RESUMO
Cancer-related metabolic features are in part maintained by hexokinase 2 upregulation, which leads to high levels of glucose-6-phosphate (G6P) and is needed to provide energy and biomass to support rapid proliferation. Using a humanized model of the yeast Saccharomyces cerevisiae, we explored how human hexokinase 2 (HK2) behaves under different nutritional conditions. At high glucose levels, yeast presents aerobic glycolysis through a regulatory mechanism known as catabolic repression, which exerts a metabolic adaptation like the Warburg effect. At high glucose concentrations, HK2 did not translocate into the nucleus and was not able to shift the metabolism toward a highly glycolytic state, in contrast to the effect of yeast hexokinase 2 (Hxk2), which is a crucial protein for the control of aerobic glycolysis in S. cerevisiae. During the stationary phase, when glucose is exhausted, Hxk2 is shuttled out of the nucleus, ceasing catabolic repression. Cells harvested at this condition display low glucose consumption rates. However, glucose-starved cells expressing HK2 had an increased capacity to consume glucose. In those cells, HK2 localized to mitochondria, becoming insensitive to G6P inhibition. We also found that the sugar trehalose-6-phosphate (T6P) is a human HK2 inhibitor, like yeast Hxk2, but was not able to inhibit human HK1, the isoform that is ubiquitously expressed in almost all mammalian tissues. In contrast to G6P, T6P inhibited HK2 even when HK2 was associated with mitochondria. The binding of HK2 to mitochondria is crucial for cancer survival and proliferation. T6P was able to reduce the cell viability of tumor cells, although its toxicity was not impressive. This was expected as cell absorption of phosphorylated sugars is low, which might be counteracted using nanotechnology. Altogether, these data suggest that T6P may offer a new paradigm for cancer treatment based on specific inhibition of HK2.
Assuntos
Hexoquinase , Fosfatos Açúcares , Animais , Humanos , Hexoquinase/metabolismo , Saccharomyces cerevisiae/metabolismo , Glicólise , Glucose/metabolismo , MamíferosRESUMO
Fructooligosaccharides (FOSs) are polymers of fructose with a prebiotic activity because of their production and fermentation by bacteria that inhabit the gastrointestinal tract and are widely used in the industry and new functional foods. Lactobacillus gasseri stands out as an important homofermentative microorganism related to FOS production, and its potential applications in the industry are undeniable. In this study, we report the production and characterization of a sucrose-6-phosphate hydrolase from L. gasseri belonging to the GH32 family. Apo-LgAs32 and LgAs32 complexed with ß-d-fructose structures were determined at a resolution of 1.94 and 1.84 Å, respectively. The production of FOS, fructans, 1-kestose, and nystose by the recombinant LgAs32, using sucrose as a substrate, shown in this study is very promising. When compared to its homologous enzyme from Lactobacillus reuteri, the production of 1-kestose by LgAs32 is increased; thus, LgAs32 can be considered as an alternative in fructan production and other industrial applications.
