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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. [ABSTRACT FROM AUTHOR]

Non-ribosomal peptide synthetases are mega-enzyme assembly lines that synthesize many clinically useful compounds. As a gatekeeper, they have an adenylation (A)-domain that controls substrate specificity and plays an important role in product structural diversity. This review summarizes the natural distribution, catalytic mechanism, substrate prediction methods, and in vitro biochemical analysis of the A-domain. Taking genome mining of polyamino acid synthetases as an example, we introduce research on mining non-ribosomal peptides based on A-domains. We discuss how non-ribosomal peptide synthetases can be engineered based on the A-domain to obtain novel non-ribosomal peptides. This work provides guidance for screening non-ribosomal peptide-producing strains, offers a method to discover and identify A-domain functions, and will accelerate the engineering and genome mining of non-ribosomal peptide synthetases. Key points: • Introducing adenylation domain structure, substrate prediction, and biochemical analysis methods • Advances in mining homo polyamino acids based on adenylation domain analysis • Creating new non-ribosomal peptides by engineering adenylation domains [ABSTRACT FROM AUTHOR]

Dithiolopyrrolone antibiotics are well known for their outstanding biological activities, and their biosynthesis has been studied vigorously. However, the biosynthesis mechanism of the characteristic bicyclic scaffold is still unknown after years of research. To uncover this mechanism, a multi-domain non-ribosomal peptide synthase DtpB from the biosynthetic gene cluster of thiolutin was selected as an object to study. We discovered that its adenylation domain not only recognized and adenylated cysteine, but also played an essential role in the formation of the peptide bond. Notably, an eight-membered ring compound was also discovered as an intermediate during the formation of the bicyclic structure. Based on these findings, we propose a new mechanism for the biosynthesis of the bicyclic scaffold of dithiolopyrrolones, and unveil additional functions of the adenylation domain. [ABSTRACT FROM AUTHOR]

Background: Brevilaterin A-E, a novel class of multi-component cationic antimicrobial lipopeptides, were biosynthesized by a non-ribosomal peptides synthetase (NRPS) in Brevibacillus laterosporus. However, the antimicrobial abilities of different brevilaterin components varied greatly, and this multi-component form was impeding the scale production of the excellent component, and a little information about the brevilaterin biosynthesis mechanism was available to apply in brevilaterin design modification. In this study, we used an accurate strategy that revealed the reason for producing multi-component was the substrate selectivity of bre2691A protein being not enough specific and pinpointed the key design sites to make the specificity of bre2691A enhanced. Results: Bioinformatic analysis revealed that the biocatalytic site of bre2691A, which was an adenylation domain catalyzed and recognized methionine, leucine, valine and isoleucine and thus introduced them into brevilaterins and caused different components (brevilaterin A-E), was consisted of A1 ~ A10 residues named specificity-conferring code. Coupling molecular docking simulations with mutation studies identified A2 and A7 as critical residues, where determined substrate-specificity and impacted activity. The in virto activity assay showed that the A2 mutant (G193A) would lose activity against methionine and have no effect on the other three amino acids, the A7 mutant (G285C) would enhance the catalytic activity against four substrates, especially against leucine at almost a double activity. When the A2 and A7 residues were synchronously mutated, this mutant would be more focused on recognizing leucine. Conclusions: An accurate strategy that combined with bioinformatics and site-directed mutation techniques revealed the pivotal site A2 and A7 positions of bre2691A protein that could be used to design and modify brevilaterins, thus further providing a reasonable direction of genetic engineering for Brevibacillus laterosporus. A deeper understanding of the function of crucial residues in the adenylation domain would make it get more accurate and highly efficient design and more fully utilized. Furthermore, it would contribute to biotechnological applications, namely for the large centralized synthesis of antimicrobial peptides, or for the optimization of their production. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an increasingly antibiotic-resistant pathogen that causes severe lung infections, burn wound infections, and diabetic foot infections. P. aeruginosa produces the siderophore pyochelin through the use of a non-ribosomal peptide synthetase (NRPS) biosynthetic pathway. Targeting members of siderophore NRPS proteins is one avenue currently under investigation for the development of new antibiotics against antibiotic-resistant organisms. Here, the crystal structure of the pyochelin adenylation domain PchD is reported. The structure was solved to 2.11 Å when co-crystallized with the adenylation inhibitor 5′-O-(N-salicylsulfamoyl)adenosine (salicyl-AMS) and to 1.69 Å with a modified version of salicyl-AMS designed to target an active site cysteine (4-cyano-salicyl-AMS). In the structures, PchD adopts the adenylation conformation, similar to that reported for AB3403 from Acinetobacter baumannii. [ABSTRACT FROM AUTHOR]

