Anacyclamide D8P, a prenylated cyanobactin from a Sphaerospermopsis sp. cyanobacterium (original) (raw)
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Sphaerocyclamide, a prenylated cyanobactin from the cyanobacterium Sphaerospermopsis sp. LEGE 00249
Scientific reports, 2018
Cyanobactins are a family of linear and cyclic peptides produced through the post-translational modification of short precursor peptides. A mass spectrometry-based screening of potential cyanobactin producers led to the discovery of a new prenylated member of this family of compounds, sphaerocyclamide (1), from Sphaerospermopsis sp. LEGE 00249. The sphaerocyclamide biosynthetic gene cluster (sph) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the sph gene cluster in Escherichia coli confirmed the connection between genomic and mass spectrometric data. Unambiguous establishment of the orientation and site of prenylation required the full structural elucidation of 1 using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound 1 was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bac...
Highly Diverse Cyanobactins in Strains of the Genus Anabaena
Applied and Environmental Microbiology, 2010
Cyanobactins are small, cyclic peptides recently found in cyanobacteria. They are formed through proteolytic cleavage and posttranslational modification of short precursor proteins and exhibit antitumor, cytotoxic, or multi-drug-reversing activities. Using genome project data, bioinformatics, stable isotope labeling, and mass spectrometry, we discovered novel cyclic peptides, anacyclamides, in 27 Anabaena strains. The lengths of the anacylamides varied greatly, from 7 to 20 amino acids. Pronounced sequence variation was also detected, and only one amino acid, proline, was present in all anacyclamides. The anacyclamides identified included unmodified proteinogenic or prenylated amino acids. We identified an 11-kb gene cluster in the genome of Anabaena sp. 90, and heterologous expression in Escherichia coli confirmed that this cluster was responsible for anacyclamide production. The discovery of anacyclamides greatly increases the structural diversity of cyanobactins. Cyanobacteria are a prolific source of secondary metabolites with potential as drug leads or useful probes for cell biology studies (23). They include biomedically interesting compounds, such as the anticancer drug lead cryptophycin (15), and environmentally problematic hepatotoxic peptides, such as microcystins and nodularins produced by bloom-forming cyanobacteria (23). Many of these compounds contain nonproteinogenic amino acids and modified peptides and are produced by nonribosomal peptide synthesis (23, 26). The cyanobactins are a new group of cyclic peptides recently found in cyanobacteria (4). They are assembled through posttranslational proteolytic cleavage and head-to-tail macrocyclization of short precursor proteins. The cyanobactins are low-molecular-weight cyclic peptides that contain heterocyclized amino acids and can be prenylated or contain D-amino acids (3, 4). The cyanobactins that contain heterocyclized amino acids include patellamides, ulithiacyclamides, trichamide, tenuecyclamides, trunkamides, patellins, and microcyclamides and are synthesized in this manner (3, 4, 20, 24, 28). They possess antitumor, cytotoxic, and multi-drug-reversing activities and have potential as drug leads (4, 18, 20). Cyanobactins containing heterocyclized amino acids are found in a variety of cyanobacteria (4). A recent study demonstrated that the cyanobactin biosynthetic pathway is prevalent in planktonic bloom-forming cyanobacteria (14). However, the products of these gene clusters encoding new cyanobactins are unknown. Here we report discovery of a novel family of low-molecular-weight cyanobactins and show that these compounds are common in strains of the genus Anabaena. These anacyclamides exhibit pronounced length and sequence variation and contain unmodified or prenylated amino acids. MATERIALS AND METHODS Cyanobacterial strains and cultivation. The Anabaena strains were grown in Z8 (12) medium lacking a source of combined nitrogen, except for a few cultures for which nitrogen was included in the medium (Table 1). Strains were grown in 40-ml cultures under continuous light with a photon irradiance of 5 to 12 mol m Ϫ2 s Ϫ1 at 20 to 25°C. Z8 medium containing 34 S was used to detect sulfurcontaining peptides. The MgSO 4 ⅐ 7 H 2 O of Z8 medium was replaced by a stable isotope of MgSO 4 (catalog no. IS7080; 90 atom% 34 S; Icon). Annotation of the acy gene cluster. We obtained the complete genome sequence of the toxic bloom-forming freshwater cyanobacterium Anabaena sp. 90. Open reading frame (ORF) predictions were made with Glimmer, and the acy gene cluster was annotated using Artemis (Sanger Institute). Functional analysis of proteins was performed using BLASTp, and