Assuntos
Hexosiltransferases , Lactobacillus gasseri , Indústria Alimentícia , Frutanos , Hexosiltransferases/genética , Oligossacarídeos , Sacarose/análogos & derivados , Fosfatos Açúcares , beta-Frutofuranosidase/genéticaRESUMO
Infectious diseases are serious public health problems, affecting a large portion of the world's population. A molecule that plays a key role in pathogenic organisms is trehalose and recently has been an interest in the metabolism of this molecule for drug development. The trehalose-6-phosphate synthase (TPS1) is an enzyme responsible for the biosynthesis of trehalose-6-phosphate (T6P) in the TPS1/TPS2 pathway, which results in the formation of trehalose. Studies carried out by our group demonstrated the inhibitory capacity of T6P in the TPS1 enzyme from Saccharomyces cerevisiae, preventing the synthesis of trehalose. By in silico techniques, we compiled sequences and experimentally determined structures of TPS1. Sequence alignments and molecular modeling were performed. The generated structures were submitted in validation of algorithms, aligned structurally and analyzed evolutionarily. Molecular docking methodology was applied to analyze the interaction between T6P and TPS1 and ADMET properties of T6P were analyzed. The results demonstrated the models created presented sequence and structural similarities with experimentally determined structures. With the molecular docking, a cavity in the protein surface was identified and the molecule T6P was interacting with the residues TYR-40, ALA-41, MET-42, and PHE-372, indicating the possible uncompetitive inhibition mechanism provided by this ligand, which can be useful in directing the molecular design of inhibitors. In ADMET analyses, T6P had acceptable risk values compared with other compounds from World Drug Index. Therefore, these results may present a promising strategy to explore to develop a broad-spectrum antibiotic of this specific target with selectivity, potency, and reduced side effects, leading to a new way to treat infectious diseases like tuberculosis and candidiasis.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Glucosiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Simulação por Computador , Inibidores Enzimáticos/química , Glucosiltransferases/química , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Fosfatos Açúcares/química , Trealose/química , Trealose/metabolismoRESUMO
Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like ß-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a ß-fructofuranosidase (E.C. 3.1.2.26, protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0-7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a ß-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal ß-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.
Assuntos
Bacillus licheniformis/enzimologia , Sacarose/análogos & derivados , Fosfatos Açúcares/metabolismo , beta-Frutofuranosidase/química , beta-Frutofuranosidase/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Especificidade por Substrato , Sacarose/metabolismo , Difração de Raios XRESUMO
Plastidial isoprenoids, such as carotenoids and tocopherols, are important anti-oxidant metabolites synthesized in plastids from precursors generated by the methylerythritol 4-phosphate (MEP) pathway. In this study, we found that irradiation of Arabidopsis thaliana plants with UV-B caused a strong increase in the accumulation of the photoprotective xanthophyll zeaxanthin but also resulted in slightly higher levels of γ-tocopherol. Plants deficient in the MEP enzymes 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase showed a general reduction in both carotenoids and tocopherols and this was associated with increased DNA damage and decreased photosynthesis after exposure to UV-B. Genetic blockage of tocopherol biosynthesis did not affect DNA damage accumulation. In contrast, lut2 mutants that accumulate ß,ß-xanthophylls showed decreased DNA damage when irradiated with UV-B. Analysis of aba2 mutants showed that UV-B protection was not mediated by ABA (a hormone derived from ß,ß-xanthophylls). Plants accumulating ß,ß-xanthophylls also showed decreased oxidative damage and increased expression of DNA-repair enzymes, suggesting that this may be a mechanism for these plants to decrease DNA damage. In addition, in vitro experiments also provided evidence that ß,ß-xanthophylls can directly protect against DNA damage by absorbing radiation. Together, our results suggest that xanthophyll-cycle carotenoids that protect against excess illumination may also contribute to protection against UV-B.
Assuntos
Arabidopsis/metabolismo , Raios Ultravioleta , Xantofilas/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Eritritol/análogos & derivados , Eritritol/deficiência , Fotossíntese/efeitos da radiação , Fosfatos Açúcares/deficiênciaRESUMO
Cancer stem cells are responsible for tumor recurrence and metastasis. A new highly reproducible procedure for human breast cancer MCF-7 stem cells (BCSC) isolation and selection was developed by using a combination of hypoxia/hypoglycemia plus taxol and adriamycin for 24h. The BCSC enriched fraction (i) expressed (2-15 times) the typical stemness protein markers CD44+, ALDH1A3 and Oct 3/4; (ii) increased its clonogenicity index (20-times), invasiveness profile (>70%), migration capacity (100%) and ability to form mammospheres, compared to its non-metastatic MCF-7 counterpart. This isolation and selection protocol was successful to obtain stem cell enriched fractions from A549, SiHa and medulloblastoma cells. Since the secretion of HPI/AMF cytokine seems involved in metastasis, the effects of erytrose-4-phosphate (E4P) and 6-phosphogluconate (6PG), potent HPI inhibitors, on the acquisition of the breast stem cell-like phenotype were also evaluated. The presence of E4P during the BCSC selection deterred the development of the stemness phenotype, whereas both extracellular E4P (5-250nM) and 6PG (1µM) as well as siRNA HPI/AMF depressed the BCSC invasiveness ability (>90%), clonogenicity index (>90%) and contents (50-96%) of stemness (CD44, ALDH1A), pluripotency (p38 MAPK, Oct3/4, wnt/ß-catenin) and EMT (SNAIL, MMP-1, vimentin) markers. The cytokine inhibitor repertaxin (10nM) or the anti-IL-8 or anti-TGF-ß monoclonal antibodies (10µg/mL) did not significantly affect the BCSC metastatic phenotype. E4P also diminished (75%) the formation and growth of MCF-7 stem cell mammospheres. These results suggested that E4P by directly interacting with extracellular HPI/AMF may be an effective strategy to deter BCSC growth and progression.