Nonribosomal peptide synthetases (NRPS) are multi-domain enzymes that have innumerably beneficial health applications. Realizing the significance of marine microorganisms in search for NRPS sequences, study was conducted for analysis of NRPS gene sequences of marine crab haemolymph bacteria for the first time. Strains belonging to five different species were found to have NRPS genes. The study generated NRPS sequences from four bacterial species, for which NRPS gene information was not available earlier. Two new putative adenylation domain signatures were identified from phylum Firmicutes. In silico analysis of amino acid sequences from four species showed less identity (42–50%) to the characterized NRPS compounds that integrate serine residue in active site, suggesting the novelty or uncharacterized nature. Altogether, the study warrants future research exploiting marine crab haemolymph bacteria, an unexplored niche of microbial genetic wealth to discover microbial novel NRPS genes and natural products using emerging tools and technologies. [ABSTRACT FROM AUTHOR]

The cyanotoxin microcystin (MC) is a secondary metabolite, synthesized by nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymes. It has many isoforms and the mechanism of its diversity is not well understood. One of the MC synthetase genes, mcyA, codes for the McyA module containing two adenylation (A) domains. The first domain, McyA-A1, generally binds to L-serine (L-ser). Then the N-methyl transferase (NMT) domain converts L-Ser into N-methyldehydroalanine (Mdha), which usually occupies position 7 on the MC molecule. However, various other amino acids (AAs) might also be present at this position. In this study, bioinformatic analyses of selected cyanobacteria were performed to understand whether genetic information in the first adenylation domain of mcyA could explain incorporation of different AAs at position 7 of the MC molecule. Binding pocket signatures of McyA-A1 and putative activated AAs were determined via various bioinformatics tools. Maximum likelihood phylogenetic trees of full length mcyA, mcyA-A1 and 16S rRNA genes were prepared in Mega 6. Phylogenetic analysis of mcyA-A1 nucleotide sequences was in agreement with the predictions of activated AAs by McyA-A1. In comparison with the 16S rRNA and full length mcyA gene trees, mcyA-A1 phylogenetic trees suggested horizontal transfer of the A domain in either Planktothrix agardhii (Gomont) Anagnostidis & Komárek or Planktothrix rubescens (De Candolle ex Gomont) Anagnostidis & Komárek strains. Predictions of activated AAs were generally in agreement with the chemically determined position 7 AAs. However, there were exceptions suggesting the multispecificity of the first A domain of McyA in some cyanobacteria. [ABSTRACT FROM AUTHOR]

