RESUMO
Sphingosine-1-phosphate (S1P) is a unique lipid ligand binding to S1P receptors to transduce various cell survival or proliferation signals via small G proteins. S1P lyase (S1PL) is the specific enzyme that degrades S1P to phosphoethanolamine and (2E)-hexadecenal and therefore regulates S1P levels. S1PL also degrades dihydrosphingosine-1-phosphate (Sa1P), with a higher affinity to produce hexadecanal. Here, we developed a newly designed assay using a C17-Sa1P substrate that degrades into pentadecanal and phosphoethanolamine. For higher sensitivity in pentadecanal analysis, we developed a quantitative protocol as well as a 5,5-dimethyl cyclohexanedione (5,5-dimethyl CHD) derivatization method. The derivatization conditions were optimized for the reaction time, temperature, and concentrations of the 5,5-dimethyl CHD reagent, acetic acid, and ammonium acetate. The S1PL reaction in the cell lysate after spiking 20 µM of C17-Sa1P for 20 min was linear to the total protein concentrations of 50 µg. The S1PL levels (4 pmol/mg/min) were readily detected in this HPLC with fluorescence detection (λex = 366 nm, λem = 455 nm). The S1PL-catalyzed reaction was linear over 30 min and yielded a Km value of 2.68 µM for C17-Sa1P. This new method was validated to measure the S1PL activity of mouse embryonal carcinoma cell lines of the standard cell (F9-0), S1PL knockdown cells (F9-2), and S1PL-overexpressed cells (F9-4). Furthermore, we treated F9-4 cells with different S1PL inhibitors such as FTY720, 4-deoxypyridoxine (DOP), and the deletion of pyridoxal-5-phosphate (P5P), an essential cofactor for S1PL activity, and observed a significant decrease in pentadecanal relative to the untreated cells. In conclusion, we developed a highly sensitive S1PL assay using a C17-Sa1P substrate for pentadecanal quantification for application in the characterization of S1PL activity in vitro.
Assuntos
Aldeído Liases/análise , Bioensaio/métodos , Aldeídos/química , Animais , Linhagem Celular Tumoral , Cromatografia/métodos , Cromatografia Líquida de Alta Pressão , Cicloexanonas/química , Etanolaminas/química , Corantes Fluorescentes/química , Ligantes , Limite de Detecção , Modelos Lineares , Camundongos , Mutação , Ligação ProteicaRESUMO
The approach of this research was to describe the biochemical transformations of minor components of virgin olive oil with high impact on its sensory quality, specifically "volatile compounds" during its extraction process. For this purpose `Picual´, `Arbequina´ and `Hojiblanca´ cultivars were processed at three different harvesting times. Their volatile profiles and levels of enzymatic activity of "hydroperoxide lyase", responsible for the biosynthesis of C6 aldehydes related to "green and fruity notes", were monitored and identified in mesocarp, paste before kneading and paste after kneading based on the catalytic action of crude enzyme extracts. Both of them, volatiles and HPL, were analyzed by incubating with their corresponding substrates and reaction products formed were analyzed by Solid Phase Microextraction-Gas Chromatography and Mass Spectrometry. The results obtained in the present research can be useful in order to develop new markers biochemical whereby improved aroma quality in olive breeding programs or in the design of new protocol of VOO extraction.