Assuntos
Neoplasias da Mama/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Hipóxia Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Transição Epitelial-Mesenquimal/genética , Feminino , Gluconatos/administração & dosagem , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Células MCF-7 , Metástase Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Paclitaxel/administração & dosagem , RNA Interferente Pequeno/genética , Fosfatos Açúcares/administração & dosagem , Sulfonamidas/administração & dosagemRESUMO
Trehalose-6-phosphate synthase OtsA from streptomycetes is unusual in that it uses GDP-glucose as the donor substrate rather than the more commonly used UDP-glucose. We now confirm that OtsA from Streptomyces venezuelae has such a preference for GDP-glucose and can utilize ADP-glucose to some extent too. A crystal structure of the enzyme shows that it shares twin Rossmann-like domains with the UDP-glucose-specific OtsA from Escherichia coli However, it is structurally more similar to Streptomyces hygroscopicus VldE, a GDP-valienol-dependent pseudoglycosyltransferase enzyme. Comparison of the donor binding sites reveals that the amino acids associated with the binding of diphosphoribose are almost all identical in these three enzymes. By contrast, the amino acids associated with binding guanine in VldE (Asn, Thr, and Val) are similar in S. venezuelae OtsA (Asp, Ser, and Phe, respectively) but not conserved in E. coli OtsA (His, Leu, and Asp, respectively), providing a rationale for the purine base specificity of S. venezuelae OtsA. To establish which donor is used in vivo, we generated an otsA null mutant in S. venezuelae The mutant had a cell density-dependent growth phenotype and accumulated galactose 1-phosphate, glucose 1-phosphate, and GDP-glucose when grown on galactose. To determine how the GDP-glucose is generated, we characterized three candidate GDP-glucose pyrophosphorylases. SVEN_3027 is a UDP-glucose pyrophosphorylase, SVEN_3972 is an unusual ITP-mannose pyrophosphorylase, and SVEN_2781 is a pyrophosphorylase that is capable of generating GDP-glucose as well as GDP-mannose. We have therefore established how S. venezuelae can make and utilize GDP-glucose in the biosynthesis of trehalose 6-phosphate.
Assuntos
Açúcares de Guanosina Difosfato/metabolismo , Streptomyces/metabolismo , Fosfatos Açúcares/biossíntese , Trealose/análogos & derivados , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Galactose/genética , Galactose/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Açúcares de Guanosina Difosfato/genética , Streptomyces/genética , Fosfatos Açúcares/genética , Trealose/biossíntese , Trealose/genéticaRESUMO
In some pathogens, trehalose biosynthesis is induced in response to stress as a protection mechanism. This pathway is an attractive target for antimicrobials as neither the enzymes, Tps1, and Tps2, nor is trehalose present in humans. Accumulation of T6P in Candida albicans, achieved by deletion of TPS2, resulted in strong reduction of fungal virulence. In this work, the effect of T6P on Tps1 activity was evaluated. Saccharomyces cerevisiae, C. albicans, and Candida tropicalis were used as experimental models. As expected, a heat stress induced both trehalose accumulation and increased Tps1 activity. However, the addition of 125 µM T6P to extracts obtained from stressed cells totally abolished or reduced in 50 and 60 % the induction of Tps1 activity in S. cerevisiae, C. tropicalis, and C. albicans, respectively. According to our results, T6P is an uncompetitive inhibitor of S. cerevisiae Tps1. This kind of inhibitor is able to decrease the rate of reaction to zero at increased concentrations. Based on the similarities found in sequence and function between Tps1 of S. cerevisiae and some pathogens and on the inhibitory effect of T6P on Tps1 activity observed in vitro, novel drugs can be developed for the treatment of infectious diseases caused by organisms whose infectivity and survival on the host depend on trehalose.