Summary: Streptomyces scabies causes potato common scab disease, which reduces the quality and market value of affected tubers. The predominant pathogenicity determinant produced by S. scabies is the thaxtomin A phytotoxin, which is essential for common scab disease development. Production of thaxtomin A involves the nonribosomal peptide synthetases (NRPSs) TxtA and TxtB, both of which contain an adenylation (A‐) domain for selecting and activating the appropriate amino acid during thaxtomin biosynthesis. The genome of S. scabies 87.22 contains three small MbtH‐like protein (MLP)‐coding genes, one of which (txtH) is present in the thaxtomin biosynthesis gene cluster. MLP family members are typically required for the proper folding of NRPS A‐domains and/or stimulating their activities. This study investigated the importance of TxtH during thaxtomin biosynthesis in S. scabies. Biochemical studies showed that TxtH is required for promoting the soluble expression of both the TxtA and TxtB A‐domains in Escherichia coli, and amino acid residues essential for this activity were identified. Deletion of txtH in S. scabies significantly reduced thaxtomin A production, and deletion of one of the two additional MLP homologues in S. scabies completely abolished production. Engineered expression of all three S. scabies MLPs could restore thaxtomin A production in a triple MLP‐deficient strain, while engineered expression of MLPs from other Streptomyces spp. could not. Furthermore, the constructed MLP mutants were reduced in virulence compared to wild‐type S. scabies. The results of our study confirm that TxtH plays a key role in thaxtomin A biosynthesis and plant pathogenicity in S. scabies. [ABSTRACT FROM AUTHOR]

Adenylation (A) domains act as the gatekeepers of non‐ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron‐chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A‐domains. We show that a single asparagine‐to‐glycine mutation in an aryl acid A‐domain leads to an enzyme that tolerates a wide range of non‐native aryl acids. The engineered catalyst is capable of activating non‐native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild‐type enzyme was observed toward these substrates. Co‐crystal structures with non‐hydrolysable aryl‐AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We used a temperature differential assay with the opportunistic fungal pathogen Cryptococcus neoformans as a simple screening platform to detect small molecules with antifungal activity in natural product extracts. By screening of a collection extracts from two different strains of the coprophilous fungus, Amphichorda felina, we detected strong, temperature-dependent antifungal activity using a two-plate agar zone of inhibition assay at 25 and 37 °C. Bioassay-guided fractionation of the crude extract followed by liquid chromatography–mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) identified cyclosporin C (CsC) as the main component of the crude extract responsible for growth inhibition of C. neoformans at 37 °C. The presence of CsC was confirmed by comparison with a commercial standard. We sequenced the genome of A. felina to identify and annotate the CsC biosynthetic gene cluster. The only previously characterized gene cluster for the biosynthesis of similar compounds is that of the related immunosuppressant drug cyclosporine A (CsA). The CsA and CsC gene clusters share a high degree of synteny and sequence similarity. Amino acid changes in the adenylation domain of the CsC nonribosomal peptide synthase’s sixth module may be responsible for the substitution of l-threonine compared to l-α-aminobutyric acid in the CsA peptide core. This screening strategy promises to yield additional antifungal natural products with a focused spectrum of antimicrobial activity. [ABSTRACT FROM AUTHOR]

We determined the crystal structure of Thr1, the self-standing adenylation domain involved in the nonribosomal-like biosynthesis of free 4-chlorothreonine in Streptomyces sp. OH-5093. Thr1 shows two monomers in the crystallographic asymmetric unit with different relative orientations of the C- and N-terminal subdomains both in the presence of substrates and in the unliganded form. Cocrystallization with substrates, adenosine 5′-triphosphate and l-threonine, yielded one monomer containing the two substrates and the other in complex with l-threonine adenylate, locked in a postadenylation state. Steady-state kinetics showed that Thr1 activates l-Thr and its stereoisomers, as well as d-Ala, l- and d-Ser, albeit with lower efficiency. Modeling of these substrates in the active site highlighted the molecular bases of substrate discrimination. This work provides the first crystal structure of a threonine-activating adenylation enzyme, a contribution to the studies on conformational rearrangement in adenylation domains and on substrate recognition in nonribosomal biosynthesis. Database Structural data are available in the Protein Data Bank under the accession number and . [ABSTRACT FROM AUTHOR]

