RESUMO
Conformational characterization of single α- and ß-epimers of galactosamine in solution still remains an intriguing task because of their flexibility and ability to interconvert. This difficulty was circumvented by recording several "snapshots" of the epimerization process by means of fast ESI vaporization of a galactosamine·HCl sample solution at different times. Consequently, the so generated gaseous mixtures were spectroscopically investigated and the specific conformational features of both α- and ß-epimers were assigned, despite the overlapping of several IR signals. Interestingly, from a comparison with time-resolved 1H-NMR data obtained for the same solutions, the catalyzing effect of the applied ESI technique in the anomerization process clearly emerges. Finally, the experimental data were supported using both the Density Functional Theory (DFT) and Block-Localized Wavefunction (BLW) approaches: the latter method was applied here for the first time for the investigation of charged species.
RESUMO
BACKGROUND: The construction of efficient cell factories for the production of metabolites requires the rational improvement/engineering of the metabolism of microorganisms. The subject of this paper is directed towards the quantitative understanding of the respiratory/fermentative Kluyveromyces lactis yeast metabolism and its rag8 casein kinase mutant, taken as a model for all rag gene mutations. METHODS: (13)C NMR spectroscopy and [1,2-(13)C2]glucose were used as metabolic stable-isotope tracer to define the metabolic profiling of a K. lactis yeast and its derivative mutants. RESULTS: Rag8 showed a decrease of all (13)C glutamate fractional enrichments, except for [4-(13)C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribution of a [4-(13)C]ketoglutarate pool not originating from mitochondria were suggested. (13)C lysine enrichments confirmed the presence of two compartmentalized α-ketoglutarate (α-KG) pools participating to glutamate and lysine synthesis. Moreover, an increased transaldolase, as compared to transketolase activity, was observed in the rag8 mutant by (13)C NMR isotopomer analysis of alanine. CONCLUSIONS: (13)C NMR-based isotopomer analysis showed the existence of different α-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, (13)C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalized α-KG pool. GENERAL SIGNIFICANCE: A compartmentalization of the α-KG pools involved in lysine biosynthesis has been revealed for the first time in K. lactis. Given its great impact in metabolic engineering field, its existence should be validated/compared with other yeasts and/or fungal species.
Assuntos
Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Ácido Glutâmico/biossíntese , Ácidos Cetoglutáricos/metabolismo , Kluyveromyces/metabolismo , Lisina/biossínteseRESUMO
Patulin is a mycotoxin that contaminates pome fruits and derived products worldwide. Basidiomycete yeasts belonging to the subphylum Pucciniomycotina have been identified to have the ability to degrade this molecule efficiently and have been explored through different approaches to understand this degradation process. In this study, Sporobolomyces sp. strain IAM 13481 was found to be able to degrade patulin to form two different breakdown products, desoxypatulinic acid and (Z)-ascladiol. To gain insight into the genetic basis of tolerance and degradation of patulin, more than 3,000 transfer DNA (T-DNA) insertional mutants were generated in strain IAM 13481 and screened for the inability to degrade patulin using a bioassay based on the sensitivity of Escherichia coli to patulin. Thirteen mutants showing reduced growth in the presence of patulin were isolated and further characterized. Genes disrupted in patulin-sensitive mutants included homologs of Saccharomyces cerevisiae YCK2, PAC2, DAL5, and VPS8. The patulin-sensitive mutants also exhibited hypersensitivity to reactive oxygen species as well as genotoxic and cell wall-destabilizing agents, suggesting that the inactivated genes are essential for tolerating and overcoming the initial toxicity of patulin. These results support a model whereby patulin degradation occurs through a multistep process that includes an initial tolerance to patulin that utilizes processes common to other external stresses, followed by two separate pathways for degradation.
Assuntos
Basidiomycota/genética , Proteínas Fúngicas/genética , Patulina/metabolismo , Saccharomyces cerevisiae/genética , Acetatos/metabolismo , Sequência de Bases , Basidiomycota/efeitos dos fármacos , Basidiomycota/crescimento & desenvolvimento , Basidiomycota/fisiologia , Caseína Quinase I/genética , Caseína Quinase I/metabolismo , Cromatografia Líquida de Alta Pressão , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Furanos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional , Estresse Oxidativo , Patulina/isolamento & purificação , Patulina/farmacologia , Pironas/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Estresse FisiológicoRESUMO
The metabolic profiling of kiwifruit (Actinidia deliciosa, Hayward cultivar) aqueous extracts and the water status of entire kiwifruits were monitored over the season (June-December) using nuclear magnetic resonance (NMR) methodologies. The metabolic profiling of aqueous kiwifruit extracts was investigated by means of high field NMR spectroscopy. A large number of water-soluble metabolites were assigned by means of 1D and 2D NMR experiments. The change in the metabolic profiles monitored over the season allowed the kiwifruit development to be investigated. Specific temporal trends of aminoacids, sugars, organic acids and other metabolites were observed. The water status of kiwifruits was monitored directly on the intact fruit measuring the T(2) spin-spin relaxation time by means of a portable unilateral NMR instrument, fully non-invasive. Again, clear trends of the relaxation time were observed during the monitoring period. The results show that the monitoring of the metabolic profiling and the monitoring of the water status are two complementary means suitable to have a complete view of the investigated fruit.