Assuntos
Aldeído Liases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Qualidade dos Alimentos , Azeite de Oliva/química , Azeite de Oliva/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Aldeído Liases/análise , Aldeído Liases/química , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/química , Cromatografia Gasosa-Espectrometria de Massas , Azeite de Oliva/análise , Microextração em Fase Sólida , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/químicaRESUMO
Chemosynthetic life was recently discovered at Chapopote, an asphalt hydrocarbon seep in the southern Gulf of Mexico. Preliminary morphological analyses indicated that one tubeworm and two mussel species colonize Chapopote. Our molecular analyses identified the tubeworm as Escarpia sp., and the mussels as Bathymodiolus heckerae and B. brooksi. Comparative 16S rRNA analysis and FISH showed that all three species harbour intracellular sulfur-oxidizing symbionts highly similar or identical to those found in the same host species from northern Gulf of Mexico (nGoM). The mussels also harbour methane-oxidizing symbionts, and these shared highly similar to identical 16S rRNA sequences to their nGoM conspecifics. We discovered a novel symbiont in B. heckerae, which is closely related to hydrocarbon-degrading bacteria of the genus Cycloclasticus. In B. heckerae, we found key genes for the use of aromatic compounds, and its stable carbon isotope values were consistently higher than B. brooksi, indicating that the novel symbiont might use isotopically heavy aromatic hydrocarbons from the asphalt seep. This discovery is particularly intriguing because until now only methane and reduced sulfur compounds have been shown to power cold-seep chemosynthetic symbioses. The abundant hydrocarbons available at Chapopote would provide these mussel symbioses with a rich source of nutrition.
Assuntos
Bactérias/classificação , Bactérias/genética , Fenômenos Fisiológicos Bacterianos , Bivalves/microbiologia , Poliquetos/microbiologia , Simbiose , Aldeído Liases/análise , Animais , Bactérias/isolamento & purificação , Biodiversidade , Bivalves/classificação , Bivalves/genética , Isótopos de Carbono/análise , Sistema Enzimático do Citocromo P-450/análise , Complexo IV da Cadeia de Transporte de Elétrons/genética , Golfo do México , Dados de Sequência Molecular , Filogenia , Poliquetos/classificação , Poliquetos/genética , RNA Ribossômico 16S/genéticaRESUMO
Sphingosine-1-phosphate lyase (SGPL1) is the last enzyme in the catabolism of sphingolipids. It catalyzes the retroaldolic cleavage of long chain base phosphates into phosphoethanolamine and a fatty aldehyde. In this article we report on an easy and sensitive procedure to determine SPL activity. The assays uses C17-sphinganine-1-phosphate as substrate and the aldehyde product, pentadecanal, is quantified as its pentafluorobenzyloxime derivative by GC/MS. Derivatization of pentadecanal is performed as a one-step reaction, and the oxime product is directly injected for GC/MS analysis without any further purification. Acquisition in selected ion monitoring mode allows very high sensitivity, with a limit of detection of 281fmol. The assay is linear with both protein concentration and incubation time up to 20µg and 40min, respectively. The K(m) value obtained (6µM) is similar to that for the natural substrate sphingosine-1-phosphate. Using this method, FTY720 and deoxypyridoxine phosphate inhibited SPL with similar potencies to those reported.
Assuntos
Aldeído Liases/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Aldeído Liases/análise , Aldeídos/análise , Aldeídos/metabolismo , Animais , Linhagem Celular , Lisofosfolipídeos/metabolismo , Camundongos , Sensibilidade e Especificidade , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
A chemical characterization of the major components, namely, triacylglycerols (TAGs), polyphenols, and tocopherols in a Sacha inchi oil derived from cold pressing of the seed, is hereby reported. To tackle such a task, high-performance liquid chromatography in combination with photodiode array (PDA), fluorescence (RF), and mass spectrometry (MS) detection was employed. The latter was interfaced with atmospheric pressure chemical ionization and with electrospray ionization for the analysis of TAGs and polyphenols, respectively, whereas RF detection was tested for the determination of tocopherol content. Furthermore, fatty acid methyl esters (FAMEs) were evaluated by gas chromatography-flame ionization detector. A 93% amount of total fatty acids was represented by unsaturated FAMEs with the greatest percentage represented by linoleic (L) and linolenic (Ln) accounting for approximately 50 and 36%, respectively. The main TAGs (>10%) were represented by LLnL, LnLnLn, and LnLLn; the latter was present in the oil sample at the highest percentage (22.2%). Among tocopherols, γ-tocopherol was detected to be the most abundant component (over 50%). The polyphenolic composition was also investigated, and a total of 15 compounds were positively identified, through the complementary analytical information coming from PDA and MS data. To the best of our knowledge, this is the first report providing a thorough chemical characterization of a Plukenetia volubilis L. oil.
