1,468 research outputs found

    A Unique Tryptophan C‚ÄźPrenyltransferase from the Kawaguchipeptin Biosynthetic Pathway

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    Acknowledgements This work was supported by funding of the Academy of Finland (259505), Helsinki University Research grant (490085) and ESCMID grant (4720572) to D.P.F., University of Pittsburgh Central Research Development Fund to X.L., Technology Strategy Board grant (131181) to W.H., M.J. and J.H.N. National Programme of Sustainability I of the Ministry of Education of the Czech Republic I grant (LO1416) to T.G.Peer reviewedPostprin

    Cyanobactins : ribosomally synthesized and post-translationally modified peptides produced by cyanobacteria

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    Ribosomal peptides are produced through the post-translational modification of precursor peptides. Cyanobactins are a family of ribosomal peptides produced by cyanobacteria. A great variety of cyclic peptides that can be assigned to the cyanobactin family have been described from cyanobacteria. In the biosynthesis, the precursor peptide is cleaved, N-to-C macrocyclized, and some amino acids are heterocyclized or prenylated. As a family, the cyanobactins possess cytotoxicity, multidrug-resistance reversing activity, anti-malarial, antimicrobial, and allelopathic activities. The aim of this study was to explore the distribution of cyanobactin pathways and determine the chemical diversity of the peptides they encode. Cyanobacterial strains were studied using a combination of bioinformatics, molecular biology, evolutionary biology, and structural chemistry. In this study, a PCR-based approach demonstrated that a gene, essential for cyanobactin biosynthesis, is common and sporadically distributed. It was found in 48 of 132 cyanobacterial strains studied. The product of a cyanobactin pathway in Anabaena sp. 90 was identified as a cyclic peptide, consisting only of proteinogenic amino acids and it was named anacyclamide. Diverse anacyclamides were identified from 27 strains of the genus Anabaena, consisting of 7-20 amino acids which were prenylated or geranylated in some strains. New cyanobactins were also found in 6 strains of the genus Microcystis based on the genetic similarity to an inactive cyanobactin gene cluster in M. aeruginosa NIES 843. These peptides, which were named piricyclamides, are similar to anacyclamides. Piricyclamides have 7 to 17 amino acids and like anacyclamides the only conserved amino acid is a single proline. Bioinformatic analysis of the cyanobactin pathway in 126 cyanobacterial genomes identified 31 cyanobactin gene clusters, demonstrating that cyanobactin pathways were widely distributed among all cyanobacteria. Surprisingly, two of the pathways identified in this study were shown to produce novel linear cyanobactins. These linear peptides of just 3 5 amino acids contain a cysteine derived thiazole and an N-terminus protected with a prenyl group and methylated C-terminus. The linear cyanobactins were named aeruginosamides B, C, and viridisamide A. Phylogenetic analyses subsequently showed that the cyanobactin pathway has a complex evolutionary history which differs from the evolutionary history of cyanobacteria. The cyanobactin family was originally defined as being cyclic peptides containing modified amino acids. In this study, altogether three types of cyanobactins were described, cyclic anacyclamides, piricyclamides, and the linear cyanobactins aeruginosamides and viridisamide A. This work broadened the cyanobactin definition to include unmodified cyclic peptides, and expanded the length variation of cyanobactins setting new upper and lower limits, 3 20, for the length of the peptides in the cyanobactin family. The definition of cyanobactin was further refined to include linear peptides with termini protected by prenylation and methylation. Overall, this study has shown that the cyanobactin pathway is common in cyanobacteria and able to produce a range of chemically diverse peptides.Bakteerit tuottavat erilaisia pieniä ribosomaalisia peptidejä, jotka on muokattu suuremmasta esipeptidistä. Syanobaktiinit ovat syanobakteerien tuottamia ribosomaalisia rengaspeptidejä, jotka sisältävät muokattuja aminohappoja. Syanobaktiinien on havaittu olevan soluille myrkyllisiä, antimikrobisia, lääkevastustuskykyä alentavia ja malariaa estäviä sekä niillä on havaittu myös olevan muiden eliöiden kasvuun vaikuttavia, allelopaattisia, ominaisuuksia. Tässä työssä selvitettiin syanobaktiinien biosynteesigeenien esiintymistä syanobakteereissa sekä syanobaktiinien kemiallisten rakenteiden monimuotoisuutta. Työssä tutkittiin syanobakteerikantoja yhdistelemällä bioinformatiikkaa, molekyylibiologiaa, evoluutiobiologiaa sekä kemiallista rakenneanalytiikkaa. Syanobaktiinien tuottamiseen tarvittavien geenien todettiin olevan yleisiä. Tutkituista 132 syanobakteerikannasta 48 löydettiin PCR-menetelmällä syanobaktiinien biosynteesille oleellinen patA-geeni. Anabaena sp. 90 -syanobakteerin genomissa on syanobaktiinien tuottamiseksi tarvittavat geenit. Geenien tuottama syanobaktiini löydettiin ja nimettiin anasyklamidiksi. Anasyklamideja löydettiin 27 Anabaena-kannasta. Anasyklamidit koostuvat 7 20 tavallisesta aminohaposta ja toisinaan jokin aminohappo on prenyloitu tai geranyloitu. Uusia syanobaktiineja löydettiin myös kuudesta Microcystis-kannasta Microcystis aeruginosa NIES 843 -kannan toimimattoman syanobaktiini geeniryhmän perusteella. Nämä syanobaktiinit nimettiin pirisyklamideiksi. Pirisyklamideissa on 7 17 aminohappoa, joista viimeinen on aina proliini, kuten anasyklamideissakin. Myös pirisyklamideissa on prenyloituja tai geranyloituja aminohappoja. Bioinformatiikan avulla löydettiin 126 syanobakteerigenomista 31 syanobaktiinien tuottamiseen tarvittavaa geeniryhmää. Näistä kahden havaittiin tuottavan lineaarisia 3 5 aminohaposta koostuvia syanobaktiineja. Löydetyissä lineaarisissa syanobaktiineissa on kysteiinistä muokattu heterosyklinen tiatsolirengas, prenyyliryhmällä suojattu aminopää sekä metyloitu karboksyylipää. Uudet lineaariset syanobaktiinit nimettiin aeruginosamidi B:ksi ja C:ksi sekä viridisamidi A:ksi. Tutkimuksen mukaan syanobaktiinien tuottamiseksi tarvittavat geeniryhmät ovat laajalle levinneitä syanobakteerien joukossa. Fylogeneettiset analyysit osoittivat, että syanobaktiineja tuottavien geenien evoluutio on monimutkainen ja se eroaa syanobakteerien evolutiivisesta historiasta. Aikaisemmin syanobaktiinit oli määritelty rengaspeptideiksi, joissa on muokattuja aminohappoja. Tässä työssä kuvattiin rengasmaiset anasyklamidit ja pirisyklamidit sekä lineaariset aeruginosamidit ja viridisamidi A. Työssä laajennettiin syanobaktiinien määritelmää käsittämään myös ainoastaan tavallisista, muokkaamattomista aminohapoista koostuvia rengaspeptidejä sekä asetettiin uudet ääripituudet syanobaktiineille (3 20). Syanobaktiinit määriteltiin nyt sisältämään myös lineaarisia peptidejä. Kaiken kaikkiaan työ osoitti, että syanobaktiinien tuottamiseksi tarvittavat geenit ovat yleisiä syanobakteereilla ja ne tuottavat suuren joukon erilaisia syanobaktiineja

