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1.
Structure ; 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-33275876

RESUMO

ETS family transcription factors of ERG and FLI1 play a key role in oncogenesis of prostate cancer and Ewing sarcoma by binding regulatory DNA sites and interfering with function of other factors. Mithramycin (MTM) is an anti-cancer, DNA binding natural product that functions as a potent antagonist of ERG and FLI1 by an unknown mechanism. We present a series of crystal structures of the DNA binding domain (DBD) of ERG/FLI1 culminating in a structure of a high-order complex of the ERG/FLI1 DBD, transcription factor Runx2, core-binding factor beta (Cbfß), and MTM on a DNA enhancer site, along with supporting DNA binding studies using MTM and its analogues. Taken together, these data provide insight into allosteric mechanisms underlying ERG and FLI1 transactions and their disruption by MTM analogues.

2.
J Med Chem ; 63(22): 14067-14086, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33191745

RESUMO

Mithramycin A (MTM) inhibits the oncogenic transcription factor EWS-FLI1 in Ewing sarcoma, but poor pharmacokinetics (PK) and toxicity limit its clinical use. To address this limitation, we report an efficient MTM 2'-oxime (MTMox) conjugation strategy for rapid MTM diversification. Comparative cytotoxicity assays of 41 MTMox analogues using E-twenty-six (ETS) fusion-dependent and ETS fusion-independent cancer cell lines revealed improved ETS fusion-independent/dependent selectivity indices for select 2'-conjugated analogues as compared to MTM. Luciferase-based reporter assays demonstrated target engagement at low nM concentrations, and molecular assays revealed that analogues inhibit the transcriptional activity of EWS-FLI1. These in vitro screens identified MTMox32E (a Phe-Trp dipeptide-based 2'-conjugate) for in vivo testing. Relative to MTM, MTMox32E displayed an 11-fold increase in plasma exposure and improved efficacy in an Ewing sarcoma xenograft. Importantly, these studies are the first to point to simple C3 aliphatic side-chain modification of MTM as an effective strategy to improve PK.

3.
Folia Microbiol (Praha) ; 65(2): 381-392, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31401763

RESUMO

Citrus black spot (CBS) and post-bloom fruit drop (PFD), caused by Phyllosticta citricarpa and Colletotrichum abscissum, respectively, are two important citrus diseases worldwide. CBS depreciates the market value and prevents exportation of citrus fruits to Europe. PFD under favorable climatic conditions can cause the abscission of flowers, thereby reducing citrus production by 80%. An ecofriendly alternative to control plant diseases is the use of endophytic microorganisms, or secondary metabolites produced by them. Strain LGMF1631, close related to Diaporthe cf. heveae 1, was isolated from the medicinal plant Stryphnodendron adstringens and showed significant antimicrobial activity, in a previous study. In view of the potential presented by strain LGMF1631, and the absence of chemical data for secondary metabolites produced by D. cf. heveae, we decided to characterize the compounds produced by strain LGMF1631. Based on ITS, TEF1, and TUB phylogenetic analysis, strain LGMF1631 was confirmed to belong to D. cf. heveae 1. Chemical assessment of the fungal strain LGMF1631 revealed one new seco-dihydroisocoumarin [cladosporin B (1)] along with six other related, already known dihydroisocoumarin derivatives and one monoterpene [(-)-(1S,2R,3S,4R)-p-menthane-1,2,3-triol (8)]. Among the isolated metabolites, compound 5 drastically reduced the growth of both phytopathogens in vitro and completely inhibited the development of CBS and PFD in citrus fruits and flowers. In addition, compound 5 did not show toxicity against human cancer cell lines or citrus leaves, at concentrations higher than used for the inhibition of the phytopathogens, suggesting the potential use of (-)-(3R,4R)-cis-4-hydroxy-5-methylmellein (5) to control citrus diseases.