Assuntos
Candida albicans/enzimologia , Candida tropicalis/enzimologia , Inibidores Enzimáticos/química , Glucosiltransferases/antagonistas & inibidores , Saccharomyces cerevisiae/enzimologia , Fosfatos Açúcares/química , Trealose/análogos & derivados , Candida albicans/patogenicidade , Candida tropicalis/patogenicidade , Candidíase/tratamento farmacológico , Candidíase/enzimologia , Inibidores Enzimáticos/farmacologia , Especificidade da Espécie , Fosfatos Açúcares/farmacologia , Trealose/química , Trealose/farmacologiaRESUMO
The GlgE pathway is thought to be responsible for the conversion of trehalose into a glycogen-like α-glucan polymer in bacteria. Trehalose is first converted to maltose, which is phosphorylated by maltose kinase Pep2 to give α-maltose 1-phosphate. This is the donor substrate of the maltosyl transferase GlgE that is known to extend α-1,4-linked maltooligosaccharides, which are thought to be branched with α-1,6 linkages. The genome of Streptomyces venezuelae contains all the genes coding for the GlgE pathway enzymes but none of those of related pathways, including glgC and glgA of the glycogen pathway. This provides an opportunity to study the GlgE pathway in isolation. The genes of the GlgE pathway were upregulated at the onset of sporulation, consistent with the known timing of α-glucan deposition. A constructed ΔglgE null mutant strain was viable but showed a delayed developmental phenotype when grown on maltose, giving less cell mass and delayed sporulation. Pre-spore cells and spores of the mutant were frequently double the length of those of the wild-type, implying impaired cross-wall formation, and spores showed reduced tolerance to stress. The mutant accumulated α-maltose 1-phosphate and maltose but no α-glucan. Therefore, the GlgE pathway is necessary and sufficient for polymer biosynthesis. Growth of the ΔglgE mutant on galactose and that of a Δpep2 mutant on maltose were analysed. In both cases, neither accumulation of α-maltose 1-phosphate/α-glucan nor a developmental delay was observed. Thus, high levels of α-maltose 1-phosphate are responsible for the developmental phenotype of the ΔglgE mutant, rather than the lack of α-glucan.
Assuntos
Glucanos/metabolismo , Glucosiltransferases/genética , Esporos Bacterianos/crescimento & desenvolvimento , Streptomyces/crescimento & desenvolvimento , Fosfatos Açúcares/metabolismo , Glicogênio/metabolismo , Maltose/metabolismo , Oligossacarídeos/metabolismo , Esporos Bacterianos/genética , Streptomyces/genética , Trealose/metabolismoRESUMO
Terpenoids constitute the main class of secondary metabolites produced in plants with industrial, pharmacological, and agricultural interests. Nicotiana sylvestris has been widely adopted as a diploid model system in plant biology for studies of terpenoid biosynthesis. In this paper, we report the isolation and analysis of the 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (CMS) gene of the MEP (methylerythritol 4-phosphate) pathway from N. sylvestris. We used homologous-based cloning with a RACE method to obtain the full-length coding sequence of the NsCMS. Then, the physical and chemical properties, function, and three-dimensional structure of the NsyCMS protein were predicted. Fluorogenic quantitative PCR was used to conduct an expression analysis at different developmental stages of various tissues of the NsyCMS. The sequence of the NsyCMS consists of a 954-bp open reading frame and encodes a predicted protein of 317 amino acids, with a molecular weight of approximately 49.6 kDa and pi of 6.92. The in vivo localization of the encoded protein was cytoplasmic with no signal peptide, whereas 2 transmembrane regions were found in NsyCMS. The conserved domains of typical 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, aminotransferase, and pyridoxal phosphate-dependent transferase were found in NsyCMS. Differential expression patterns of the NsyCMS were observed throughout the different developmental stages and tissues. NsyCMS messenger RNA was expressed in all tissues, with the highest level of expression in the seedling leaves. NsyMK was expressed at a higher level in the resettling roots. The results from our study set the foundation for exploring the terpenoid biosynthetic pathways in N. sylvestris.