Assuntos
Euphorbiaceae/química , Óleos de Plantas/química , Sementes/química , Aldeído Liases/análise , Cromatografia Líquida de Alta Pressão/métodos , Sistema Enzimático do Citocromo P-450/análise , Polifenóis/análise , Espectrometria de Massas por Ionização por Electrospray , Tocoferóis/análise , Triglicerídeos/análiseRESUMO
Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an omega-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a K(m) of 35 microM for BODIPY-sphingosine 1-phosphate.
Assuntos
Aldeído Liases/análise , Compostos de Boro/química , Corantes Fluorescentes/química , Lisofosfolipídeos/química , Esfingosina/análogos & derivados , Aldeído Liases/química , Animais , Catálise , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Humanos , Métodos , Camundongos , Esfingosina/químicaRESUMO
Pantothenamides have been the subject of much study as potential inhibitors of CoA and carrier protein dependent biosynthetic pathways. Based on an initial observation of growth inhibition in Escherichia coli by 3, we have synthesized a small panel of pantetheine analogues and re-examined the inhibitory properties of this class of antibiotics with an emphasis on understanding the ability of these compounds to act as substrates of native CoA and carrier protein utilizing biosynthetic pathways. Our findings suggest that a secondary structure-activity relationship is an important factor in the antibiotic activity of these compounds.
Assuntos
Antimetabólitos/metabolismo , Coenzima A/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Panteteína/análogos & derivados , Aldeído Liases/análise , Aldeído Liases/metabolismo , Antibacterianos/metabolismo , Técnicas de Química Combinatória , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/metabolismo , Estrutura Molecular , Panteteína/síntese química , Panteteína/química , Panteteína/metabolismo , Relação Estrutura-AtividadeRESUMO
We have undertaken a comprehensive analysis of cytoplasmic protein phosphorylation in Campylobacter jejuni by mass spectrometric identification of phosphoproteins and localization of the sites of modification by phosphopeptide analyses. Cell extracts, enriched for phosphoproteins using Fe(III) IMAC or commercial phosphoprotein purification kits, were analyzed by 1-D and 2-D SDS-PAGE and subjected to mass fingerprinting by in-gel tryptic digestion and MALDI-TOF MS. Fifty-eight phosphopeptides were identified from 1-D gel bands by nano-LC-MS/MS and automated searching in a C. jejuni ORF database resulting in the unequivocal identification of 36 phosphoproteins of diverse function. In addition to elongation factors and chaperonins, which have been reported to be phosphorylated in other bacteria, the major phosphoproteins included bacterioferritin and superoxide dismutase. The sequences around the phosphorylated Ser and Thr residues are indicative of specific kinases being responsible for some of the modifications. However, many of the other identified proteins are enzymes that have phosphorylated substrates, including ATP, hence other modifications may arise from autophosphorylation. Comparative analyses of IMAC extracts from the Escherichia coli strain AD202 and Helicobacter pylori resulted in the identification of homologs of six of the C. jejuni phosphoproteins, though their overall phosphoproteome maps were distinctly different.
