and nonribosomal peptide synthetase genes in Antarctic sediment

The modular polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) have been found to be involved in natural product synthesis in many microorganisms. Study on their diversities in natural environment may provide important ecological insights, in addition to opportunities for antibacterial drugs development. In this study, the PKS and NRPS gene diversities in two coast sediments near China Zhongshan Station were studied. The phylogenetic analysis of amino acid (AA) sequences indicated that the identified ketosynthase (KS) domains were clustered with those from diverse bacterial groups, including Proteobacteria, Firmicutes, Planctomycetes, Cyanobacteria, Actinobacteria, and some uncultured symbiotic bacteria. One new branch belonging to hybrid PKS/NRPS enzyme complexes and five independent clades were found on the phylogenetic tree. The obtained adenylation (A) domains were mainly clustered within the Cyanobacteria and Proteobacteria group. Most of the identified KS and A domains showed below 80 and 60% identities at the AA level to their closest matches in GenBank, respectively. The diversities of both KS and A domains in natural environmental sample were different from those in sewage-contaminated sample. These results revealed the great diversity and novelty of both PKS and NRPS genes in Antarctic sediment.     

The sypA,sypS, and sypC synthetase genes encode twenty-two modules involved in the nonribosomal peptide synthesis of syringopeptin by Pseudomonas syringae pv. syringae B301D

Syringopeptin is a necrosis-inducing phytotoxin, composed of 22 amino acids attached to a 3-hydroxy fatty acid tail. Syringopeptin, produced by Pseudomonas syringae pv. syringae, functions as a virulence determinant in the plant-pathogen interaction. A 73,800-bp DNA region was sequenced, and analysis identified three large open reading frames, sypA, sypB, and sypC, that are 16.1, 16.3, and 40.6 kb in size. Sequence analysis of the putative SypA, SypB, and SypC sequences determined that they are homologous to peptide synthetases, containing five, five, and twelve amino acid activation modules, respectively. Each module exhibited characteristic domains for condensation, aminoacyl adenylation, and thiolation. Within the aminoacyl adenylation domain is a region responsible for substrate specificity. Phylogenetic analysis of the substrate-binding pockets resulted in clustering of the 22 syringopeptin modules into nine groups. This clustering reflects the substrate amino acids predicted to be recognized by each of the respective modules based on placement of the syringopeptin NRPS (nonribosomal peptide synthetase) system in the linear (type A) group. Finally, SypC contains two C-terminal thioesterase domains predicted to catalyze the release of syringopeptin from the synthetase and peptide cyclization to form the lactone ring. The syringopeptin synthetases, which carry 22 NRPS modules, represent the largest linear NRPS system described for a prokaryote.     

Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome

Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications.     

Genetic variation of adenylation domains of the anabaenopeptin synthesis operon and evolution of substrate promiscuity

Anabaenopeptins (AP) are bioactive cyclic hexapeptides synthesized nonribosomally in cyanobacteria. APs are characterized by several conserved motifs, including the ureido bond, N-methylation in position 5, and d-Lys in position 2. All other positions of the AP molecule are variable, resulting in numerous structural variants. We have identified a nonribosomal peptide synthetase (NRPS) operon from Planktothrix agardhii strain CYA126/8 consisting of five genes (apnA to apnE) encoding six NRPS modules and have confirmed its role in AP synthesis by the generation of a mutant via insertional inactivation of apnC. In order to correlate the genetic diversity among adenylation domains (A domains) with AP structure variation, we sequenced the A domains of all six NRPS modules from seven Planktothrix strains differing in the production of AP congeners. It is remarkable that single strains coproduce APs bearing either of the chemically divergent amino acids Arg and Tyr in exocyclic position 1. Since the A domain of the initiation module (the ApnA A₁ domain) has been proposed to activate the amino acid incorporated into exocyclic position 1, we decided to analyze this domain both biochemically and phylogenetically. Only ApnA A₁ enzymes from strains producing AP molecules containing Arg or Tyr in position 1 were found to activate these two chemically divergent amino acids in vitro. Phylogenetic analysis of apn A domain sequences revealed that strains with a promiscuous ApnA A₁ domain are derived from an ancestor that activates only Arg. Surprisingly, positive selection appears to affect only three codons within the apnA A₁ gene, suggesting that this remarkable promiscuity has evolved from point mutations only.     

Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product

Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically important antibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid, followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, the knowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of the ever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thus deciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is the substrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtH homolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequenced genome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPS gene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.     