    Cyclic peptide production using a macrocyclase with enhanced substrate promiscuity and relaxed recognition determinants

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    This project was supported by grants from the ERC (no. 339367, MJ), BBSRC IBCatalyst (no. BB/M028526/1, MJ, WEH), BBSRC FoF (no. BB/M013669/1, MJ, WEH), IBioIC Exemplar (no. 2014-2-4, MJ, WEH), an AstraZeneca studentship (MJ, WEH, LT, KR), the Academy of Finland (no. 259505, DPF) and the SULSA leaders award (WEH). The authors like to thank the Aberdeen Proteomics Facility and the Aberdeen School of Natural and Computing Sciences MS Facility for LCMS analysis. Electronic supplementary information (ESI) available: Experimental section, Fig. S1‚ÄďS60 and Tables S1‚ÄďS3. See DOI: 10.1039/c7cc05913bPeer reviewedPublisher PD

    Doctor of Philosophy

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    dissertationCyanobactins are N-to-C macrocyclic peptides that contain diverse modifications such as heterocyclization of Cys, Ser, or Thr, and isoprenylation of Ser, Thr, and Tyr. Although the above can be inferred to occur based on the final products, none of the enzymatic steps en route to cyanobactin biosynthesis had been characterized prior to this work. Indeed, until very recently, nothing at all was known about cyanobactin biosynthesis. Only after cloning and sequencing of the genetic elements required for cyanobactin biosynthesis was their biosynthetic origin deduced. Surprisingly, these complex natural products are biosynthesized by extensive posttranslational modification of ribosomally synthesized precursor peptides. As noted, these precursors are initially synthesized on the ribosome. Following ribosomal synthesis, various modifying enzymes carry out posttranslational modification of the aforementioned amino acids, as well as proteolysis of the precursor peptide to liberate 6-12 amino acid peptidyl groups, which are then macrocyclized. However, the manner in which the disparate genetic components required for cyanobactin biosynthesis functioned enzymatically to create these highly diverse and medicinally interesting compounds was unknown prior to this work. Herein the results of several studies that elucidate the steps en route to cyanobactin biosynthesis are described. The characterized steps include: N- and C-terminal proteolysis, macrocyclization, heterocyclization of Cys, Ser, and Thr, and prenylation of Ser, Thr and Tyr

