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1.
J Biol Inorg Chem ; 25(6): 875-885, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32719971

RESUMO

L-ascorbic acid 2-phosphate magnesium (APMg) salt is a vitamin C derivative frequently used as a raw material in cell and tissue therapy. APMg is not only used as a replacement of the unstable ascorbate, but also shows additional cell-biological functionalities. However, its unknown structural characteristics hamper the mechanistic elucidation of its biological role. Therefore, different techniques were applied for APMg structure characterization. Firstly, the stoichiometric composition was characterized by its solvent, ligand and magnesium content. No crystals of APMg could be obtained; however, a single crystal of APNa, the sodium salt of l-ascorbic acid 2-phosphate, was successfully obtained and its crystal structure was elucidated. FT-IR was applied to further clarify the structure of solid APMg. Finally, the structure of APMg in aqueous solution was explored by potentiometric titration as well as FT-IR.

2.
New Phytol ; 2020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-33616937

RESUMO

Plant roots are specialized belowground organs that spatiotemporally shape their development in function of varying soil conditions. This root plasticity relies on intricate molecular networks driven by phytohormones, such as auxin and jasmonate (JA). Loss-of-function of the NOVEL INTERACTOR OF JAZ (NINJA), a core component of the JA signaling pathway, leads to enhanced triterpene biosynthesis, in particular of the thalianol gene cluster, in Arabidopsis thaliana roots. We have investigated the biological role of thalianol and its derivatives by focusing on Thalianol Synthase (THAS) and Thalianol Acyltransferase 2 (THAA2), two thalianol cluster genes that are upregulated in the roots of ninja mutant plants. THAS and THAA2 activity was investigated in yeast, and metabolite and phenotype profiling of thas and thaa2 loss-of-function plants was carried out. THAA2 was shown to be responsible for the acetylation of thalianol and its derivatives, both in yeast and in planta. In addition, THAS and THAA2 activity was shown to modulate root development. Our results indicate that the thalianol pathway is not only controlled by phytohormonal cues, but also may modulate phytohormonal action itself, thereby affecting root development and interaction with the environment.

3.
Chembiochem ; 20(19): 2513-2518, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31062451

RESUMO

Proline is often found as a turn inducer in peptide or protein domains. Exploitation of its restricted conformational freedom led to the development of the d-Pro-l-Pro (corresponding to (R)-Pro-(S)-Pro) segment as a "templating" unit, frequently used in the design of ß-hairpin peptidomimetics, in which conformational stability is, however, inherently linked to the cis-trans isomerization of the prolyl amide bonds. In this context, the stereoelectronic properties of the CF3 group can aid in conformational control. Herein, the impact of α-trifluoromethylated proline analogues is examined for the design of enhanced ß-turn inducers. A theoretical conformational study permitted the dipeptide (R)-Pro-(R)-TfmOxa (TfmOxa: 2-trifluoromethyloxazolidine-2-carboxylic acid) to be selected as a template with an increased trans-cis rotational energy barrier. NMR spectroscopic analysis of the Ac-(R)-Pro-(R)-TfmOxa-(S)-Val-OtBu ß-turn model, obtained through an original synthetic pathway, validated the prevalence of a major trans-trans conformer and indicated the presence of an internal hydrogen bond. Altogether, it was shown that the (R)-Pro-(R)-TfmOxa template fulfilled all crucial ß-turn-inducer criteria.


Assuntos
Ácidos Carboxílicos/química , Dipeptídeos/química , Oxazolona/análogos & derivados , Prolina/análogos & derivados , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Metilação , Oxazolona/química , Conformação Proteica , Termodinâmica
4.
Nucleic Acids Res ; 46(22): 11671-11686, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30418582

RESUMO

Previous investigations of the impact of an imidazole-tethered thymidine in synthetic DNA duplexes, monitored using UV and NMR spectroscopy, revealed a base context dependent increase in thermal stability of these duplexes and a striking correlation with the imidazolium pKa. Unrestrained molecular dynamics (MD) simulations demonstrated the existence of a hydrogen bond between the imidazolium and the Hoogsteen side of a nearby guanosine which, together with electrostatic interactions, form the basis of the so-called pKa-motif responsible for these duplex-stabilizing and pKa-modulating properties. Here, the robustness and utility of this pKa-motif was explored by introducing multiple imidazole-tethered thymidines at different positions on the same dsDNA duplex. For all constructs, sequence based expectations as to pKa-motif formation were supported by MD simulations and experimentally validated using NOESY. Based on the analysis of the pKa values and melting temperatures, guidelines are formulated to assist in the rational design of oligonucleotides modified with imidazolium-tethered thymidines for increased thermal stability that should be generally applicable, as demonstrated through a triply modified construct. In addition, a proof-of-principle study demonstrating enhanced stability of the l-argininamide binding aptamer modified with an imidazole-tethered thymidine in the presence and absence of ligand, demonstrates its potential for the design of more stable aptamers.