Assuntos
Proteínas de Bactérias/análise , Campylobacter jejuni/química , Fosfoproteínas/análise , Proteoma/análise , Aldeído Liases/análise , Aldeído Liases/química , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Campylobacter jejuni/metabolismo , Grupo dos Citocromos b/análise , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/metabolismo , Ferritinas/análise , Ferritinas/química , Ferritinas/metabolismo , Helicobacter pylori/química , Helicobacter pylori/metabolismo , Modelos Moleculares , Fosfopeptídeos/análise , Fosfopeptídeos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteoma/genética , Proteoma/metabolismo , Espectrometria de Massas em TandemRESUMO
Sphingosine-1-phosphate (S1P) lyase (SPL) catalyzes the conversion of S1P to ethanolamine phosphate and hexadecenal. This enzyme plays diverse roles in physiology and disease and, thus, may be useful as a disease marker and/or drug target. Unfortunately, the radioisotope-based assay currently used to quantify SPL activity is suboptimal. We have devised an assay using a commercially available omega(7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro (NBD)-labeled fluorescent substrate. Alternatively, we provide a method for synthesis of the substrate from NBD-sphingosine. Enzyme activity is determined by following the formation of NBD-aldehyde product, which is isolated from unreacted substrate by lipid extraction and quantified after separation by HPLC using a C18 column. A fluorescent NBD-C18-sphingosine internal standard is used to control for extraction efficiency. The reaction is linear over 20 min and total protein concentrations of 20-200 mg/l. The sensitivity of the fluorescence assay is comparable to or better than that of the radioactive assay, and SPL levels as low as 8 pmol/mg/min were readily detected. Semicarbazide, a nonspecific SPL inhibitor, reduced SPL activity in vitro by approximately 70% using both standard and fluorescence methods. Product inhibition was not observed using ethanolamine phosphate and a commercially available source of hexadecenal. This method is suitable for quantifying SPL activity in a variety of cell and tissue sources.
Assuntos
Aldeído Liases/análise , Espectrometria de Fluorescência/métodos , Células 3T3-L1 , Aldeído Liases/química , Aldeído Liases/metabolismo , Animais , Catecóis/química , Linhagem Celular Tumoral , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Compostos Organometálicos/químicaRESUMO
The lipoxygenase pathway is responsible for the production of oxylipins, which are important compounds for plant defence responses. Jasmonic acid, the final product of the allene oxide synthase/allene oxide cyclase branch of the pathway, regulates wound-induced gene expression. In contrast, C6 aliphatic aldehydes produced via an alternative branch catalysed by hydroperoxide lyase, are themselves toxic to pests and pathogens. Current evidence on the subcellular localization of the lipoxygenase pathway is conflicting, and the regulation of metabolic channelling between the two branches of the pathway is largely unknown. It is shown here that while a 13-lipoxygenase (LOX H3), allene oxide synthase and allene oxide cyclase proteins accumulate upon wounding in potato, a second 13-lipoxygenase (LOX H1) and hydroperoxide lyase are present at constant levels in both non-wounded and wounded tissues. Wound-induced accumulation of the jasmonic acid biosynthetic enzymes may thus commit the lipoxygenase pathway to jasmonic acid production in damaged plants. It is shown that all enzymes of the lipoxygenase pathway differentially localize within chloroplasts, and are largely found associated to thylakoid membranes. This differential localization is consistently observed using confocal microscopy of GFP-tagged proteins, chloroplast fractionation, and western blotting, and immunodetection by electron microscopy. While LOX H1 and LOX H3 are localized both in stroma and thylakoids, both allene oxide synthase and hydroperoxide lyase protein localize almost exclusively to thylakoids and are strongly bound to membranes. Allene oxide cyclase is weakly associated with the thylakoid membrane and is also detected in the stroma. Moreover, allene oxide synthase and hydroperoxide lyase are differentially distributed in thylakoids, with hydroperoxide lyase localized almost exclusively to the stromal part, thus closely resembling the localization pattern of LOX H1. It is suggested that, in addition to their differential expression pattern, this segregation underlies the regulation of metabolic fluxes through the alternative branches of the lipoxygenase pathway.