Assuntos
Ascomicetos/efeitos dos fármacos , Citrus/microbiologia , Fungicidas Industriais/farmacologia , Isocumarinas/farmacologia , Saccharomycetales/química , Ascomicetos/fisiologia , Colletotrichum/efeitos dos fármacos , Colletotrichum/fisiologia , Fabaceae/microbiologia , Frutas/microbiologia , Fungicidas Industriais/química , Fungicidas Industriais/metabolismo , Isocumarinas/química , Isocumarinas/metabolismo , Filogenia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Saccharomycetales/classificação , Saccharomycetales/genética , Saccharomycetales/isolamento & purificação
4.
Angew Chem Int Ed Engl ; 59(2): 826-832, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31702856

RESUMO

MtmOIV and MtmW catalyze the final two reactions in the mithramycin (MTM) biosynthetic pathway, the Baeyer-Villiger opening of the fourth ring of premithramycin B (PMB), creating the C3 pentyl side chain, strictly followed by reduction of the distal keto group on the new side chain. Unexpectedly this results in a C2 stereoisomer of mithramycin, iso-mithramycin (iso-MTM). Iso-MTM undergoes a non-enzymatic isomerization to MTM catalyzed by Mg2+ ions. Crystal structures of MtmW and its complexes with co-substrate NADPH and PEG, suggest a catalytic mechanism of MtmW. The structures also show that a tetrameric assembly of this enzyme strikingly resembles the ring-shaped ß subunit of a vertebrate ion channel. We show that MtmW and MtmOIV form a complex in the presence of PMB and NADPH, presumably to hand over the unstable MtmOIV product to MtmW, yielding iso-MTM, as a potential self-resistance mechanism against MTM toxicity.

5.
Fitoterapia ; 138: 104273, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31344395

RESUMO

Endophytic fungi have been considered a rich source for bioactive secondary metabolites with novel chemical structures. A high diverse group of endophytes, isolated from different medicinal plants, belongs to the genus Diaporthe. In a previously study performed by our group the crude extract of strain LGMF1583 showed considerable antibacterial activity mainly against Gram-negative bacteria. Based on ITS phylogeny analysis, strain LGMF1583 was identified as belonging to Diaporthe genus and may represent a new species. In the present study, we described the new species Diporthe vochysiae based on multilocus phylogeny analysis and morphological characteristics. The species name refers to the host, from which strain LGMF1583 was isolated, the medicinal plant Vochysia divergens. In view of the biotechnological potential of strain LGMF1583, we have also characterized the secondary metabolites produced by D. vochysiae. Chemical assessment of the D. vochysiae LGMF1583 revealed two new carboxamides, vochysiamides A (1) and B (2), in addition to the known metabolite, 2,5-dihydroxybenzyl alcohol (3). In the biological activity analysis, vochysiamide B (2) displayed considerable antibacterial activity against the Gram-negative bacterium Klebsiella pneumoniae (KPC), a producer of carbapenemases, MIC of 80 µg/mL. Carbapenemases are considered a major antimicrobial resistance threat, and infections caused by KPC have been considered a public health problem worldwide, and new compounds with activity against this bacterium are nowadays even more required.


Assuntos
Amidas/farmacologia , Antibacterianos/farmacologia , Ascomicetos/química , Myrtales/microbiologia , Plantas Medicinais/microbiologia , Amidas/isolamento & purificação , Antibacterianos/isolamento & purificação , Ascomicetos/classificação , Brasil , Linhagem Celular Tumoral , Endófitos/química , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Estrutura Molecular , Filogenia
6.
Medchemcomm ; 10(5): 735-741, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31191864

RESUMO

An aureolic acid natural product mithramycin (MTM) has been known for its potent antineoplastic properties. MTM inhibits cell growth by binding in the minor groove of double-stranded DNA as a dimer, in which the two molecules of MTM are coordinated to each other through a divalent metal ion. A crystal structure of an MTM analogue, MTM SA-Phe, in the active metal ion-coordinated dimeric form demonstrates how the stereochemical features of MTM define the helicity of the dimeric scaffold for its binding to a right-handed DNA double helix. We also show crystallographically and biochemically that MTM, but not MTM SA-Phe, can be inactivated by boric acid through formation of a large macrocyclic species, in which two molecules of MTM are crosslinked to each other through 3-side chain-boron-sugar intermolecular bonds. We discuss these structural and biochemical properties in the context of MTM biosynthesis and the design of MTM analogues as anticancer therapeutics.