Nonribosomal peptide synthetases (NRPS) are multifunctional proteins that catalyze the synthesis of the peptide products with enormous biological potential. The process of biosynthesis starts with the adenylation (A) domain, which during the catalytic cycle undergoes extensive structural rearrangements. In this paper, we present the first study of the tyrocidine synthetase 1 A-domain (TycA-A) fluorescence properties. The TycA-A protein contains five potentially fluorescent Trp residues at positions 227, 301, 323, 376 and 406. The contribution of each Trp to the TycA-A emission was determined using protein variants bearing single Trp to Phe substitutions. The accessibility of the Trp side chains during adenylation showed that only W227 is affected by substrate binding. The protein variant containing solely fluorescent W227 residue was constructed and further used as a probe to explore the binding effect of different non-cognate amino acid substrates. The results indicate a different accessibility of W227 residue in the presence of non-cognate amino acids, which might offer an explanation for the higher aminoacyl-adenenylate leakage. Overall, our results suggest that intrinsic tryptophan fluorescence could be used as a method to probe the effect of substrate binding on the local structure in NRPS adenylation domains. [ABSTRACT FROM AUTHOR]

Three different deep sea sponge species, Inflatella pellicula, Poecillastra compressa, and Stelletta normani comprising seven individual samples, retrieved from depths of 760-2900 m below sea level, were investigated using 454 pyrosequencing for their secondary metabolomic potential targeting adenylation domain and ketosynthase domain sequences. The data obtained suggest a diverse microbial origin of nonribosomal peptide synthetases and polyketide synthase fragments that in part correlates with their respective microbial community structures that were previously described and reveals an untapped source of potential novelty. The sequences, especially the ketosynthase fragments, display extensive clade formations which are clearly distinct from sequences hosted in public databases, therefore highlighting the potential of the microbiome of these deep sea sponges to produce potentially novel small-molecule chemistry. Furthermore, sequence similarities to gene clusters known to be involved in the production of many classes of antibiotics and toxins including lipopeptides, glycopeptides, macrolides, and hepatotoxins were also identified. [ABSTRACT FROM AUTHOR]

In the biosynthesis of diverse natural bioactive products the adenylation domains (ADs) of nonribosomal peptide synthetases select specific precursors from the cellular pool and activate them for further incorporation into the scaffold of the final compound. Therefore, the drug discovery programs employing PCR-based screening studies of microbial collections or metagenomic libraries often use AD-coding genes as markers of relevant biosynthetic gene clusters. However, due to significant sequence diversity of ADs, the conventional approach using only one primer pair in a single screening experiment could be insufficient for maximal coverage of AD abundance. In this study, the widely used primer pair A3F/A7R was compared with the newly designed aa194F/aa413R one by 454 pyrosequencing of two sets of actinomycete strains from highly dissimilar environments: subseafloor sediments and forest soil. Individually, none of the primer pairs was able to cover the overall diversity of ADs. However, due to slightly shifted specificity of the primer pairs, the total number and diversity of identified ADs were noticeably extended when both primer pairs were used in a single assay. Additionally, the efficiency of AD detection by different primer combinations was confirmed on the model of Salinispora tropica genomic DNA of known sequence. [ABSTRACT FROM AUTHOR]

Salinispora arenicola CNS-205 was a first-isolated obligate marine actinomycete. A gene ( sare0357), annotated as ''amino acid adenylation domain'' located on the genome of Salinispora arenicola CNS-205, was cloned and characterized. The recombinant target protein Sare0357 was expressed in E. coli. Sare0357 specifically recognized and activated tryptophan (Trp) and phenylalanine (Phe). The basic kinetic parameters of Sare0357 for Trp were K = 0.04 mM, V = 2.1 μM/min, k = 14.2 min, and for Phe were K = 0.03 mM, V = 1.6 μM/min, k = 10.4 min. Our data elucidated Sare0357 biological role and biochemical properties as a Trp and Phe-activating adenylation domain. [ABSTRACT FROM AUTHOR]

Metabolites nonribosomal peptide synthetases and Adenylation domain of Marine actinomycete were summarized. [ABSTRACT FROM AUTHOR]

