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1.
Plant J ; 95(1): 126-137, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29681129

RESUMO

Starch phosphorylation by starch-related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50-kDa starch-binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α-glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfotransferases (Aceptores Pareados)/metabolismo , Amido/metabolismo , Arabidopsis/enzimologia , Clonagem Molecular , Fosforilação
2.
PLoS One ; 12(11): e0187985, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29155859

RESUMO

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5-7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.


Assuntos
Proteínas de Arabidopsis/genética , Cloroplastos/genética , Sistema da Enzima Desramificadora do Glicogênio/genética , Fosfotransferases (Aceptores Pareados)/genética , Proteínas Tirosina Fosfatases/genética , Amido/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Cloroplastos/ultraestrutura , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/efeitos da radiação , Grânulos Citoplasmáticos/ultraestrutura , Expressão Gênica , Genótipo , Sistema da Enzima Desramificadora do Glicogênio/deficiência , Hidrólise , Luz , Mutação , Fenótipo , Fosfotransferases (Aceptores Pareados)/deficiência , Fotoperíodo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Folhas de Planta/ultraestrutura , Proteínas Tirosina Fosfatases/deficiência , Amido/biossíntese
3.
Sci Rep ; 7(1): 9863, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28852191

RESUMO

ABSTARCT: Regulation of storage root development by source strength remains largely unknown. The cassava storage root delay (srd) T-DNA mutant postpones storage root development but manifests normal foliage growth as wild-type plants. The SRD gene was identified as an orthologue of α-glucan, water dikinase 1 (GWD1), whose expression is regulated under conditions of light/dark cycles in leaves and is associated with storage root development. The GWD1-RNAi cassava plants showed both retarded plant and storage root growth, as a result of starch excess phenotypes with reduced photosynthetic capacity and decreased levels of soluble saccharides in their leaves. These leaves contained starch granules having greatly increased amylose content and type C semi-crystalline structures with increased short chains that suggested storage starch. In storage roots of GWD1-RNAi lines, maltose content was dramatically decreased and starches with much lower phosphorylation levels showed a drastically reduced ß-amylolytic rate. These results suggested that GWD1 regulates transient starch morphogenesis and storage root growth by decreasing photo-assimilation partitioning from the source to the sink and by starch mobilization in root crops.


Assuntos
Metabolismo dos Carboidratos , Glucanos/metabolismo , Manihot/metabolismo , Fosfotransferases (Aceptores Pareados)/metabolismo , Raízes de Plantas/metabolismo , Amido/metabolismo , DNA Bacteriano , Regulação da Expressão Gênica de Plantas , Manihot/genética , Mutação , Fenótipo , Fosforilação , Fosfotransferases (Aceptores Pareados)/genética , Fotossíntese , Análise de Sequência de DNA
4.
Sci Rep ; 7(1): 3339, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28611462

RESUMO

Starch phosphorylation occurs naturally during starch metabolism in the plant and is catalysed by glucan water dikinases (GWD1) and phosphoglucan water dikinase/glucan water dikinase 3 (PWD/GWD3). We generated six stable individual transgenic lines by over-expressing the potato GWD1 in rice. Transgenic rice grain starch had 9-fold higher 6-phospho (6-P) monoesters and double amounts of 3-phospho (3-P) monoesters, respectively, compared to control grain. The shape and topography of the transgenic starch granules were moderately altered including surface pores and less well defined edges. The gelatinization temperatures of both rice flour and extracted starch were significantly lower than those of the control and hence negatively correlated with the starch phosphate content. The 6-P content was positively correlated with amylose content and relatively long amylopectin chains with DP25-36, and the 3-P content was positively correlated with short chains of DP6-12. The starch pasting temperature, peak viscosity and the breakdown were lower but the setback was higher for transgenic rice flour. The 6-P content was negatively correlated with texture adhesiveness but positively correlated with the cohesiveness of rice flour gels. Our data demonstrate a way forward to employ a starch bioengineering approach for clean modification of starch, opening up completely new applications for rice starch.


Assuntos
Amilopectina/metabolismo , Amilose/metabolismo , Oryza/genética , Fosfotransferases (Aceptores Pareados)/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Amilopectina/genética , Amilose/genética , Grão Comestível/genética , Fosforilação , Fosfotransferases (Aceptores Pareados)/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/genética
5.
Physiol Plant ; 160(4): 447-457, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28303594

RESUMO

The genome of Arabidopsis thaliana encodes three glucan, water dikinases. Glucan, water dikinase 1 (GWD1; EC 2.7.9.4) and phosphoglucan, water dikinase (PWD; EC 2.7.9.5) are chloroplastic enzymes, while glucan, water dikinase 2 (GWD2) is cytosolic. Both GWDs and PWD catalyze the addition of phosphate groups to amylopectin chains at the surface of starch granules, changing its physicochemical properties. As a result, GWD1 and PWD have a positive effect on transitory starch degradation at night. Because of its cytosolic localization, GWD2 does not have the same effect. Single T-DNA mutants of either GWD1 or PWD or GWD2 have been analyzed during the entire life cycle of A. thaliana. We report that the three dikinases are all important for proper seed development. Seeds from gwd2 mutants are shrunken, with the epidermal cells of the seed coat irregularly shaped. Moreover, gwd2 seeds contain a lower lipid to protein ratio and are impaired in germination. Similar seed phenotypes were observed in pwd and gwd1 mutants, except for the normal morphology of epidermal cells in gwd1 seed coats. The gwd1, pwd and gwd2 mutants were also very similar in growth and flowering time when grown under continuous light and all three behaved differently from wild-type plants. Besides pinpointing a novel role of GWD2 and PWD in seed development, this analysis suggests that the phenotypic features of the dikinase mutants in A. thaliana cannot be explained solely in terms of defects in leaf starch degradation at night.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Amido/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Metabolismo dos Carboidratos , Cloroplastos/metabolismo , Citosol/metabolismo , Luz , Mutação , Fosforilação , Fosfotransferases (Aceptores Pareados)/genética , Fosfotransferases (Aceptores Pareados)/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Isoformas de Proteínas
6.
Arch Biochem Biophys ; 606: 26-33, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27431058