Assuntos
Cloroplastos/enzimologia , Lipoxigenase/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/enzimologia , Aldeído Liases/análise , Aldeído Liases/metabolismo , Western Blotting , Fracionamento Celular , Cloroplastos/ultraestrutura , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Fluorescência Verde/análise , Oxirredutases Intramoleculares/análise , Oxirredutases Intramoleculares/metabolismo , Lipoxigenase/análise , Microscopia Confocal , Dados de Sequência Molecular , Proteínas de Plantas/análise , Solanum tuberosum/ultraestrutura , Tilacoides/enzimologia , Tilacoides/ultraestruturaRESUMO
The degradation of polyunsaturated fatty acids through the lipoxygenase pathway is responsible for the production of volatile compounds that confer green sensory notes to the aroma of fruits and vegetables. The peroxidation of free linoleic or linolenic acid by action of lipoxygenase and then the lysis of the resulting hydroperoxides, through a reaction catalyzed by the hydroperoxide lyase, are the most determinant steps of this pathway. This work analyzes the impact of the hydroperoxide lyase depletion on the volatile composition of leaves from Arabidopsis thaliana that is used as model system. The work involves the characterization of the volatile profiles of the Arabidopsis plants followed by a study of the metabolism of radio-labeled linoleic acid and determinations of lipoxygenase activity. Hydroperoxide lyase-knockout plants show similar levels of C6 compounds, but the total amount of C5 compounds is 4-fold higher in mutant plants. The perspectives of production of vegetable products with a modified aroma by genetically engineering the lipoxygenase pathway were discussed with respect to the statistical results.
Assuntos
Aldeído Liases/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Sistema Enzimático do Citocromo P-450/genética , Mutação , Folhas de Planta/metabolismo , Aldeído Liases/análise , Aldeído Liases/metabolismo , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/metabolismo , Peróxido de Hidrogênio/metabolismo , Ácido Linoleico/metabolismo , Lipoxigenase/metabolismo , Mutagênese Insercional , Odorantes/análise , Folhas de Planta/química , Folhas de Planta/enzimologia , VolatilizaçãoRESUMO
A polyphasic taxonomic approach was used to identify a major atypical group of gas-forming, arginine-negative lactic acid bacteria associated with spoilage of whole (nonsliced) refrigerated (4 degrees C) cooked hams produced in two Greek industrial meat plants. Biochemical characterization revealed that the ham isolates shared their phenotypic properties with Leuconostoc carnosum, Weissella viridescens, and Weissella hellenica. However, gas chromatographic analysis of cellular fatty acids clearly differentiated the ham isolates from the Weissella spp. None of the isolates contained eicosenoic acid (n-C20:1), which is typically synthesized by W. viridescens, but all strains contained high amounts of C 19cycl acid, which is absent in W. hellenica and has been found in trace amounts in W. viridescens. All strains had similar cellular fatty acid profiles, which were qualitatively similar to those of the cellular fatty acids of L. carnosum. In addition to the phenotypic and chemotaxonomic tests, three representative isolates were studied using a lactic acid bacteria database, which employs 16S and 23S HindIII restriction fragment length polymorphism patterns as operational taxonomic units in a numerical analysis. The isolate patterns were identical to those of the L. carnosum type strain, NCFB 2776T. Based on the polyphasic taxonomic approach, the dominating lactic acid bacteria group was identified as L. carnosum.
Assuntos
Aldeído Liases/análise , Sistema Enzimático do Citocromo P-450/análise , Contaminação de Alimentos/análise , Conservação de Alimentos/métodos , Leuconostoc/crescimento & desenvolvimento , Produtos da Carne/microbiologia , Filogenia , Animais , Qualidade de Produtos para o Consumidor , Conservação de Alimentos/normas , Grécia , Humanos , Lactobacillus/classificação , Lactobacillus/genética , Lactobacillus/crescimento & desenvolvimento , Leuconostoc/classificação , Leuconostoc/genética , Polimorfismo de Fragmento de Restrição , Refrigeração , Ribotipagem , Especificidade da Espécie , SuínosRESUMO
Policosanols, long-chain alcohols, have many beneficial physiological activities. Contents and compositions in perilla seeds (Perilla frutescens) produced in Korea and China were determined. Waxy materials were extracted from perilla seeds using hot hexane. Yield of the waxy materials from perilla seeds was 72.1 mg/100 g of dry weight. Contents and compositions of the waxy materials and policosanols were identified and quantified by TLC, HPLC, and GC. Major components of the waxy materials from Korean and Chinese perilla seeds were policosanols (25.5 and 34.8%, respectively), hydrocarbons (18.8 and 10.5%), wax esters, steryl esters and aldehydes (53.0 and 49.8%), acids (1.7 and 2.1%), and triacylglycerols (1.0 and 2.9%), determined by HPLC. For comparison, waxy materials of sesame seeds were also analyzed. Yield of the waxy materials from sesame seeds were 8.6 mg/100 g. Less than 5% policosanols were detected in the waxy materials extracted from sesame seeds produced in Korea and China. Wax esters or steryl esters accounted for 93-95% of the sesame waxy materials. Policosanols in the perilla seeds were composed of 67-68% octacosanol, 16-17% hexacosanol, 6-9% triacontanol, and others.