7.
Biomed Chromatogr ; 33(8): e4544, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30927450

RESUMO

Mithramycin (MTM) has potent anticancer activity, but severe toxicities restrict its clinical use. Semi-synthetic approaches have yielded novel MTM analogs with potentially lower toxicity and similar efficacy. In an effort to transition these analogs into in vivo models, a bioanalytical method was developed for their quantification in mouse plasma. Here we present the validation of the method for the quantitation of mithramycin SA-tryptophan (MTMSA-Trp) as well as the applicability of the methodology for assaying additional analogs, including MTM, mithramycin SK (MTMSK) and mithramycin SA-phenylalanine (MTMSA-Phe) with run times of 6 min. Assay linearity ranged from 5 to 100 ng/mL. Accuracies of calibration standards and quality control samples were within 15% of nominal with precision variability of <20%. MTMSA-Trp was stable for 30 days at -80°C and for at least three freeze-thaw cycles. Methanol (-80°C) extraction afforded 92% of MTMSA-Trp from plasma. Calibration curves for MTM and analogs were also linear from ≤5 to 100 ng/mL. This versatile method was used to quantitate MTM analogs in plasma samples collected during preclinical pharmacokinetic studies.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Plicamicina/análogos & derivados , Plicamicina/sangue , Animais , Antibióticos Antineoplásicos , Estabilidade de Medicamentos , Feminino , Limite de Detecção , Modelos Lineares , Camundongos , Plicamicina/química , Plicamicina/farmacocinética , Reprodutibilidade dos Testes
8.
J Antibiot (Tokyo) ; 72(5): 306-310, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30792517

RESUMO

The isolation and structure elucidation of one new fungal metabolite, phenguignardic acid butyl ester (1a), and four previously reported metabolites (1b, 2a, 3-4) from the citrus phytopathogen Phyllosticta citricarpa LGMF06 are described. The new dioxolanone phenguignardic acid butyl ester (1a) had low phytotoxic activity in citrus leaves and fruits (at dose of 100 µg), and its importance as virulence factor in citrus black spot disease needs to be further addressed. Beside the phytotoxic analysis, we also evaluated the antibacterial (against methicillin sensitive and resistant Staphylococcus aureus) and cytotoxic (A549 non-small cell lung cancer, PC3 prostate cancer and HEL 299 normal epithelial lung) activities of the isolated compounds, which revealed that compounds 1a, 1b and 2a were responsible for the antibacterial activity of this strain.


Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Ascomicetos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citrus/microbiologia , Humanos , Estrutura Molecular , Doenças das Plantas/microbiologia
9.
Folia Microbiol (Praha) ; 64(3): 453-460, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30565048

RESUMO

The citrus black spot (CBS), caused by Phyllosticta citricarpa, is one of the most important citrus diseases in subtropical regions of Africa, Asia, Oceania, and the Americas, and fruits with CBS lesions are still subject to quarantine regulations in the European Union. Despite the high application of fungicides, the disease remains present in the citrus crops of Central and South America. In order to find alternatives to help control CBS and reduce the use of fungicides, we explored the antifungal potential of endophytic actinomycetes isolated from the Brazilian medicinal plant Vochysia divergens found in the Pantanal biome. Two different culture media and temperatures were selected to identify the most efficient conditions for the production of active secondary metabolites. The metabolites produced by strain Microbacterium sp. LGMB471 cultured in SG medium at 36 °C considerably inhibited the development of P. citricarpa. Three isoflavones and five diketopiperazines were identified, and the compounds 7-O-ß-D-glucosyl-genistein and 7-O-ß-D-glucosyl-daidzein showed high activity against P. citricarpa, with the MIC of 33 µg/mL and inhibited the production of asexual spores of P. citricarpa on leaves and citrus fruits. Compounds that inhibit conidia formation may be a promising alternative to reduce the use of fungicides in the control of CBS lesions, especially in regions where sexual reproduction does not occur, as in the USA. Our data suggest the use of Microbacterium sp. LGMB471 or its metabolites as an ecological alternative to be used in association with the fungicides for the control of CBS disease.