Assuntos
Nicotiana/enzimologia , Fósforo-Oxigênio Liases/genética , Proteínas de Plantas/genética , Terpenos/metabolismo , Clonagem Molecular , Eritritol/análogos & derivados , Eritritol/biossíntese , Eritritol/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Redes e Vias Metabólicas , Modelos Moleculares , Fósforo-Oxigênio Liases/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Fosfatos Açúcares/metabolismo , Nicotiana/genéticaRESUMO
Xanthomonas citri subsp. citri (Xcc) is a bacterial pathogen that causes citrus canker in susceptible Citrus spp. The Xcc genome contains genes encoding enzymes from three separate pathways of trehalose biosynthesis. Expression of genes encoding trehalose-6-phosphate synthase (otsA) and trehalose phosphatase (otsB) was highly induced during canker development, suggesting that the two-step pathway of trehalose biosynthesis via trehalose-6-phosphate has a function in pathogenesis. This pathway was eliminated from the bacterium by deletion of the otsA gene. The resulting XccΔotsA mutant produced less trehalose than the wild-type strain, was less resistant to salt and oxidative stresses, and was less able to colonize plant tissues. Gene expression and proteomic analyses of infected leaves showed that infection with XccΔotsA triggered only weak defence responses in the plant compared with infection with Xcc, and had less impact on the host plant's metabolism than the wild-type strain. These results suggested that trehalose of bacterial origin, synthesized via the otsA-otsB pathway, in Xcc, plays a role in modifying the host plant's metabolism to its own advantage but is also perceived by the plant as a sign of pathogen attack. Thus, trehalose biosynthesis has both positive and negative consequences for Xcc. On the one hand, it enables this bacterial pathogen to survive in the inhospitable environment of the leaf surface before infection and exploit the host plant's resources after infection, but on the other hand, it is a tell-tale sign of the pathogen's presence that triggers the plant to defend itself against infection.
Assuntos
Citrus/microbiologia , Trealose/fisiologia , Fatores de Virulência/metabolismo , Xanthomonas/patogenicidade , Vias Biossintéticas/genética , Citrus/metabolismo , Citrus/fisiologia , Resistência à Doença , Mutação , Estresse Oxidativo , Fotossíntese , Doenças das Plantas , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Folhas de Planta/fisiologia , Proteoma , Cloreto de Sódio/metabolismo , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Trealose/biossíntese , Trealose/metabolismo , Trealose/farmacologia , Fatores de Virulência/genética , Xanthomonas/enzimologia , Xanthomonas/genéticaRESUMO
The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.