Salinispora arenicola CNS-205 was a firstly-isolated obligate marine actinomycete. Several bioactive metabolites, including NRP compounds, have been isolated from this strain. Existence of a high number of secondary metabolites biosynthetic clusters on its genome revealed its strong potential to accumulate more natural products. Nonribosomal peptide synthetases (NRPSs) are critical enzymes for the biosynthesis of nonribosomal peptide (NRP) compounds, a large class of biologically active natural products. Amino acid adenylation domains (A domains) are essential components within NRPSs and recognize specifically amino acid building blocks to be incorporated during the biosynthesis of NRPSs. a gene sare0357 annotated as "amino acid adenylation domain" located on the genome of Salinispora arenicola CNS-205, was cloned and characterized. The recombinant target protein Sare0357 was expressed efficiently but as inclusion bodies in a prokaryotic system. After denaturation and rena-turation and purification, soluble protein was obtained. [ABSTRACT FROM AUTHOR]

Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes that produce a wide array of bioactive peptides. Here we show that a single tryptophan-to-serine mutation in phenylalanine-specific NRPS adenylation domains enables the efficient activation of non-natural aromatic amino acids functionalized with azide and alkyne groups. The resulting 105-fold switch in substrate specificity was achieved without appreciable loss of catalytic efficiency. Moreover, the effective communication of the modified A domains with downstream modules in dipeptide synthetases permitted incorporation of O-propargyl- L-tyrosine into diketopiperazines both in vitro and in vivo, even in the presence of competing phenylalanine. Because azides and alkynes readily undergo bioorthogonal click reactions, reprogramming NRPSs to accept non-natural amino acids that contain these groups provides a potentially powerful means of isolating, labeling, and modifying biologically active peptides. [ABSTRACT FROM AUTHOR]

Uba5 is the smallest ubiquitin-like molecule-activating enzyme and contains an adenylation domain and a C-terminal region. This enzyme only exists in multicellular organisms. The mechanism through which the enzyme recognizes and activates ubiquitin-fold modifier 1 (Ufm1) remains unknown. In this study, Uba5 adenylation domains with different C-terminal region lengths were cloned, expressed and purified. The results of an in vitro truncation assay suggest that Uba5 residues 57-363 comprise the minimal fragment required for the high-efficiency activation of Ufm1. Crystallization of Uba5 residues 57-363 was performed at 277 K using PEG 3350 as the precipitant, and crystals optimized by microseeding diffracted to 2.95 Å resolution, with unit-cell parameters a = b = 97.66, c = 144.83 Å, α = β = 90, γ = 120°. There is one molecule in the asymmetric unit; the Matthews coefficient and the solvent content were calculated to be 2.93 Å3 Da−1 and 58.1%, respectively. [ABSTRACT FROM AUTHOR]

Mammalian ACSF4-U26 (Acyl CoA synthetase family member 4), a protein of unknown function, comprises a putative adenylation domain ( AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site, and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 show greater identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26, and prediction of its specificity suggests that it activates β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was not formed in a point mutant lacking the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was almost specific for β-alanine, and a KM of ~ 5 μ m was calculated for this reaction. The loaded enzyme was used to study the formation of a potential end product. Among the 20 standard amino acids, only cysteine stimulated unloading of the enzyme. This effect was mimicked by cysteamine and dithiothreitol, and was unaffected by absence of the PQQDH-related domain, suggesting that β-alanine transfer onto thiols is catalysed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine-activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification. [ABSTRACT FROM AUTHOR]