Assuntos
Álcoois Graxos/análise , Perilla/química , Sementes/química , Aldeído Liases/análise , Ésteres/análise , Hidrocarbonetos/análise , Ceras/análiseRESUMO
The gas-phase fragmentation reactions of a series of site-directed mutagenesis products of Staphylococcus aureus dihydroneopterin aldolase have been examined by multistage tandem mass spectrometry (MS/MS and MS(3)) in a linear quadrupole ion trap in order to explore the utility of this instrumentation for routine 'top-down' recombinant protein characterization. Following a rapid low resolution survey of the fragmentation behavior of the precursor ions from the wild type (WT) protein, selected charge states were subjected to detailed structural characterization by using high resolution 'zoom' and 'ultrazoom' resonance ejection MS/MS product ion scans. Dissociation of the [M + 18H](18+) charge state yielded a range of product ions from which extensive sequence information could be derived. In contrast, dissociation of the [M + 20H](20+) charge state resulted in a single dominant y(96) product ion formed by fragmentation between adjacent Ile/Gly residues, with only limited sequence coverage. Further extensive sequence information was readily obtained however, by MS(3) dissociation of this initial product. From the combined MS/MS and MS(3) spectra an overall sequence coverage of 66.9%, with fragmentation of 85 of the 127 amide bonds within the WT protein, was obtained. MS/MS and MS(3) of three of the four site-directed mutagenesis products (E29A), (Y61F) and (E81A) were found to yield essentially identical product ion spectra to the WT protein, indicating that these modifications had no significant influence on the fragmentation behavior. The specific site of modification could be unambiguously determined in each case by characterization of product ions resulting from fragmentation of amide bonds on either side of the mutation site. In contrast, MS/MS and MS(3) of the K107A mutant led to significantly different product ion spectra dominated by cleavages occurring N-terminal to proline, which restricted the ability to localize the modification site to within only an 8 amino acid region of the sequence. This work highlights the need for further studies to characterize the charge state, sequence and structural dependence to the low energy collision induced dissociation reactions of multiply protonated intact protein ions.
Assuntos
Aldeído Liases/análise , Espectrometria de Massas/métodos , Mutagênese Sítio-Dirigida , Staphylococcus aureus/enzimologia , Aldeído Liases/genética , Cromatografia Gasosa-Espectrometria de Massas/métodos , Proteínas Recombinantes/análiseRESUMO
The hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a 'promiscuous' non-phosphorylative variant of the Entner-Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of D-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose.
Assuntos
Aldeído Liases/metabolismo , Archaea/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sulfolobus solfataricus/enzimologia , Sulfolobus solfataricus/metabolismo , Trifosfato de Adenosina/metabolismo , Aldeído Liases/análise , Aldeído Liases/química , Aldeído Liases/genética , Aldeído Liases/isolamento & purificação , Biotransformação , Clonagem Molecular , Escherichia coli/genética , Galactose/metabolismo , Genes Bacterianos , Genoma Bacteriano , Glucose/metabolismo , Cinética , Modelos Biológicos , Peso Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/análise , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Sulfolobus solfataricus/genética , TemperaturaRESUMO
The activity of the enzymes of the central metabolic pathways has been the subject of intensive analysis; however, the Entner-Doudoroff (ED) pathway has only recently begun to attract attention. The metabolic response to edd gene knockout in Escherichia coli JM101 and PTS- Glc+ was investigated in gluconate and glucose batch cultures and compared with other pyruvate kinase and PTS mutants previously constructed. Even though the specific growth rates between the strain carrying the edd gene knockout and its parent JM101 and PTS- Glc+ edd and its parent PTS- Glc+ were very similar, reproducible changes in the specific consumption rates and biomass yields were obtained when grown on glucose. These results support the participation of the ED pathway not only on gluconate metabolism but on other metabolic and biochemical processes in E. coli. Despite that gluconate is a non-PTS carbohydrate, the PTS- Glc+ and derived strains showed important reductions in the specific growth and gluconate consumption rates. Moreover, the overall activity of the ED pathway on gluconate resulted in important increments in PTS- Glc+ and PTS- Glc+ pykF mutants. Additional results obtained with the pykA pykF mutant indicate the important contribution of the pyruvate kinase enzymes to pyruvate synthesis and energy production in both carbon sources.