the predictions for start sites of the genes were checked manually. A putative peptide precursor gene was identified using BLASTp searches and homology of the N-terminal sequence of the 49-amino-acid AcyE protein and the precursor proteins of patellamide, tenuecyclamide, trichamide, and microcyclamide (4, 20, 24, 28). DNA extraction, amplification, and sequencing. Genomic DNA was extracted from the strains of Anabaena using an E.Z.N.A. plant DNA mini kit (Omega Bio-Tek, Doraville, GA). We prescreened 74 Anabaena strains for the presence of cyanobactin biosynthetic genes using a PCR approach based on the conserved subtilisin-like protease gene, as previously described (14). In order to identify an Anabaena strain producing a cyanobactin containing a sulfur amino acid, the acyE peptide precursor gene was amplified using primers preRNAF (5ЈGAAG AACATCCGCCCCCAACAAGTTG3Ј) and preRNAR (5ЈCTCCGCGTCGTC GCCTGCAAAAGG-3Ј) and primers PreF (5Ј-GCCTTCACCAAACCAGTCT TCTTCAT-3Ј) and PreR (5Ј-CATCGAGGCGAACCGTGCGCCAAGGGAT-3Ј) from the genomic DNA of Anabaena strains that were selected based on the presence of a cyanobactin biosynthetic gene fragment. PCRs were performed as previously described (14). Sequencing was carried out with an ABI 3730xl automated sequencer (Applied Biosystems) by Macrogen (Seoul, Korea). Sequences were checked and edited by using the Chromas 2.2 program (Technelysium Pty. Ltd.) and were aligned by using the Bioedit sequence alignment editor. Cloning the acy biosynthetic gene cluster. The entire 11-kb acy gene cluster was amplified from genomic DNA of Anabaena sp. 90 by PCR using primers patex2f (5Ј-ATGGATCCTGATGGACTGTAGTGTGAG-3Ј) and patex5r (5Ј-TACTCGAGAGGTTTTGGGACTCTTTAG-3Ј) in three 60-l reaction mixtures containing 1ϫ PCR buffer for Super Taq Plus (HT Biotechnology Ltd.), 200 mol of each nucleotide (Finnzymes), 0.75 M of each primer, 0.8 U Super Taq Plus proofreading polymerase (HT Biotechnology Ltd.), and 100 ng of Anabaena sp. 90 template DNA. The thermocycling conditions were 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 56.4°C for 30 s, and 68°C for 9 min and then a final extension at 68°C for 20 min. The PCR products were separated on a 0.6% agarose gel containing 0.5ϫ Tris-acetate-EDTA and run for 30 min at 100 V. The gel was stained using SYBR Safe DNA gel stain (Invitrogen) and was visualized using a Dark reader (Clare Chemical Research Inc.
Highly Diverse Cyanobactins in Strains of the Genus Anabaena
Applied and Environmental Microbiology, 2010
Cyanobactins are small, cyclic peptides recently found in cyanobacteria. They are formed through proteolytic cleavage and posttranslational modification of short precursor proteins and exhibit antitumor, cytotoxic, or multi-drug-reversing activities. Using genome project data, bioinformatics, stable isotope labeling, and mass spectrometry, we discovered novel cyclic peptides, anacyclamides, in 27 Anabaena strains. The lengths of the anacylamides varied greatly, from 7 to 20 amino acids. Pronounced sequence variation was also detected, and only one amino acid, proline, was present in all anacyclamides. The anacyclamides identified included unmodified proteinogenic or prenylated amino acids. We identified an 11-kb gene cluster in the genome of Anabaena sp. 90, and heterologous expression in Escherichia coli confirmed that this cluster was responsible for anacyclamide production. The discovery of anacyclamides greatly increases the structural diversity of cyanobactins.
Cyanobactins—ribosomal cyclic peptides produced by cyanobacteria
Applied Microbiology and Biotechnology, 2010
Cyanobactins are small cyclic peptides that are produced by a diverse selection of cyanobacteria living in symbioses as well as terrestrial, marine, or freshwater environments. They include compounds with antimalarial, antitumor, and multidrug reversing activities and potential as pharmaceutical leads. Cyanobactins are produced through the proteolytic cleavage and cyclization of precursor peptides coupled with further posttranslational modifications such as heterocyclization, oxidation, or prenylation of amino acids. Cyanobactin gene clusters encode two proteases which cleave and cyclisize the precursor peptide as well as proteins participating in posttranslational modifications. The bioinformatic mining of cyanobacterial genomes has led to the discovery of novel cyanobactins. Heterologous expression of these gene clusters provided insights into the role of the genes participating in the biosynthesis of cyanobactins and facilitated the rational design of novel peptides. Enzymes participating in the biosynthesis of cyanobactins may prove useful as catalysts for producing novel cyclic peptides in the future. The recent discovery of the cyanobactin biosynthetic pathway in cyanobacteria extends our knowledge of their potential as producers of interesting metabolites.