Assuntos
Actinomycetales/química , Ascomicetos/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Ásia , Brasil , Citrus/microbiologia , Meios de Cultura , Dicetopiperazinas/isolamento & purificação , Dicetopiperazinas/farmacologia , Fungicidas Industriais/isolamento & purificação , Isoflavonas/isolamento & purificação , Isoflavonas/farmacologia , Testes de Sensibilidade Microbiana , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Metabolismo Secundário , Esporos Fúngicos/efeitos dos fármacos , Estados Unidos
10.
J Med Chem ; 61(17): 8001-8016, 2018 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-30114371

RESUMO

Mithramycin A (1) was identified as the top potential inhibitor of the aberrant ETS transcription factor EWS-FLI1, which causes Ewing sarcoma. Unfortunately, 1 has a narrow therapeutic window, compelling us to seek less toxic and more selective analogues. Here, we used MTMSA (2) to generate analogues via peptide coupling and fragment-based drug development strategies. Cytotoxicity assays in ETS and non-ETS dependent cell lines identified two dipeptide analogues, 60 and 61, with 19.1- and 15.6-fold selectivity, respectively, compared to 1.5-fold for 1. Importantly, the cytotoxicity of 60 and 61 is <100 nM in ETS cells. Molecular assays demonstrated the inhibitory capacity of these analogues against EWS-FLI1 mediated transcription in Ewing sarcoma. Structural analysis shows that positioning the tryptophan residue in a distal position improves selectivity, presumably via interaction with the ETS transcription factor. Thus, these analogues may present new ways to target transcription factors for clinical use.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Desenvolvimento de Medicamentos , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Plicamicina/análogos & derivados , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ets/antagonistas & inibidores , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Sarcoma de Ewing/tratamento farmacológico , Antibióticos Antineoplásicos/química , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Estrutura Molecular , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Células Tumorais Cultivadas
11.
Artigo em Inglês | MEDLINE | ID: mdl-29735559

RESUMO

Muraymycins are antibacterial natural products from Streptomyces spp. that inhibit translocase I (MraY), which is involved in cell wall biosynthesis. Structurally, muraymycins consist of a 5'-C-glycyluridine (GlyU) appended to a 5″-amino-5″-deoxyribose (ADR), forming a disaccharide core that is found in several peptidyl nucleoside inhibitors of MraY. For muraymycins, the GlyU-ADR disaccharide is further modified with an aminopropyl-linked peptide to generate the simplest structures, annotated as the muraymycin D series. Two enzymes encoded in the muraymycin biosynthetic gene cluster, Mur29 and Mur28, were functionally assigned in vitro as a Mg·ATP-dependent nucleotidyltransferase and a Mg·ATP-dependent phosphotransferase, respectively, both modifying the 3″-OH of the disaccharide. Biochemical characterization revealed that both enzymes can utilize several nucleotide donors as cosubstrates and the acceptor substrate muraymycin also behaves as an inhibitor. Single-substrate kinetic analyses revealed that Mur28 preferentially phosphorylates a synthetic GlyU-ADR disaccharide, a hypothetical biosynthetic precursor of muraymycins, while Mur29 preferentially adenylates the D series of muraymycins. The adenylated or phosphorylated products have significantly reduced (170-fold and 51-fold, respectively) MraY inhibitory activities and reduced antibacterial activities, compared with the respective unmodified muraymycins. The results are consistent with Mur29-catalyzed adenylation and Mur28-catalyzed phosphorylation serving as complementary self-resistance mechanisms, with a distinct temporal order during muraymycin biosynthesis.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Nucleosídeos/biossíntese , Nucleosídeos/química , Nucleotidiltransferases/química , Peptídeos/química , Fosfotransferases/química , Streptomyces/metabolismo , Transferases/antagonistas & inibidores , Antibacterianos/biossíntese , Nucleotídeos/biossíntese , Nucleotidiltransferases/genética , Fosforilação , Fosfotransferases/genética
12.
Folia Microbiol (Praha) ; 63(4): 499-505, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29497981