Assuntos
Escherichia coli/metabolismo , Gluconatos/metabolismo , Glucose/metabolismo , Aldeído Liases/análise , Biomassa , Escherichia coli/crescimento & desenvolvimento , Deleção de Genes , Hidroliases/análise , Hidroliases/genética , Fosfotransferases/genética , Piruvato Quinase/genéticaRESUMO
We report a novel application for the operator-repressor titration (ORT) plasmid maintenance system. The ability of ORT to maintain a plasmid during production of DNA has been demonstrated previously. In this study, we have used the ORT system to maintain a plasmid during high cell density cultivation and expression of a recombinant protein. No evidence of plasmid loss was seen during protein expression at high cell densities. In addition, the quantity of protein produced using this system was similar to traditional plasmid maintenance systems.
Assuntos
Aldeído Liases/biossíntese , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Regiões Operadoras Genéticas , Plasmídeos , Proteínas Repressoras/genética , Aldeído Liases/análise , Animais , Escherichia coli/enzimologia , Fermentação , Regulação Bacteriana da Expressão Gênica/genética , Microbiologia Industrial/métodos , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Repressoras/metabolismoRESUMO
An enzyme-based assay was developed for the detection of bifidobacteria in infant faeces. Ninety-five samples from 51 breast-fed infants in the age between 3 and 276 days were investigated. Bifidobacteria and other bacterial groups were determined by cultivation and fluorescence in situ hybridisation (FISH). Faecal samples were examined for the activity of fructoso-6-phosphate phosphoketolase (F6PPK) and for other enzymatic reactions using the API-ZYM kit. Twenty-nine infants had high numbers of bifidobacteria (usually higher than 9 log CFU/g) in their faeces. Seventeen infants (35%) did not contain detectable amounts of bifidobacteria in their faecal samples. The remaining five individuals had low counts of bifidobacteria (3-6 log CFU/g). Most negative infants possessed major amounts of clostridia in their faecal flora. There were no significant differences among bifidobacterial counts obtained by cultivation and FISH, detection of F6PPK, alpha-galactosidase and alpha-glucosidase activities could routinely be used for the rapid and simple detection of bifidobacteria in infant faecal samples. Bifidobacterial colonies were identified using enzymatic tests and PCR procedure based on 16S rRNA gene sequences species-specific primers. In 14 samples, the identifications of individual isolates were compared with direct analyses of faeces using the nested PCR-denaturing gradient gel electrophoresis (nested DGGE) procedure. The results obtained in several cases are not identical. Bifidobacterium longum and Bifidobacterium breve were most frequently identified. Bifidobacteria-positive samples had high activities of alpha-galactosidase and alpha-glucosidase. On the contrary, negative samples missed either one or both of these enzymatic activities. While all positive samples tested showed distinctive fructose-6-phosphate phosphoketolase activity (F6PPK), none of the negative samples expressed F6PPK activity.