The nonribosomal peptide synthetases (NRPSs) are a family of modular proteins that contain multiple catalytic domains joined in a single protein. Together, these domains work to produce chemically diverse peptides, including compounds with antibiotic activity or that play a role in iron acquisition. Understanding the structural mechanisms that govern the domain interactions has been a long-standing goal. During NRPS synthesis, amino-acid substrates are loaded onto integrated carrier protein domains through the activity of NRPS adenylation domains. The structures of two adenylation domain-carrier protein domain complexes have recently been determined in an effort that required the use of a mechanism-based inhibitor to trap the domain interaction. Here, the continued analysis of these proteins is presented, including a higher resolution structure of an engineered di-domain protein containing the EntE adenylation domain fused with the carrier protein domain of its partner EntB. The protein crystallized in a novel space group in which molecular replacement and refinement were challenged by noncrystallographic pseudo-translational symmetry. The structure determination and how the molecular packing impacted the diffraction intensities are reported. Importantly, the structure illustrates that in this new crystal form the functional interface between the adenylation domain and the carrier protein domain remains the same as that observed previously. At a resolution that allows inclusion of water molecules, additional interactions are observed between the two protein domains and between the protein and its ligands. In particular, a highly solvated region that surrounds the carrier protein cofactor is described. [ABSTRACT FROM AUTHOR]

Background: Many important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly. Results: Here, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu1] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA2 adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA2 adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu1] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met1] microcystin-LR. Conclusions: Phylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu1] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations. [ABSTRACT FROM AUTHOR]

Fusaricidins produced by Paenibacillus polymyxa DBB1709 are lipopeptide antibiotics active against fungi and Gram-positive bacteria. The cyclic hexapeptide structures of fusaricidins are synthesized by fusaricidin synthetase, a non-ribosomal peptide synthetase. The adenylation domain of the third module (FusA-A3) can recruit l-Tyr, l-Val, l-Ile, l-allo-Ile, or l-Phe, which diversifies the fusaricidin structures. Since the l-Phe-incorporated fusaricidin analog (LI-F07) exhibits more potent antimicrobial activity than other analogs, we modified a specificity-conferring sequence in the substrate binding pocket of FusA-A3 to direct the enhanced production of LI-F07. Base on comparison to the adenylation domain of gramicidin S synthetase 1 and tyrocidine synthetase 1, both of which mainly activate l-Phe, six mutant strains with altered FusA-A3 were generated using site-directed mutagenesis. M3 (I239W, I299V), M5 (I299V, G322A, V330I), and M6 (S239W, I299V, G322A, V330I) mutants produced significantly more LI-F07 than the wild-type strain. [ABSTRACT FROM AUTHOR]

Molecular screening using degenerate PCR to determine the presence of secondary metabolite genes in cyanobacteria was performed. This revealed 18 NRPS and 19 PKS genes in the 21 new cyanobacterial strains examined, representing three families of cyanobacteria ( Nostocales, Chroococales and Oscillatoriales). A BLAST analysis shows that these genes have similarities to known cyanobacterial natural products. Analysis of the NRPS adenylation domain indicates the presence of novel features previously ascribed to both proteobacteria and cyanobacteria. Furthermore, binding-pocket predictions reveal diversity in the amino acids used during the biosynthesis of compounds. A similar analysis of the PKS ketosynthase domain shows significant structural diversity and their presence in both mixed modules with NRPS domains and individually as part of a PKS module. We have been able to classify the NRPS genes on the basis of their binding-pockets. Further, we show how this data can be used to begin to link structure to function by an analysis of the compounds Scyptolin A and Hofmannolin from Scytonema sp. PCC 7110. [ABSTRACT FROM AUTHOR]

Abstract: Adenylate forming enzymes play an important role in nature as they are involved in a number of essential biochemical pathways. In this study, we investigated the ability of a set of structurally related recombinant bacterial adenylate forming enzymes derived from nonribosomal peptide synthetases for their ability to synthesize acyl-CoAs in vitro. Adenylation-domains normally transfer their reactive aminoacyl-adenylates onto the covalently attached 4′-phosphopantetheine moiety of small carrier proteins. In detail, DltA, DhbE, GrsA-A, TycB3-A, and TycC3-A were investigated for their ability to synthesize acyl-CoAs. As reference, acetyl-CoA-synthetase (Acs) of B. subtilis was utilized, which naturally synthesizes acetyl-CoA from acetate, CoA-SH and ATP. Interestingly, all enzymes were capable of producing acyl-CoAs, albeit with differing efficiencies. Surprisingly, both CoA-SH and ATP were observed to inhibit the adenylation reaction at higher concentrations. Product quantification for kinetic determination was carried out by ESI-SIM-MS. Our results allow speculation as to evolutionary relationships within the large class of adenylate forming enzymes. [Copyright &y& Elsevier]