RESUMO

Antibiotic-resistant bacteria have been observed with increasing frequency over the past decades, driving the search for new drugs and stimulating the interest in natural products sources. Endophytic fungi from medicinal plants represent a great source of novel bioactive compounds useful to pharmaceutical and agronomical purposes. Diaporthe terebinthifolii is an endophytic species isolated from Schinus terebinthifolius, a plant used in popular medicine for several health problems. The strain D. terebinthifolii LGMF907 was previously reported by our group to produce secondary metabolites with biological activity against phytopathogens. Based on these data, strain LGMF907 was chosen for bioprospecting against microorganisms of clinical importance and for characterization of major secondary metabolites. In this study, different culture conditions were evaluated and the biological activity of this strain was expanded. The crude extracts demonstrated high antibacterial activity against Escherichia coli, Micrococcus luteus, Saccharomyces cerevisiae, methicillin-sensitive Staphylococcus aureus, and methicillin-resistant S. aureus. The compounds diaporthin and orthosporin were characterized and also showed activity against the clinical microorganisms evaluated. This study discloses the first isolation of diaporthin and orthosporin from D. terebinthifolii, and revealed the potential of this endophytic fungus to produce secondary metabolites with antimicrobial activity.


Assuntos
Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Bioprospecção , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomycetales/metabolismo , Anti-Infecciosos/química , Meios de Cultura , Endófitos/química , Endófitos/metabolismo , Escherichia coli/efeitos dos fármacos , Fermentação , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium/efeitos dos fármacos , Saccharomycetales/química , Staphylococcus aureus/efeitos dos fármacos
13.
Sci Rep ; 8(1): 3122, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29449610

RESUMO

Microorganisms associated with plants are highly diverse and can produce a large number of secondary metabolites, with antimicrobial, anti-parasitic and cytotoxic activities. We are particularly interested in exploring endophytes from medicinal plants found in the Pantanal, a unique and widely unexplored wetland in Brazil. In a bio-prospecting study, strains LGMF1213 and LGMF1215 were isolated as endophytes from Vochysia divergens, and by morphological and molecular phylogenetic analyses were characterized as Phaeophleospora vochysiae sp. nov. The chemical assessment of this species reveals three major compounds with high biological activity, cercoscosporin (1), isocercosporin (2) and the new compound 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone (3). Besides the isolation of P. vochysiae as endophyte, the production of cercosporin compounds suggest that under specific conditions this species causes leaf spots, and may turn into a pathogen, since leaf spots are commonly caused by species of Cercospora that produce related compounds. In addition, the new compound 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone showed considerable antimicrobial activity and low cytotoxicity, which needs further exploration.


Assuntos
Bactérias/isolamento & purificação , Myrtales/metabolismo , Myrtales/microbiologia , Antibacterianos/metabolismo , Anti-Infecciosos/metabolismo , Ascomicetos/metabolismo , Bactérias/metabolismo , Brasil , Endófitos/metabolismo , Magnoliopsida/metabolismo , Magnoliopsida/microbiologia , Testes de Sensibilidade Microbiana , Perileno/análogos & derivados , Filogenia , Plantas Medicinais/metabolismo
14.
Front Microbiol ; 8: 1642, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28932210

RESUMO

Endophytic actinomycetes from medicinal plants produce a wide diversity of secondary metabolites (SM). However, to date, the knowledge about endophytes from Brazil remains scarce. Thus, we analyzed the antimicrobial potential of 10 actinomycetes isolated from the medicinal plant Vochysia divergens located in the Pantanal sul-mato-grossense, an unexplored wetland in Brazil. Strains were classified as belonging to the Aeromicrobium, Actinomadura, Microbacterium, Microbispora, Micrococcus, Sphaerisporangium, Streptomyces, and Williamsia genera, through morphological and 16S rRNA phylogenetic analyzes. A susceptibility analysis demonstrated that the strains were largely resistant to the antibiotics oxacillin and nalidixic acid. Additionally, different culture media (SG and R5A), and temperatures (28 and 36°C) were evaluated to select the best culture conditions to produce the active SM. All conditions were analyzed for active metabolites, and the best antibacterial activity was observed from metabolites produced with SG medium at 36°C. The LGMB491 (close related to Aeromicrobium ponti) extract showed the highest activity against methicillin-resistant Staphylococcus aureus (MRSA), with a MIC of 0.04 mg/mL, and it was selected for SM identification. Strain LGMB491 produced 1-acetyl-ß-carboline (1), indole-3-carbaldehyde (2), 3-(hydroxyacetyl)-indole (4), brevianamide F (5), and cyclo-(L-Pro-L-Phe) (6) as major compounds with antibacterial activity. In this study, we add to the knowledge about the endophytic community from the medicinal plant V. divergens and report the isolation of rare actinomycetes that produce highly active metabolites.