Assuntos
Bifidobacterium/enzimologia , Bifidobacterium/isolamento & purificação , Fezes/microbiologia , Aldeído Liases/análise , Bifidobacterium/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese , Feminino , Glucuronidase/análise , Glicosídeo Hidrolases/análise , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Masculino , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , alfa-Galactosidase/análise , beta-Galactosidase/análiseRESUMO
The extraction of 13-hydroperoxide-lyase activity from mint leaves as well as its use for C6-aldehyde production was studied in this work. The enzyme cleaves 13(S)-hydroperoxy-C18 fatty acids into C6-aldehyde and C12-oxo-acid. Two mint species were tested: Mentha veridis and Mentha pulegium. The headspace injection method coupled to gas chromatography was used for volatile compound analysis. The optimal conditions for temperature and pH were, respectively, 15 and 7 degrees C. We also studied the specific synthesis of hexanal and hexenals respectively from 13(S)-hydroperoxy-linoleic acid and 13(S)-hydroperoxy-linolenic acid. Considerable quantities of aldehyde (up to 2.58 micromol) were produced after 15 min of cleavage reaction in 2 ml stirred at 100 rpm, especially in presence of extract of M. veridis. The conversion yields decreased from 52.5% as maximum to 3.3% when using initial hydroperoxide concentrations between 0.2 and 15 mM. An unsaturated aldehyde, the 3(Z)-hexenal was produced from 13(S)-hydroperoxy-linolenic acid. The 3(Z)-isomer was unstable and isomerized in part to 2(E)-hexenal. In this work, we observed a very limited isomerization of 3(Z)-hexenal to 2(E)-hexenal, since the reaction and the volatile purge were carried out successively in the same flask without delay or any contact with the atmosphere. These aldehydes contribute to the fresh green odor in plants and are widely used in perfumes and in food technology. Their importance increases especially when the starting materials are of natural biological origin as used in this work. GC-MS analysis allowed the identification of the products.
Assuntos
Aldeído Liases/química , Aldeídos/síntese química , Sistema Enzimático do Citocromo P-450/química , Peróxidos Lipídicos/química , Mentha/enzimologia , Extratos Vegetais/química , Folhas de Planta/enzimologia , Aldeído Liases/análise , Aldeído Liases/classificação , Sistema Enzimático do Citocromo P-450/análise , Sistema Enzimático do Citocromo P-450/classificação , Ativação Enzimática , Mentha pulegium/enzimologia , Especificidade da Espécie , VolatilizaçãoRESUMO
Tetrahydrofolate (vitamin B9) and its folate derivatives are essential cofactors in one-carbon (C1) transfer reactions and absolutely required for the synthesis of a variety of different compounds including methionine and purines. Most plants, microbial eukaryotes, and prokaryotes synthesize folate de novo. We have characterized an important enzyme in this pathway, the Saccharomyces cerevisiae FOL1 gene. Expression of the budding yeast gene FOL1 in Escherichia coli identified the folate biosynthetic enzyme activities dihydroneopterin aldolase (DHNA), 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (HPPK), and dihydropteroate synthase (DHPS). All three enzyme activities were also detected in wild-type yeast strains, whereas fol1Delta deletion strains only showed background activities, thus demonstrating that Fol1p catalyzes three sequential steps of the tetrahydrofolate biosynthetic pathway and thus is the central enzyme of this pathway, which starting from GTP consists of seven enzymatic reactions in total. Fol1p is exclusively localized to mitochondria as shown by fluorescence microscopy and immune electronmicroscopy. FOL1 is an essential gene and the nongrowth phenotype of the fol1 deletion leads to a recessive auxotrophy for folinic acid (5'-formyltetrahydrofolate). Growth of the fol1Delta deletion strain on folinic acid-supplemented rich media induced a dimorphic switch with haploid invasive and filamentous pseudohyphal growth in the presence of glucose and ammonium, which are known suppressors of filamentous and invasive growth. The invasive growth phenotype induced by the depletion of C1 carrier is dependent on the transcription factor Ste12p and the flocullin/adhesin Flo11p, whereas the filamentation phenotype is independent of Ste12p, Tec1p, Phd1p, and Flo11p, suggesting other signaling pathways as well as other adhesion proteins.