Lysobacter lactamgenus produces cephabacins, a class of β-lactam antibiotics which have an oligopeptide moiety attached to the cephem ring at the C-3 position. The nonribosomal peptide synthetase (NRPS) system, which comprises four distinct modules, is required for the biosynthesis of this short oligopeptide, when one takes the chemical structure of these antibiotics into consideration. The cpbI gene, which has been identified in a region upstream of the pcbAB gene, encodes the NRPS – polyketide synthase hybrid complex, where NRPS is composed of three modules, while the cpbK gene – which has been reported as being upstream of cpbI– comprises a single NRPS module. An in silico protein analysis was able to partially reveal the specificity of each module. The four recombinant adenylation (A) domains from each NRPS module were heterologously expressed in Escherichia coli and purified. Biochemical data from ATP–PPi exchange assays indicated thatl-arginine was an effective substrate for the A1 domain, while the A2, A3 and A4 domains activatedl-alanine. These findings are in an agreement with the known chemical structure of cephabacins, as well as with the anticipated substrate specificity of the NRPS modules in CpbI and CpbK, which are involved in the assembly of the tetrapeptide at the C-3 position. [ABSTRACT FROM AUTHOR]

Lysergyl peptide synthetase 1 catalyzes the assembly of toxic ergopeptines from activated D-lysergic acid and three amino acids. The gene encoding this enzyme in the endophytic fungus Neotyphodium lolii was analyzed and compared to a homologous gene from the ergot fungus Claviceps purpurea . Each gene contained two introns, which were found in the same relative position within two modules of the gene. The 5′ ends of the two genes were unusually divergent. Signature sequences determining substrate specificity were similar in adenylation domains that recognized identical amino acids but differed within the adenylation domain for the amino acid that varies between the major ergopeptines of the two fungi. Homologues were detected in several related endophytic fungi; the tall fescue endophyte Neotyphodium coenophialum contained a divergent, second copy of the gene. Our results provide new information on the structure and distribution of this important peptide synthetase involved in ergot alkaloid biosynthesis. [ABSTRACT FROM AUTHOR]

Background: Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis. Results: Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined. Conclusions: We describe the NRPS-t1PKS cluster 'BIIRfg' compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene. [ABSTRACT FROM AUTHOR]

Microcystins are a family of chemically diverse hepatotoxins produced by distantly related cyanobacteria and are potent inhibitors of eukaryotic protein phosphatases 1 and 2A. Here we provide evidence for the biosynthesis of rare variants of microcystin that contain a selection of homo-amino acids by the benthic cyanobacterium Phormidium sp. LP904c. This strain produces at least 16 microcystin chemical variants many of which contain homophenylalanine or homotyrosine. We retrieved the complete 54.2 kb microcystin (mcy) gene cluster from a draft genome assembly. Analysis of the substrate specificity of McyB1 and McyC adenylation domain binding pockets revealed divergent substrate specificity sequences, which could explain the activation of homo-amino acids which were present in 31% of the microcystins detected and included variants such as MC-LHty, MC-HphHty, MC-LHph and MC-HphHph. The mcy gene cluster did not encode enzymes for the synthesis of homo-amino acids but may instead activate homo-amino acids produced during the synthesis of anabaenopeptins. We observed the loss of microcystin during cultivation of a closely related strain, Phormidium sp. DVL1003c. This study increases the knowledge of benthic cyanobacterial strains that produce microcystin variants and broadens the structural diversity of known microcystins. [ABSTRACT FROM AUTHOR]