15.
ACS Chem Biol ; 12(10): 2529-2534, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28892347

RESUMO

Glycosyltransferases are key enzymes involved in the biosynthesis of valuable natural products providing an excellent drug-tailoring tool. Herein, we report the identification of two cooperative glycosyltransferases from the sqn gene cluster directing the biosynthesis of saquayamycins in Streptomyces sp. KY40-1: SqnG1 and SqnG2. Gene inactivation of sqnG1 leads to 50-fold decrease in saquayamycin production, while inactivation of sqnG2 leads to complete production loss, suggesting that SqnG2 acts as dual O- and C-glycosyltransferase. Gene inactivation of a third putative glycosyltransferase-encoding gene, sqnG3, does not affect saquayamycin production in a major way, suggesting that SqnG3 has no or a supportive role in glycosylation. The data indicate that SqnG1 and SqnG2 are solely and possibly cooperatively responsible for the sugar diversity observed in saquayamycins 1-7. This is the first evidence of a glycosyltransferase system showing codependence to achieve dual O- and C-glycosyltransferase activity, utilizing NDP-activated d-olivose, l-rhodinose, as well as an unusual amino sugar, presumably 3,6-dideoxy-l-idosamine.


Assuntos
Antraquinonas/metabolismo , Carboidratos/biossíntese , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glicosiltransferases/metabolismo , Streptomyces/enzimologia , Configuração de Carboidratos , Glicosiltransferases/genética
16.
mBio ; 8(5)2017 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-28951478

RESUMO

Glycosylation is a universal strategy to posttranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial translation elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-ß-l-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design.IMPORTANCE The structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP not only provides the first molecular insights into arginine glycosylation but also lays the basis for targeted-inhibitor design against Pseudomonas aeruginosa infection.


Assuntos
Arginina/metabolismo , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Pseudomonas putida/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Modelos Moleculares , Fatores de Alongamento de Peptídeos/genética , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Pseudomonas putida/química , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Ribossomos/genética
17.
Cell Chem Biol ; 24(7): 881-891.e4, 2017 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-28712746

RESUMO

Bacterial aromatic polyketides are a group of natural products synthesized by polyketide synthases (PKSs) that show diverse structures and biological activities. They are structurally subclassified into linear, angular, and discoid aromatic polyketides, the formation of which is commonly determined by the shaping and folding of the poly-ß-keto intermediates under the concerted actions of the minimal PKSs, cyclases and ketoreductases. Murayaquinone, found in several streptomycetes, possesses an unusual tricyclic angular aromatic polyketide core containing a 9,10-phenanthraquinone. In this study, genes essential for murayaquinone biosynthesis were identified, and a linear anthraoxirene intermediate was discovered. A unique biosynthetic model for the angular aromatic polyketide formation was discovered and confirmed through in vivo and in vitro studies. Three oxidoreductases, MrqO3, MrqO6, and MrqO7, were identified to catalyze the conversion of the linear aromatic polyketide intermediate into the final angularly arranged framework, which exemplifies a novel strategy for the biosynthesis of angular aromatic polyketides.