Assuntos
Carotenoides/biossíntese , Eritritol/análogos & derivados , Herbicidas/farmacologia , Isoxazóis/farmacologia , Oxazolidinonas/farmacologia , Fosfatos Açúcares/biossíntese , Bioensaio , Vias Biossintéticas , Relação Dose-Resposta a Droga , Descoberta de Drogas , Eritritol/biossíntese , Hordeum/efeitos dos fármacos , Hordeum/metabolismoRESUMO
SnRK1 activity is developmentally regulated in bean seeds and exhibits a transient increase with the highest value at 20 days after anthesis (DAA), which coincides with the beginning of protein and starch accumulation. The catalytic subunit of SnRK1 shows a consistent decrease throughout the seed development period. However, by 15 DAA a significant proportion of the catalytic subunit appears phosphorylated. The increase in activity and phosphorylation of the catalytic subunit coincides with a decrease in hexoses. However, SnRK1 activity is differentially regulated in the cotyledon and embryo axe, where a larger proportion of the catalytic subunit is phosphorylated. SnRK1 obtained from endosperm extract is inhibited by T6P and to a lesser extent by ADPG and UDPG, whereas the enzyme isolated from embryo is virtually insensitive to T6P but exhibits some inhibition by ADPG and UDPG. In cotyledon extracts, the effects of T6P and ADPG on SnRK1 activity are additive, whereas in embryo extract, T6P inhibits the enzyme only when ADPG is present. After fractionation on Sephacryl-S300, SnRK1 activity obtained from cotyledon extracts is detected as a single peak associated with a molecular weight of 250 kDa whereas that obtained form embryo axe extracts detected as 2 peaks associated with molecular weight of 250 and 180 kDa. In both cases, the catalytic subunit exhibits a wide distribution but is concentrated in the fractions with the highest activity. To analyse the composition of the complex, cotyledon and embryo extracts were treated with a reversible crosslinker (DSP). DSP induced the formation of complexes with molecular weights of 97 and 180 kDa in the cotyledon and embryo extracts, respectively. Since all the phosphorylated catalytic subunit is present in the complexes induced by DSP, it appears that the phosphorylation favors its interaction with other proteins.
Assuntos
Cotilédone/metabolismo , Phaseolus/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Regulação da Expressão Gênica de Plantas , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Trealose/metabolismoRESUMO
The cytosolic mevalonate (MVA) pathway in Hevea brasiliensis latex is the conventionally accepted pathway which provides isopentenyl diphosphate (IPP) for cis-polyisoprene (rubber) biosynthesis. However, the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway may be an alternative source of IPP since its more recent discovery in plants. Quantitative RT-PCR (qRT-PCR) expression profiles of genes from both pathways in latex showed that subcellular compartmentalization of IPP for cis-polyisoprene synthesis is related to the degree of plastidic carotenoid synthesis. From this, the occurrence of two schemes of IPP partitioning and utilization within one species is proposed whereby the supply of IPP for cis-polyisoprene from the MEP pathway is related to carotenoid production in latex. Subsequently, a set of latex unique gene transcripts was sequenced and assembled and they were then mapped to IPP-requiring pathways. Up to eight such pathways, including cis-polyisoprene biosynthesis, were identified. Our findings on pre- and post-IPP metabolic routes form an important aspect of a pathway knowledge-driven approach to enhancing cis-polyisoprene biosynthesis in transgenic rubber trees.
Assuntos
Eritritol/análogos & derivados , Expressão Gênica/genética , Hevea/metabolismo , Látex/análise , Ácido Mevalônico/metabolismo , Borracha/metabolismo , Fosfatos Açúcares/metabolismo , Sequência de Bases , Carotenoides/metabolismo , Eritritol/metabolismo , Biblioteca Gênica , Genes de Plantas/genética , Hevea/genética , Dados de Sequência Molecular , RNA de Plantas/genética , Análise de Sequência de DNA , Terpenos/metabolismo , TranscriptomaRESUMO
Elicitors are compounds or factors capable of triggering a defense response in plants. This kind of response involves signal transduction pathways, second messengers and events such as Reactive Oxygen Species (ROS) generation, proline accumulation and secondary metabolite production. Anthraquinone (AQs) biosynthesis in Rubia tinctorum L. involves different metabolic routes, including shikimate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP+ generated by this cycle could act as a cofactor of the first enzymes of the PPP. The end-product of this pathway is erithrose-4-phosphate, which becomes the substrate of the shikimate pathway. The aim of this work was to study the effect of methyl jasmonate (MeJ), a well-known endogenous elicitor, on the PPP, the proline cycle and AQs production in R. tinctorum cell suspension cultures, and to elucidate the role of ROS in MeJ elicitation. Treatment with MeJ resulted in AQs as well as proline accumulation, which was mimicked by the treatment with a H2O2-generating system. Both MeJ-induced effects were abolished in the presence of diphenyliodonium (DPI), a NADPH oxidase inhibitor (main source of ROS). Treatment with the elicitor failed to induce PPP; therefore, this route did not turn out to be limiting the carbon flux to the shikimate pathway.