Specificity prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using transductive support vector machines (TSVMs)

We present a new support vector machine (SVM)-based approach to predict the substrate specificity of subtypes of a given protein sequence family. We demonstrate the usefulness of this method on the example of aryl acid-activating and amino acid-activating adenylation domains (A domains) of nonribosomal peptide synthetases (NRPS). The residues of gramicidin synthetase A that are 8 A around the substrate amino acid and corresponding positions of other adenylation domain sequences with 397 known and unknown specificities were extracted and used to encode this physico-chemical fingerprint into normalized real-valued feature vectors based on the physico-chemical properties of the amino acids. The SVM software package SVM(light) was used for training and classification, with transductive SVMs to take advantage of the information inherent in unlabeled data. Specificities for very similar substrates that frequently show cross-specificities were pooled to the so-called composite specificities and predictive models were built for them. The reliability of the models was confirmed in cross-validations and in comparison with a currently used sequence-comparison-based method. When comparing the predictions for 1230 NRPS A domains that are currently detectable in UniProt, the new method was able to give a specificity prediction in an additional 18% of the cases compared with the old method. For 70% of the sequences both methods agreed, for <6% they did not, mainly on low-confidence predictions by the existing method. None of the predictive methods could infer any specificity for 2.4% of the sequences, suggesting completely new types of specificity.     

Bioinformatics and molecular approaches to detect NRPS genes involved in the biosynthesis of kurstakin from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Degenerated primers designed for the detection by polymerase chain reaction of nonribosomal peptide synthetases (NRPS) genes involved in the biosynthesis of lipopeptides were used on genomic DNA from a new isolate of Bacillus thuringiensis CIP 110220. Primers dedicated to surfactin and bacillomycin detection amplified sequences corresponding respectively to the surfactin synthetase operon and to a gene belonging to a new NRPS operon identified in the genome of B. thuringiensis serovar pondicheriensis BSCG 4BA1. A bioinformatics analysis of this operon led to the prediction of an NRPS constituted of seven modules beginning with a condensation starter domain and which could be involved in the biosynthesis of a heptalipopeptide similar to kurstakin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) performed on whole cells of B. thuringiensis CIP 110220 confirmed the production of kurstakin by this strain. The kurstakin operon was thus used to design a new set of degenerated primers specifically to detect kurstakin genes. These primers were used to screen kurstakin producers in a collection of nine B. thuringiensis strains isolated from different areas in Algeria and two from the Pasteur Institute collection. For eight among the 11 tested strains, the amplified fragment matched with an operon similar to the kurstakin operon and found in the newly sequenced genome of Bacillus cereus or B. thuringiensis serovar pulsiensis, kurstaki, and thuringiensis. Kurstakin production was detected by MALDI-ToF-MS on whole cells for six strains. This production was compared with the spreading of the strains and their antimicrobial activity. Only the spreading can be correlated with the kurstakin production.     

Structural aspects of non-ribosomal peptide biosynthesis

Small peptides have powerful biological activities ranging from antibiotic to immune suppression. These peptides are synthesized by non-ribosomal peptide synthetases (NRPS). Structural understanding of NRPS took a huge leap forward in 2002; this information has led to several detailed biochemical studies and further structural studies. NRPS are complex molecular machines composed of multiple modules and each module contains several autonomously folded catalytic domains. Structural studies have largely focused on individual domains, isolated from the context of the multienzyme. Biochemical studies have looked at individual domains, isolated whole modules and intact NRPS, and the combined data begin to allow us to visualize the process of peptide assembly by NRPS.     

Designing and Implementing an Assay for the Detection of Rare and Divergent NRPS and PKS Clones in European,Antarctic and Cuban Soils

The ever increasing microbial resistome means there is an urgent need for new antibiotics. Metagenomics is an underexploited tool in the field of drug discovery. In this study we aimed to produce a new updated assay for the discovery of biosynthetic gene clusters encoding bioactive secondary metabolites. PCR assays targeting the polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) were developed. A range of European soils were tested for their biosynthetic potential using clone libraries developed from metagenomic DNA. Results revealed a surprising number of NRPS and PKS clones with similarity to rare Actinomycetes. Many of the clones tested were phylogenetically divergent suggesting they were fragments from novel NRPS and PKS gene clusters. Soils did not appear to cluster by location but did represent NRPS and PKS clones of diverse taxonomic origin. Fosmid libraries were constructed from Cuban and Antarctic soil samples; 17 fosmids were positive for NRPS domains suggesting a hit rate of less than 1 in 10 genomes. NRPS hits had low similarities to both rare Actinobacteria and Proteobacteria; they also clustered with known antibiotic producers suggesting they may encode for pathways producing novel bioactive compounds. In conclusion we designed an assay capable of detecting divergent NRPS and PKS gene clusters from the rare biosphere; when tested on soil samples results suggest the majority of NRPS and PKS pathways and hence bioactive metabolites are yet to be discovered.     