Assuntos
Oxirredutases/metabolismo , Fenantrenos/metabolismo , Policetídeos/metabolismo , Quinonas/metabolismo , Antracenos/química , Antracenos/metabolismo , Biocatálise , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Família Multigênica , Oxirredutases/genética , Fenantrenos/química , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/química , Quinonas/química , Streptomyces/química , Streptomyces/metabolismo , Streptomyces lividans/metabolismo
18.
J Nat Prod ; 80(4): 1141-1149, 2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28358212

RESUMO

The structures of 12 new "enantiomeric"-like abyssomicin metabolites (abyssomicins M-X) from Streptomyces sp. LC-6-2 are reported. Of this set, the abyssomicin W (11) contains an unprecedented 8/6/6/6 tetracyclic core, while the bicyclic abyssomicin X (12) represents the first reported naturally occurring linear spirotetronate. Metabolite structures were determined based on spectroscopic data and X-ray crystallography, and Streptomyces sp. LC-6-2 genome sequencing also revealed the corresponding putative biosynthetic gene cluster.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/isolamento & purificação , Compostos de Espiro/isolamento & purificação , Streptomyces/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Carvão Mineral , Cristalografia por Raios X , Conformação Molecular , Estrutura Molecular , Família Multigênica , Ressonância Magnética Nuclear Biomolecular , Compostos de Espiro/química , Streptomyces/genética
19.
Free Radic Biol Med ; 106: 134-147, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28189848

RESUMO

Landomycin E (LE) is an angucycline antibiotic produced by Streptomyces globisporus. Previously, we have shown a broad anticancer activity of LE which is, in contrast to the structurally related and clinically used anthracycline doxorubicin (Dx), only mildly affected by multidrug resistance-mediated drug efflux. In the present study, cellular and molecular mechanisms underlying the anticancer activity of landomycin E towards Jurkat T-cell leukemia cells were dissected focusing on the involvement of radical oxygen species (ROS). LE-induced apoptosis distinctly differed in several aspects from the one induced by Dx. Rapid generation of both extracellular and cell-derived hydrogen peroxide already at one hour drug exposure was observed in case of LE but not found before 24h for Dx. In contrast, Dx but not LE induced production of superoxide radicals. Mitochondrial damage, as revealed by JC-1 staining, was weakly enhanced already at 3h LE treatment and increased significantly with time. Accordingly, activation of the intrinsic apoptosis pathway initiator caspase-9 was not detectable before 12h exposure. In contrast, cleavage of the down-stream caspase substrate PARP-1 was clearly induced already at the three hour time point. Out of all caspases tested, only activation of effector caspase-7 was induced at this early time points paralleling the LE-induced oxidative burst. Accordingly, this massive cleavage of caspase-7 at early time points was inhibitable by the radical scavenger N-acetylcysteine (NAC). Additionally, only simultaneous inhibition of multiple caspases reduced LE-induced apoptosis. Specific scavengers of both H2O2 and OH• effectively decreased LE-induced ROS production, but only partially inhibited LE-induced apoptosis. In contrast, NAC efficiently blocked both parameters. Summarizing, rapid H2O2 generation and a complex caspase activation pattern contribute to the antileukemic effects of LE. As superoxide generation is considered as the main cardiotoxic mechanism of Dx, LE might represent a better tolerable drug candidate for further (pre)clinical development.


Assuntos
Aminoglicosídeos/administração & dosagem , Antibióticos Antineoplásicos/administração & dosagem , Células Jurkat/metabolismo , Leucemia/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Acetilcisteína/administração & dosagem , Apoptose/efeitos dos fármacos , Caspase 7/metabolismo , Caspase 9/metabolismo , Doxorrubicina/administração & dosagem , Humanos , Peróxido de Hidrogênio/toxicidade , Células Jurkat/efeitos dos fármacos , Células Jurkat/patologia , Leucemia/metabolismo , Leucemia/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Streptomyces/química , Superóxidos/toxicidade
20.
Org Lett ; 19(3): 540-543, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-28102686

RESUMO

Early acting cyclases play critical roles in programming the polyketide biosynthesis toward certain, distinguished scaffolds. Starting from acetyl-CoA and malonyl-CoA, a one-pot enzymatic total synthesis of an anthracyclinone scaffold, presteffimycinone, was achieved by mixing polyketide synthase (PKS) and early post-PKS enzymes from the biosynthetic pathways of three different types of type II-PKS driven anticancer antibiotics, namely, the mithramycin (aureolic acid-type), gilvocarcin (rearranged angucycline-type), and steffimycin (anthracycline) pathways.


Assuntos
Antraciclinas/metabolismo , Antibacterianos , Estrutura Molecular , Policetídeo Sintases , Streptomyces
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