    Sphaerocyclamide, a prenylated cyanobactin from the cyanobacterium Sphaerospermopsis sp. LEGE 00249

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    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 bacterium Halomonas aquamarina CECT 5000.Peer reviewe

    Identifiering av biosyntesvägen för muscorid leder till upptäckt av en ny antimikrobiell naturprodukt

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    Bacteria are a great source of natural products with complex chemical structures and diverse biological activities. Many have therapeutic properties and half of drugs in clinical use today are derived directly or indirectly from natural products. The pharmaceutical industry stopped investing in drug development from natural resources, due to perceived limitations in chemical space, and difficulties in rediscovery of known compounds and in obtaining sufficient quantities of natural products for clinical trials. There is now renewed interest in natural products as drug leads driven by technological advances in genome sequencing and analytical chemistry. Cyanobacteria produce a variety of natural products with therapeutic potential. Muscoride A is an unusual peptide alkaloid produced by a terrestrial freshwater cyanobacterium with reported antimicrobial activity. The aim of this study was to characterize the biosynthetic origin and biological activity of muscoride A. I identified the 12.7 kb muscoride (mus) biosynthetic gene cluster from a draft genome of Nostoc sp. PCC 7906 using bioinformatics analysis. The mus biosynthetic gene cluster encoded enzymes for the heterocyclization, oxidation and prenylation of a precursor protein. Comparative genomics identified a mus biosynthetic gene cluster in the unpublished draft genome of Nostoc sp. UHCC sp. 0398 encoding a novel muscoride. This novel muscoride, muscoride B, was detected from Nostoc sp. UHCC 0398 based on this analysis. Muscoride B was purified using solid phase extraction and high-performance liquid chromatography and the chemical structure was verified by combining nuclear magnetic resonance and mass spectrometry data. Furthermore, the function and evolutionary history of the muscoride prenyltransferases were studied. A significant finding was that the biosynthetic pathway encodes two regiospecific prenyltransferases, catalyzing the C- and N-terminal prenylation of muscoride. An antimicrobial activity screening showed that muscoride B had antimicrobial activity against Bacillus cereus. Here I report the discovery of the muscoride biosynthetic pathway and the discovery of a novel antimicrobial peptide from cyanobacteria through genome mining. The results show that the variant is a novel muscoride, a linear bis-prenylated polyoxazole pentapeptide with antimicrobial activity.Bakterier är en bra källa till naturprodukter med komplexa kemiska strukturer och olika biologiska aktivitet. Många har terapeutiska egenskaper och hälften av läkemedel i kliniskt bruk idag, är direkt eller indirekt härledda ur naturprodukter. Läkemedelsindustrin slutade att investera i läkemedelsutveckling från naturresurser på grund av uppfattade begränsningar i kemiskt utrymme och svårigheter att återupptäcka kända föreningar och erhålla tillräckliga mängder av naturprodukter för kliniska prövningar. Det finns nu ett förnyat intresse för naturprodukter som läkemedelskandidat som drives av tekniska framsteg inom genomsekvensering och analytisk kemi. Cyanobakterier producerar en mängd naturprodukter med terapeutisk potential. Muscorid A är en ovanlig peptidalkaloid producerad av en markbunden sötvattens cyanobakterie med rapporterad antimikrobiell aktivitet. Syfte med denna studien var att karakterisera det biosyntetiska ursprunget och bioaktiviteten av muscorid A. Jag identifierade det 12.7 kb muscorid (mus) biosyntetiska genklustret från utkast genomet av Nostoc sp. PCC 7960 med bioinformatiska analyser. Det biosyntetiska genklustret mus kodade enzymer för heterocyklisering, oxidering och prenylering av en prekursorpeptid. Jämförande genomik identifierade det biosyntestiska genklustret mus från ett opublicerat utkast genom av Nostoc sp. UHCC 0398 som kodar för en ny muscorid. Denna nya muscorid, muscorid B, upptäcktes från Nostoc sp. UHCC 0398 baserat på denna analys. Muscorid B extraherades med fastfasextraktion och högupplösande vätskekromatografi och den kemiska strukturen bestämdes kombinerat med kärnmagnetisk resonansspektroskopi och masspektrometri. Dessutom studerades muscorid prenyltransferasens funktion och evolutionära historia. Ett signifikant fynd var att biosyntesvägen kodar för två regiospecifika prenyltransferas som katalyserar prenyleringen av N- och C-terminalen för muscorid. Screening av antimikrobiell aktivitet visade att muscorid B hade antimikrobiell aktivitet mot Bacillus cereus. Här rapporterar jag upptäckten av biosyntesvägen för muscorid och fyndet av en ny antimikrobiell peptid från cyanobakterier genom genomutvinning (eng. genome mining). Resultaten visar att varianten är en ny muscorid, en linjär bis-prenylerad polyoxazol pentapeptid med antimikrobiell aktivitet