Assuntos
Aptâmeros de Nucleotídeos/química , Imidazóis/química , Oligodesoxirribonucleotídeos/química , Timidina/análogos & derivados , Uridina/análogos & derivados , Aptâmeros de Nucleotídeos/síntese química , Arginina/análogos & derivados , Arginina/química , Sequência de Bases , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Eletricidade Estática , Termodinâmica , Raios Ultravioleta
5.
J Exp Bot ; 68(15): 4185-4203, 2017 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-28922768

RESUMO

The volatile two-carbon hormone ethylene acts in concert with an array of signals to affect etiolated seedling development. From a chemical screen, we isolated a quinoline carboxamide designated ACCERBATIN (AEX) that exacerbates the 1-aminocyclopropane-1-carboxylic acid-induced triple response, typical for ethylene-treated seedlings in darkness. Phenotypic analyses revealed distinct AEX effects including inhibition of root hair development and shortening of the root meristem. Mutant analysis and reporter studies further suggested that AEX most probably acts in parallel to ethylene signaling. We demonstrated that AEX functions at the intersection of auxin metabolism and reactive oxygen species (ROS) homeostasis. AEX inhibited auxin efflux in BY-2 cells and promoted indole-3-acetic acid (IAA) oxidation in the shoot apical meristem and cotyledons of etiolated seedlings. Gene expression studies and superoxide/hydrogen peroxide staining further revealed that the disrupted auxin homeostasis was accompanied by oxidative stress. Interestingly, in light conditions, AEX exhibited properties reminiscent of the quinoline carboxylate-type auxin-like herbicides. We propose that AEX interferes with auxin transport from its major biosynthesis sites, either as a direct consequence of poor basipetal transport from the shoot meristematic region, or indirectly, through excessive IAA oxidation and ROS accumulation. Further investigation of AEX can provide new insights into the mechanisms connecting auxin and ROS homeostasis in plant development and provide useful tools to study auxin-type herbicides.


Assuntos
Aminoácidos Cíclicos/metabolismo , Arabidopsis/metabolismo , Herbicidas/química , Ácidos Indolacéticos/metabolismo , Quinolonas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Etilenos/metabolismo , Expressão Gênica , Homeostase , Plântula/metabolismo
6.
Nat Commun ; 8: 14153, 2017 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-28165039

RESUMO

Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Evolução Molecular , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/metabolismo , Triterpenos/metabolismo , Biodiversidade , Sistema Enzimático do Citocromo P-450/genética , Filogenia , Proteínas de Plantas/genética
7.
Biotechnol Bioeng ; 113(12): 2644-2651, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27317616

RESUMO

Bola-amphiphilic surfactants are molecules with fascinating properties. Their unique configuration consisting of a long hydrophobic spacer connecting two hydrophilic entities renders the molecule more water soluble than the average surfactant, but still allows formation of supramolecular structures. These properties make them extremely suitable for applications in in nanotechnology, electronics, and gene and drug delivery. In general, these compounds are obtained by chemical synthesis. We report here an efficient microbial production process for the fully green synthesis of bolaform surfactants. A sophorolipid-producing Starmerella bombicola yeast strain was disabled in its sophorolipid acetyltransferase and lactone esterase, which should logically result in synthesis of non-acetylated acidic sophorolipids; molecules with the classic amphiphilic structure. Surprisingly, also bolaform glycolipids were obtained, with an additional sophorose linked to the free carboxyl end of the acidic sophorolipids as confirmed by MS and NMR analysis. The obtained titers of 27.7 g/L total product are comparable to wild type values, and the novel molecules account for at least 74% of this. Bola-amphiphile biosynthesis proved to be attributed to the promiscuous activity of both UDP-glucosyltransferases UGTA1 and UGTB1 from the core sophorolipid pathway, displaying activity toward non-acetylated intermediates. The absence of acetyl groups seems to trigger formation of bolaform compounds starting from acidic sophorolipids. Hence, wild type S. bombicola produces these compounds only at marginal amounts in general not reaching detection limits. We created a strain knocked-out in its sophorolipid acetyltransferase and lactone esterase able to produce these novel compounds in economical relevant amounts, opening doors for the application of biological-derived bolaform structures. Biotechnol. Bioeng. 2016;113: 2644-2651. © 2016 Wiley Periodicals, Inc.


Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Melhoramento Genético/métodos , Tensoativos/metabolismo , Acetiltransferases/genética , Ascomicetos/classificação , Esterases/genética , Técnicas de Silenciamento de Genes/métodos , Química Verde/métodos , Especificidade da Espécie , Tensoativos/isolamento & purificação
8.
Fungal Genet Biol ; 81: 88-97, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26051490

RESUMO

The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS-non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange-red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.


Assuntos
Aspergillus flavus/enzimologia , Aspergillus flavus/metabolismo , Família Multigênica , Peptídeo Sintases/metabolismo , Policetídeo Sintases/metabolismo , Piridonas/metabolismo , Aspergillus flavus/genética , Regulação Fúngica da Expressão Gênica , Peptídeo Sintases/genética , Pigmentos Biológicos/análise , Policetídeo Sintases/genética , Metabolismo Secundário
9.
Nucleic Acids Res ; 43(1): 51-62, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25520197

RESUMO

The predictable 3D structure of double-stranded DNA renders it ideally suited as a template for the bottom-up design of functionalized nucleic acid-based active sites. We here explore the use of a 14mer DNA duplex as a scaffold for the precise and predictable positioning of catalytic functionalities. Given the ubiquitous participation of the histidine-based imidazole group in protein recognition and catalysis events, single histidine-like modified duplexes were investigated. Tethering histamine to the C5 of the thymine base via an amide bond, allows the flexible positioning of the imidazole function in the major groove. The mutual interactions between the imidazole and the duplex and its influence on the imidazolium pKaH are investigated by placing a single modified thymine at four different positions in the center of the 14mer double helix. Using NMR and unrestrained molecular dynamics, a structural motif involving the formation of a hydrogen bond between the imidazole and the Hoogsteen side of the guanine bases of two neighboring GC base pairs is established. The motif contributes to a stabilization against thermal melting of 6°C and is key in modulating the pKaH of the imidazolium group. The general features, prerequisites and generic character of the new pKaH-regulating motif are described.


Assuntos
DNA/química , Imidazóis/química , Pareamento Incorreto de Bases , Sequência de Bases , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Timina/química
10.
Proc Natl Acad Sci U S A ; 111(4): 1634-9, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24434554

RESUMO

The saikosaponins comprise oleanane- and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1, encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16α hydroxylation of oleanane- and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated ß-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.


Assuntos
Bupleurum/enzimologia , Técnicas de Química Combinatória , Saccharomyces cerevisiae/metabolismo , Sapogeninas/metabolismo , Saponinas/biossíntese , Esteroide 16-alfa-Hidroxilase/genética , Meios de Cultura , Hidroxilação , Dados de Sequência Molecular , RNA Mensageiro/genética
11.
Chem Commun (Camb) ; 49(28): 2927-9, 2013 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-23460127

RESUMO

A general method for the facile and versatile decoration of peptides is proposed exploiting furan based cycloaddition and electrophilic aromatic substitution reactions. Given the commercial availability of furylalanine derivatives for peptide synthesis, the current work significantly enlarges the toolbox of available methodologies for site specific labeling and conjugation of peptide probes.


Assuntos
Furanos/química , Oligopeptídeos/química , Sequência de Aminoácidos , Coloração e Rotulagem
12.
Nat Chem Biol ; 8(6): 583-9, 2012 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-22561410

RESUMO

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Carbocianinas/química , Membrana Celular/metabolismo , Colestanóis/metabolismo , Endocitose , Corantes Fluorescentes/química , Proteínas Quinases/metabolismo , Transdução de Sinais , Arabidopsis/enzimologia , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Brassinosteroides/química , Brassinosteroides/metabolismo , Membrana Celular/ultraestrutura , Colestanóis/química , Relação Dose-Resposta a Droga , Endossomos/enzimologia , Endossomos/metabolismo , Endossomos/ultraestrutura , Proteínas de Fluorescência Verde/genética , Cinética , Meristema/enzimologia , Meristema/metabolismo , Meristema/ultraestrutura , Microscopia Confocal , Estrutura Molecular , Reguladores de Crescimento de Planta , Proteínas Quinases/genética , Transporte Proteico , Plântula/enzimologia , Plântula/metabolismo , Plântula/ultraestrutura , Vacúolos/enzimologia , Vacúolos/metabolismo , Vacúolos/ultraestrutura
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