Assuntos
Actinidia/metabolismo , Análise de Alimentos/métodos , Frutas/química , Espectroscopia de Ressonância Magnética/métodos , Água/análise , Actinidia/crescimento & desenvolvimento , Aminoácidos/análise , Carboidratos/análise , Ácidos Carboxílicos/análise , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Itália , Extratos Vegetais/química , Estações do AnoRESUMO
An NMR and chemometric analytical approach to classify extra virgin olive oils according to their geographical origin was developed within the European TRACE project (FP6-2003-FOOD-2-A, contract number: 0060942). Olive oils (896 samples) of three consecutive harvesting years (2005, 2006, and 2007) coming from Mediterranean areas were analyzed by (1)H NMR spectroscopy. Olive oil samples from Liguria, an Italian region, were chosen as a case study and PLS-DA and SIMCA modeling analyses were used to build up statistical models both to discriminate between Ligurian and non-Ligurian olive oils and to define the Ligurian olive oil class to confirm the declared provenience.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Óleos de Plantas/classificação , Europa (Continente) , Análise dos Mínimos Quadrados , Azeite de Oliva , Óleos de Plantas/análiseRESUMO
The nuclear magnetic resonance (NMR) technique was used as analytical tool to determine the complete metabolic profiling of sea bass extracts: water-soluble metabolites belonging to different classes such as sugars, amino acids, dipeptides and organic acids as well as metabolites soluble in organic solvent such as lipids, sterols and fatty acids were identified. The metabolite profiling together with a suitable statistical analysis were used to discriminate between wild and cultured sea bass samples. Preliminary results show that discrimination between wild and cultured sea bass was obtained not only using fatty acid composition but also cholesterol and phosphatidylethanolamine and some water-soluble metabolites such as choline, trimethylamine oxide, glutamine, fumaric and malic acids.
Assuntos
Bass/metabolismo , Pesqueiros , Espectroscopia de Ressonância Magnética/métodos , Extratos de Tecidos/química , Aminoácidos/metabolismo , Animais , Metabolismo dos Carboidratos , Metabolismo dos Lipídeos , Peptídeos/metabolismoRESUMO
Two binary aqueous mixtures which contain the small amphiphilic molecules TMAO (trimethylamine-N-oxide) and TBA (tert-butyl alcohol) have been investigated by molecular dynamics simulations and NMR chemical shift and self-diffusion measurements. TMAO is an osmolyte, while TBA is a monohydrate alcohol. Both possess bulky hydrophobic groups and polar heads, namely, NO in TMAO and OH in TBA. The hydrophilic/hydrophobic content of these isosteric molecules strongly modulates the structure and dynamics of the hydration shell, which is thought to be responsible for the effects observed on proteins and phospholipids. Simulation results, especially on hydrogen-bond networking, spatial correlations, and self-diffusivity, are consistent with NMR data and agree well with previous numerical studies on similar solutions. The methods employed allow the elucidation of the microscopic features of the solutions. For TBA solutions, the hydration shell is found to have a low density and a large spatial spread, and thus, above the molar fraction of 0.03, reduction of hydrophobic hydration drives self-aggregation of the solute. This effect does not take place in TMAO solutions, where the hydration shell is more compact and stable, maintaining its structure over a wider range of solute concentrations.