Tetrahedron, 2002
During our biosynthesis study of cyanobacterial peptides including microcystins, we investigated the metabolic peptides in the hepatotoxic cyanobacteria, Anabaena sp. strains 90 and 202A2, in which the genetic analysis of the peptide synthetase had been carried out. For the exhaustive screening of cyanobacterial peptides, an analytical method using ESI-LC/MS on-line photodiode array detection was successfully developed and applied. Based on the analytical results, two groups of peptides, the cyclic depsipeptides having a 3-amino-6hydroxy-2-piperidone moiety, anabaenopeptilides, and the cyclic peptides possessing an ureido linkage, anabaenopeptins, were isolated together with microcystins from both strains. Consequently, we confirmed the structures including the stereochemistry of the anabaenopeptilides encoded by the sequencing peptide synthetase genes in Anabaena strain 90. q 2002 Elsevier Science Ltd. All rights reserved.
Three novel anabaenopeptins from the cyanobacterium Anabaena sp
Tetrahedron, 2008
Three new cyclic peptides, anabaenopeptins NZ825, NZ841, and NZ857, were isolated from the hydrophilic extract of the cultured cyanobacterium Anabaena sp. The planar structure of the compounds was determined by homonuclear and inverse-heteronuclear 2D-NMR techniques as well as high-resolution mass spectrometry. The absolute configuration of the asymmetric centers was studied using Marfey's method for HPLC. This is the first report of anabaenopeptins that contain N-methyl glycine instead of the common N-methyl alanine. The incorporation of N-methyl glycine into the cyclic portion of the compounds results in their appearance as a mixture of two, equally stable, conformers, instead of the one distinct conformer in anabaenopeptins that contain N-methyl alanine or N-methyl homotyrosine. The three compounds were tested for inhibition of serine proteases and found to be not active.
Cyanobactins from Cyanobacteria: Current Genetic and Chemical State of Knowledge
Marine Drugs, 2015
Cyanobacteria are considered to be one of the most promising sources of new, natural products. Apart from non-ribosomal peptides and polyketides, ribosomally synthesized and post-translationally modified peptides (RiPPs) are one of the leading groups of bioactive compounds produced by cyanobacteria. Among these, cyanobactins have sparked attention due to their interesting bioactivities and for their potential to be prospective candidates in the development of drugs. It is assumed that the primary source of cyanobactins is cyanobacteria, although these compounds have also been isolated from marine animals such as ascidians, sponges and mollusks. The aim of this review is to update the current knowledge of cyanobactins, recognized as being produced by cyanobacteria, and to emphasize their genetic clusters and chemical structures as well as their bioactivities, ecological roles and biotechnological potential.
Cyanobacterial peptides — Nature's own combinatorial biosynthesis
FEMS Microbiology Reviews, 2006
Cyanobacterial secondary metabolites have attracted increasing scientific interest due to bioactivity of many compounds in various test systems. Among the known structures, oligopeptides are often found with many congeners sharing conserved substructures, while being highly variable in others. A major part of known oligopeptides are of non-ribosomal origin and can be grouped into classes with conserved structural properties. Thus, the overall structural diversity of cyanobacterial oligopeptides only seemingly suggests an equally high diversity of biosynthetic pathways and respective genes. For each class of peptides, some of which have been found in all major branches of the cyanobacterial evolutionary tree, homologous synthetases and genes can be inferred. This implies that nonribosomal peptide synthetase genes are a very ancient part of the cyanobacterial genome and presumably have evolved by recombination and duplication events to reach the present structural diversity of cyanobacterial oligopeptides. In addition, peptide synthetases would appear to be an essential part of the cyanobacterial evolution and physiology. The present review presents an overview of the biosynthesis of cyanobacterial peptides and corresponding gene clusters, the structural diversity of structural types and structural variations within peptide classes, and implications for the evolution and plasticity of biosynthetic genes and the potential function of cyanobacterial peptides.
Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis
Journal of the American Chemical Society, 2018
Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C geranyl group. Here we characterize a novel geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr O-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic bi...
PLoS ONE, 2012
Cyanobactins are cyclic peptides assembled through the cleavage and modification of short precursor proteins. An inactive cyanobactin gene cluster has been described from the genome Microcystis aeruginosa NIES843. Here we report the discovery of active counterparts in strains of the genus Microcystis guided by this silent cyanobactin gene cluster. The end products of the gene clusters were structurally diverse cyclic peptides, which we named piricyclamides. Some of the piricyclamides consisted solely of proteinogenic amino acids while others contained disulfide bridges and some were prenylated or geranylated. The piricyclamide gene clusters encoded between 1 and 4 precursor genes. They encoded highly diverse core peptides ranging in length from 7-17 amino acids with just a single conserved amino acid. Heterologous expression of the pir gene cluster from Microcystis aeruginosa PCC7005 in Escherichia coli confirmed that this gene cluster is responsible for the biosynthesis of piricyclamides. Chemical analysis demonstrated that Microcystis strains could produce an array of piricyclamides some of which are geranylated or prenylated. The genetic diversity of piricyclamides in a bloom sample was explored and 19 different piricyclamide precursor genes were found. This study provides evidence for a stunning array of piricyclamides in Microcystis, a worldwide occurring bloom forming cyanobacteria.