    Biochemical characterization of a cyanobactin arginine-N-prenylase from the autumnalamide biosynthetic pathway

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    Cyanobactins are linear and cyclic post-translationally modified peptides. Here we show that the prenyl-D-Arg-containing autum-nalamide A is a member of the cyanobactin family. Biochemical assays demonstrate that the AutF prenyltransferase targets the guanidinium moiety in arginine and homoarginine and is a useful tool for biotechnological applications.Peer reviewe

    Genomics-driven and biochemical approaches to expand the spectrum of natural products

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    Natural products (NPs) derived from secondary metabolism of living organisms play a pivotal role in drug discovery, especially for the treatment of cancer and infectious diseases. Their versatile skeletons are synthesized by different biosynthetic enzymes including polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), terpene cyclases, and hybrid enzymes. Further modifications by tailoring enzymes such as oxidoreductases, cytochrome P450 enzymes, and prenyltransferases (PTs) increase the structural diversity and improve their biological and pharmacological activities. With the advances in genome sequencing and analytical technologies, many strategies have been applied for exploring the promising categories for drug discovery. Due to low or no expression of the majority of biosynthetic gene clusters (BGCs) in the native genomes, it is of great significance to activate such promiscuous BGCs for new drug leads. In recent years, genome mining of novel BGCs has achieved significant progress in NP discovery. In addition to secondary metabolite (SM) exploration in microorganisms, the substrate-based enzymatic reactions have also been proven to be a useful tool for enriching the chemical database. The members of dimethylallyl tryptophan synthase (DMATS) superfamily as important biocatalysts were widely used for structural modification of diverse small molecules. Vast of new prenylated structures have been obtained through chemoenzymatic synthesis. In this thesis, we identified an őĪ-pyrone derivative by genome mining of a NRPS-PKS gene in Penicillium crustosum and obtained a series of prenylated cyclodipeptide (CDP) analogs through chemoenzymatic synthesis of different DMATSs. In the first project, a novel NRPS-PKS hybrid gene pcr10109 from Penicillium crustosum PRB-2 was chosen for detailed investigation by Dr. Jie Fan. She cloned the gene into the expression vector for heterologous expression in Aspergillus nidulans. Analysis of the SMs and structure elucidation proved its responsibility for 4-hydroxy-6-(4-hydroxyphenyl)-2H-pyran-2-one production. Further isotopic feeding experiments revealed its biosynthetic pathway. Para-hydroxybenzoic acid (PHBA) as the precursor and two acetate molecules are assembled for final product formation. To increase the product yield, we fed PHBA in the cultures and the product yield reached a maximum of 51 mg/kg rice culture, which is five-fold higher than that obtained without feeding. This provides another method to increase product formation by supplementing of special substrates. In the second project, we mainly focused on the production of diprenylated cyclo-L-Trp-L-Pro (cWP). At first, we intended to follow the nature¬īs biosynthetic machinery by utilizing C2-PT EchPT1 as the first biocatalyst. However, the C2-prenylated cWP could not be accepted by C4-, C5-, C6-, and C7-PTs for further prenylation. Dr. Lindsay Coby found that the C2-PT EchPT1 can also catalyze prenylations of monoprenylated cyclodipeptides. Then we changed the strategy and firstly obtained the C4-, C5-, C6-, C7-monoprenylated cWP in high product yields. After that, the monoprenylated derivatives were incubated with EchPT1 for the reverse C2-prenylation. Large scale enzyme assays and NMR analysis proved the products to be C2,C4-, C2,C5-, N1,C6-, and C2,C7-diprenylated cWP. This is the first report that EchPT1 can also catalyze the prenylation at the N1 position of the indole ring. In the third project, a similar method was used for the production of prenylated tryptophan-containing dimeric CDPs. We chose different dimeric CDPs and PTs for the enzyme activity test. cyclo-L-Trp-L-Trp (cWW) dimers tetratryptomycins A ‚ąí C were well accepted by EchPT1 in the presence of DMAPP. Tetratryptomycins A and C are better substrates of EchPT1 for prenylation compared with tetratryptomycin B. Compound isolation and NMR analysis determined the products as C2- (and C2¬ī-) prenylated tetratryptomycins, which is consistent with EchPT1-catalyzed reactions. Further kinetic parameter determination revealed that the values are in the range of EchPT1-catalyzed reactions toward most CDPs
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