Assuntos
Simulação por Computador , Espectroscopia de Ressonância Magnética/métodos , Metilaminas/química , Modelos Químicos , Tensoativos/química , terc-Butil Álcool/química , Espectroscopia de Ressonância Magnética/normas , Padrões de Referência , Sensibilidade e Especificidade , Soluções/química , Água/químicaRESUMO
1H high-field nuclear magnetic resonance (NMR) was used to analyze 216 extra virgin olive oils collected in three years (1996, 1997, and 1998) in different Italian areas in order to evaluate the potential contribution of this technique to the geographical characterization of olive oils. A statistical procedure performed on the intensity of selected NMR peaks has been proposed. Tree clustering analysis of NMR data performed without any a priori hypothesis showed the existence of reliable parameters able to group the olive oils according to the location of olive oil production. Linear discriminant analysis applied to selected NMR parameters of olive oils of the same year of production allowed the grouping of samples according to their geographical origin with only very few errors. Moreover, a satisfactory grouping is reached by combining the NMR data of olive oils from two different years (1996 and 1997). Operating on appropriate sampling, a careful analysis of data yielded the conclusion that the place of olive production could be singled out as a discriminating factor regardless of the cultivars from which the olive oils are derived.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Óleos de Plantas/análise , Itália , Azeite de Oliva , Fatores de Tempo , Topografia MédicaRESUMO
Aqueous solutions of lactose and lysine were refluxed for up to 4 h without pH control. Samples were collected every hour, and the reaction was monitored by measuring the pH, the optical density at 420 nm, and the relative antioxidative efficiency (RAE). The greatest change in optical density and antioxidative efficiency occurred for the mixture heated for 4 h. The 4 h solution was separated into three fractions according to the molecular weights of the components and tested for RAE. The high molecular weight fraction was more colored, and it had the highest antioxidative activity. The low molecular weight fraction was separated by high-performance liquid chromatography (HPLC). RAE values were measured for each purified compound. HPLC coupled with diode array and electrospray mass spectrometry allowed a rapid screening of the solutions and a tentative identification of several peaks. Nuclear magnetic resonance analysis allowed the identification of galactosylisomaltol and pyrraline. The resonance assignments for these compounds were revised.
Assuntos
Lactose/química , Lisina/química , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Reação de Maillard , Espectrometria de Massas , Modelos QuímicosRESUMO
Fusaproliferin is a toxic sesterterpene isolated from Fusarium proliferatum, a widespread pathogen of cereals. Its absolute configuration has been determined by single crystal X-ray diffraction analysis. Fusaproliferin is considered to be a sesterterpene with a new ring skeleton having four C = C double bonds and four chiral atoms. The configurations of the four chiral atoms C10, C14, C15, and C19 are (R), (S), (R), and (S), respectively. In the solid state the macrolide shows a concave hydrophobic surface and hydrophilic convex face. The absolute configuration of C14 and C15 is the same as that observed for retigeranic acid, consistent with fusaproliferin being formed via a sesterterpenic-type biosynthetic pathway.
Assuntos
Fusarium/química , Micotoxinas/química , Terpenos/química , Animais , Artemia/efeitos dos fármacos , Cromatografia em Camada Fina , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Conformação Molecular , Micotoxinas/isolamento & purificação , Micotoxinas/toxicidade , Estereoisomerismo , Terpenos/metabolismo , Terpenos/toxicidadeRESUMO
Extracts from autoclaved maize culture of Fusarium tumidum strain R-5823 were toxic towards Artemia salina. Bioassay-guided fractionation of the organic extract led to the isolation of the toxic compound that was identified as the trichothecene toxin neosolaniol (NEOS) by 1H, 13C nuclear magnetic resonance spectroscopy and low-resolution electronic impact mass spectrometry. The amount of NEOS produced by the strain R-5823 was 300 mg/kg maize culture. NEOS was also detected by HPLC in cultures of four out of seven additional strains of F. tumidum and Gibberella tumida with different origin, in amounts ranging from 1 to 311 mg/kg. This is the first report on the production of a trichothecene toxin by F. tumidum.
Assuntos
Fusarium/química , Tricotecenos/análise , Animais , Artemia/efeitos dos fármacos , Extratos Celulares , Fusarium/crescimento & desenvolvimento , Gibberella/química , Tricotecenos/isolamento & purificação , Tricotecenos/toxicidade , Zea mays/microbiologiaRESUMO
A new toxic sesterterpene, named fusaproliferin, was purified from corn kernel cultures (120 mg/kg dry culture) of a strain of Fusarium proliferatum isolated from corn ear rot in northern Italy. The stain, designated ITEM-1494, also produced fumonisin B1 (1.500 mg/kg dry culture) and beauvericin (90 mg/kg dry culture), but not moniliformin. To monitor toxicity, the brine shrimp assay was used throughout the isolation procedure. Fusaproliferin had a molecular formula of C27H40O5, and it is the first sesterterpene isolated from a Fusarium species.
Assuntos
Depsipeptídeos , Fumonisinas , Fusarium/metabolismo , Micotoxinas/isolamento & purificação , Peptídeos , Terpenos/isolamento & purificação , Animais , Antibacterianos/análise , Antibacterianos/isolamento & purificação , Artemia/fisiologia , Carcinógenos Ambientais/análise , Carcinógenos Ambientais/isolamento & purificação , Meios de Cultura , Ciclobutanos/análise , Ciclobutanos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Micotoxinas/análise , Terpenos/análise , Zea maysRESUMO
The synthesis of 2'-heptylcarbamoyloxy-2-methyl-6,7-benzomorphan is described. The compound is structurally related to the cholinesterase inhibitor heptylphysostigmine (MF 201) because the angular methyl group of the esoroline nucleus has been changed into a bridging carbon and the anilinic nitrogen has been replaced by a methylene group. This compound proved to be a potent cholinesterase in vitro inhibitor.