Assuntos
Acetatos/farmacologia , Antraquinonas/metabolismo , Ciclopentanos/farmacologia , Oxilipinas/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Prolina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rubia/metabolismo , Antraquinonas/análise , Compostos de Bifenilo/farmacologia , Ciclo do Carbono , Sobrevivência Celular , Células Cultivadas , Eritritol/análogos & derivados , Eritritol/metabolismo , Glutamato Desidrogenase/efeitos dos fármacos , Glutamato Desidrogenase/metabolismo , Peróxido de Hidrogênio/metabolismo , Isocitrato Desidrogenase/efeitos dos fármacos , Isocitrato Desidrogenase/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Via de Pentose Fosfato/efeitos dos fármacos , Imunidade Vegetal , Prolina/análise , Prolina/efeitos dos fármacos , Rubia/citologia , Rubia/enzimologia , Rubia/crescimento & desenvolvimento , Transdução de Sinais , Fosfatos Açúcares/metabolismo , Fatores de TempoRESUMO
D. salina is one of the recognized natural sources to produce beta-carotene, and an useful model for studying the role of inhibitors and enhancers of carotenogenesis. However there is little information in D. salina regarding whether the isoprenoid substrate can be influenced by stress factors (carotenogenic) or selective inhibitors which in turn may further contribute to elucidate the early steps of carotenogenesis and biosynthesis of beta-carotene. In this study, Dunaliella salina (BC02) isolated from La Salina BC Mexico, was subjected to the method of isoprenoids-beta-carotene interference in order to promote the interruption or accumulation of the programmed biosynthesis of carotenoids. When Carotenogenic and non-carotenogenic cells of D. salina BC02 were grown under photoautotrophic growth conditions in the presence of 200 microM fosmidomycin, carotenogenesis and the synthesis of beta-carotene were interrupted after two days in cultured D. salina cells. This result is an indirect consequence of the inhibition of the synthesis of isoprenoids and activity of the recombinant DXR enzyme thereby preventing the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol (MEP) and consequently interrupts the early steps of carotenogenesis in D. salina. The effect at the level of proteins and RNA was not evident. Mevinolin treated D. salina cells exhibited carotenogenesis and beta-carotene levels very similar to those of control cell cultures indicating that mevinolin not pursued any indirect action in the biosynthesis of isoprenoids and had no effect at the level of the HMG-CoA reductase, the key enzyme of the Ac/MVA pathway.
Assuntos
Carotenoides/biossíntese , Clorófitas/isolamento & purificação , Terpenos/metabolismo , California , Células Cultivadas , Clorófitas/crescimento & desenvolvimento , Clorófitas/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Fosfomicina/análogos & derivados , Fosfomicina/farmacologia , Lovastatina/farmacologia , México , Pentosefosfatos/metabolismo , Fosfatos Açúcares/metabolismo , beta Caroteno/biossínteseRESUMO
Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-(13)C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.