New PCR Primers for the Screening of NRPS and PKS-I Systems in Actinomycetes: Detection and Distribution of These Biosynthetic Gene Sequences in Major Taxonomic Groups

Nonribosomal peptide synthetases (NRPS) and type I polyketide synthases (PKS-I) are biosynthetic systems involved in the synthesis of a large number of important biologically active compounds produced by microorganisms, among others by actinomycetes. In order to assess the occurrence of these biosynthetic systems in this metabolically active bacterial group, we designed new PCR primers targeted to specifically amplify NRPS and PKS-I gene sequences from actinomycetes. The sequence analysis of amplified products cloned from two model systems and used to validate these molecular tools has shown the extreme richness of NRPS or PKS-I-like sequences in the actinomycete genome. When these PCR primers were tested on a large collection of 210 reference strains encompassing all major families and genera in actinomycetes, we observed that the wide distribution of these genes in the well-known productive Streptomyces species is also extended to other minor lineages where in some cases very few bioactive compounds have been identified to date.     

Analysis of the linker region joining the adenylation and carrier protein domains of the modular nonribosomal peptide synthetases

Nonribosomal peptide synthetases (NRPSs) are multimodular proteins capable of producing important peptide natural products. Using an assembly line process, the amino acid substrate and peptide intermediates are passed between the active sites of different catalytic domains of the NRPS while bound covalently to a peptidyl carrier protein (PCP) domain. Examination of the linker sequences that join the NRPS adenylation and PCP domains identified several conserved proline residues that are not found in standalone adenylation domains. We examined the roles of these proline residues and neighboring conserved sequences through mutagenesis and biochemical analysis of the reaction catalyzed by the adenylation domain and the fully reconstituted NRPS pathway. In particular, we identified a conserved LPxP motif at the start of the adenylation‐PCP linker. The LPxP motif interacts with a region on the adenylation domain to stabilize a critical catalytic lysine residue belonging to the A10 motif that immediately precedes the linker. Further, this interaction with the C‐terminal subdomain of the adenylation domain may coordinate movement of the PCP with the conformational change of the adenylation domain. Through this work, we extend the conserved A10 motif of the adenylation domain and identify residues that enable proper adenylation domain function. Proteins 2014; 82:2691–2702. © 2014 Wiley Periodicals, Inc.     

Evolution of Chemical Diversity in Echinocandin Lipopeptide Antifungal Metabolites

The echinocandins are a class of antifungal drugs that includes caspofungin, micafungin, and anidulafungin. Gene clusters encoding most of the structural complexity of the echinocandins provided a framework for hypotheses about the evolutionary history and chemical logic of echinocandin biosynthesis. Gene orthologs among echinocandin-producing fungi were identified. Pathway genes, including the nonribosomal peptide synthetases (NRPSs), were analyzed phylogenetically to address the hypothesis that these pathways represent descent from a common ancestor. The clusters share cooperative gene contents and linkages among the different strains. Individual pathway genes analyzed in the context of similar genes formed unique echinocandin-exclusive phylogenetic lineages. The echinocandin NRPSs, along with the NRPS from the inp gene cluster in Aspergillus nidulans and its orthologs, comprise a novel lineage among fungal NRPSs. NRPS adenylation domains from different species exhibited a one-to-one correspondence between modules and amino acid specificity that is consistent with models of tandem duplication and subfunctionalization. Pathway gene trees and Ascomycota phylogenies are congruent and consistent with the hypothesis that the echinocandin gene clusters have a common origin. The disjunct Eurotiomycete-Leotiomycete distribution appears to be consistent with a scenario of vertical descent accompanied by incomplete lineage sorting and loss of the clusters from most lineages of the Ascomycota. We present evidence for a single evolutionary origin of the echinocandin family of gene clusters and a progression of structural diversification in two fungal classes that diverged approximately 290 to 390 million years ago. Lineage-specific gene cluster evolution driven by selection of new chemotypes contributed to diversification of the molecular functionalities.     

PCR detection of novel non-ribosomal peptide synthetase genes in lipopeptide-producing Pseudomonas

Bacterial lipopeptides (LPs) are a diverse group of secondary metabolites synthesized through one or more non-ribosomal peptide synthetases (NRPSs). In certain genera, such as Pseudomonas and Bacillus, these enzyme systems are often involved in synthesizing biosurfactants or antimicrobial compounds. Several different types of LPs have been reported for non-pathogenic plant-associated Pseudomonas. Focusing on this group of bacteria, we devised and validated a PCR method to detect novel LP-synthesizing NRPS genes by targeting their lipoinitiation and tandem thioesterase domains, thus avoiding amplification of genes for non-LP metabolites, such as the pyoverdine siderophores present in all fluorescent Pseudomonas. This approach enabled detection of as yet unknown NRPS genes in strains producing viscosin, viscosinamide, WLIP, or lokisin. Furthermore, it proved valuable to identify novel candidate LP producers among Pseudomonas rhizosphere isolates. By phylogenetic analysis of these amplicons, several of the corresponding NRPS genes can be tentatively assigned to the viscosin, amphisin, or entolysin biosynthetic groups, while some others may represent novel NRPS systems.