Assuntos
Eritritol/análogos & derivados , Euphorbiaceae/genética , Euphorbiaceae/metabolismo , Genes de Plantas/genética , Hevea/genética , Borracha/metabolismo , Fosfatos Açúcares/metabolismo , Sequência de Aminoácidos , Isótopos de Carbono , Clonagem Molecular , Bases de Dados Genéticas , Eritritol/metabolismo , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica de Plantas , Hevea/metabolismo , Dados de Sequência Molecular , Mutação , Filogenia , Reação em Cadeia da Polimerase , Plântula/genética , Plântula/metabolismo , Coloração e Rotulagem , Xilulose/análogos & derivados , Xilulose/metabolismoRESUMO
Biocatalytic procedures offer a good alternative to the chemical synthesis of nucleosides since biocatalyzed reactions are regio- and stereoselective and afford reduced by-products contents. Among them, enzymatic transglycosylation between a pyrimidine nucleoside and a purine base catalyzed by nucleoside phosphorylases or microorganisms that contain them, has attracted considerable attention. In addition, the combination to other enzymatic steps has been explored. In this work we investigate the coupled action of nucleoside phosphorylases with other enzymatic activities: deaminase and phosphopentomutase. Unlike the preparation of other purine nucleosides, transglycosylation from a pyrimidine nucleoside and guanine is difficult because of the low solubility of this base. Therefore, another strategy, based on microbial transglycosylation followed by deamination, is here explored. The direct use of furanose 1-phosphate, the intermediate in the transglycosylation reaction, is an attractive alternative when pyrimidine nucleosides are not available. Its preparation from the more stable furanose 5-phosphate and phosphopentomutase is here applied to different sugars and bases.
Assuntos
Nucleosídeos/biossíntese , Nucleosídeos/química , Adenosina Desaminase/metabolismo , Arthrobacter/enzimologia , Catálise , Pentosiltransferases/metabolismo , Fosfotransferases/metabolismo , Fosfatos Açúcares/químicaRESUMO
In Plasmodium falciparum, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, central intermediates in the biosynthesis of isoprenoids, occurs via the methylerythritol phosphate (MEP) pathway. Fosmidomycin is a specific inhibitor of the second enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase. We analyzed the effect of fosmidomycin on the levels of each intermediate and its metabolic requirement for the isoprenoid biosynthesis, such as dolichols and ubiquinones, throughout the intraerythrocytic cycle of P. falciparum. The steady-state RNA levels of the MEP pathway-associated genes were quantified by real-time polymerase chain reaction and correlated with the related metabolite levels. Our results indicate that MEP pathway metabolite peak precede maximum transcript abundance during the intraerythrocytic cycle. Fosmidomycin-treatment resulted in a decrease of the intermediate levels in the MEP pathway as well as in ubiquinone and dolichol biosynthesis. The MEP pathway associated transcripts were modestly altered by the drug, indicating that the parasite is not strongly responsive at the transcriptional level. This is the first study that compares the effect of fosmidomycin on the metabolic and transcript profiles in P. falciparum, which has only the MEP pathway for isoprenoid biosynthesis.
Assuntos
Eritritol/análogos & derivados , Eritrócitos/parasitologia , Fosfomicina/análogos & derivados , Plasmodium falciparum/efeitos dos fármacos , Fosfatos Açúcares/metabolismo , Animais , Eritritol/metabolismo , Fosfomicina/farmacologia , Genes de Protozoários , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Reação em Cadeia da PolimeraseRESUMO
The biosynthesis of (2S)-2-methyl-2-(4'-methyl-3'-pentenyl)-8-(3''-methyl-2-butenyl)-2H-1-benzopyran-6-carboxylic acid (gaudichaudianic acid), the major metabolite in leaves and roots of Piper gaudichaudianum Kunth (Piperaceae), has been investigated employing [1-(13)C]-D-glucose as precursor. The labelling pattern in the isolated gaudichaudianic acid was determined by quantitative (13)C NMR spectroscopy analysis and was consistent with involvement of both mevalonic acid and 2-C-methyl-D-erythritol-4-phosphate pathways in the formation of the dimethylallyl- and geranyl-derived moieties. The results confirmed that both plastidic and cytoplasmic pathways are able to provide isopentenyl diphosphate units for prenylation of p-hydroxybenzoic acid.