RESUMO

Although oxidative stress is known to impede the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, the nutritionally-versatile microbe, Pseudomonas fluorescens has been shown to proliferate in the presence of hydrogen peroxide (H2O2) and nitrosative stress. In this study we demonstrate the phospho-transfer system that enables this organism to generate ATP was similar irrespective of the carbon source utilized. Despite the diminished activities of enzymes involved in the TCA cycle and in the electron transport chain (ETC), the ATP levels did not appear to be significantly affected in the stressed cells. Phospho-transfer networks mediated by acetate kinase (ACK), adenylate kinase (AK), and nucleoside diphosphate kinase (NDPK) are involved in maintaining ATP homeostasis in the oxidatively-challenged cells. This phospho-relay machinery orchestrated by substrate-level phosphorylation is aided by the up-regulation in the activities of such enzymes like phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), and phosphoenolpyruvate synthase (PEPS). The enhanced production of phosphoenolpyruvate (PEP) and pyruvate further fuel the synthesis of ATP. Taken together, this metabolic reconfiguration enables the organism to fulfill its ATP need in an O2-independent manner by utilizing an intricate phospho-wire module aimed at maximizing the energy potential of PEP with the participation of AMP.


Assuntos
Trifosfato de Adenosina/química , Pseudomonas fluorescens/metabolismo , Monofosfato de Adenosina/química , Ciclo do Ácido Cítrico , Densitometria , Transporte de Elétrons , Homeostase , Peróxido de Hidrogênio/química , Lipídeos/química , Oxirredução , Fosforilação Oxidativa , Estresse Oxidativo , Oxigênio/química , Fosfoenolpiruvato/química , Fosforilação , Fosfotransferases (Aceptores Pareados)/metabolismo , Piruvato Ortofosfato Diquinase/metabolismo , Espécies Reativas de Oxigênio/metabolismo
7.
Plant Biotechnol J ; 14(1): 398-408, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25989474

RESUMO

Starch phosphate ester content is known to alter the physicochemical properties of starch, including its susceptibility to degradation. Previous work producing wheat (Triticum aestivum) with down-regulated glucan, water dikinase, the primary gene responsible for addition of phosphate groups to starch, in a grain-specific manner found unexpected phenotypic alteration in grain and growth. Here, we report on further characterization of these lines focussing on mature grain and early growth. We find that coleoptile length has been increased in these transgenic lines independently of grain size increases. No changes in starch degradation rates during germination could be identified, or any major alteration in soluble sugar levels that may explain the coleoptile growth modification. We identify some alteration in hormones in the tissues in question. Mature grain size is examined, as is Hardness Index and starch conformation. We find no evidence that the increased growth of coleoptiles in these lines is connected to starch conformation or degradation or soluble sugar content and suggest these findings provide a novel means of increasing coleoptile growth and early seedling establishment in cereal crop species.


Assuntos
Cotilédone/crescimento & desenvolvimento , Endosperma/enzimologia , Germinação , Glucanos/metabolismo , Fosfotransferases (Aceptores Pareados)/metabolismo , Sementes/anatomia & histologia , Triticum/enzimologia , Água/metabolismo , Amilopectina/metabolismo , Dureza , Modelos Biológicos , Tamanho do Órgão , Fosfatos/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas , Plantas Geneticamente Modificadas , Plântula/crescimento & desenvolvimento , Amido/metabolismo , Transgenes , Triticum/anatomia & histologia , Triticum/embriologia , alfa-Amilases/metabolismo
8.
Ukr Biochem J ; 87(2): 66-75, 2015.
Artigo em Ucraniano | MEDLINE | ID: mdl-26255340

RESUMO

It has been established that in cells of Nocardia vaccinii IMB B-7405 (surfactant producer) glucose catabolism is performed through pentose phosphate cycle as well as through gluconate (activity of NAD+-dependent glucose-6-phosphate dehydrogenase and FAD+-dependent glucose dehydrogenase 835 ± 41 and 698 ± 35 nmol.min-1.mg-1 of protein respectively). 6-Phosphogluconate formed in the gluconokinase reaction is involved in the pentose phosphate cycle (activity of constitutive NADP+-dependent 6-phosphogluconate dehydrogenase 357 ± 17 nmol.min-1.mg-1 of protein). Glycerol catabolism to dihydroxyacetonephosphate (the intermediate of glycolysis) may be performed in two ways: through glycerol-3-phosphate (glycerol kinase activity 244 ± 12 nmol.min-1.mg-1 of protein) and through dihydroxyacetone. Replenishment of the C4-dicarboxylic acids pool in N. vaccinii IMV B-7405 grown on glucose and glycerol occurs in the phosphoenolpyruvate(PEP)carboxylase reaction (714-803 nmol.min-1.mg-1 of protein). 2-Oxoglutarate was involved in tricarboxylic acid cycle by alternate pathway with the participation of 2-oxoglutarate synthase. The observed activity of both key enzymes of gluconeogenesis (PEP-carboxykinase and PEP-synthase), trehalose phosphate synthase and NADP+-dependent glutamate dehydrogenase confirmed the ability of IMV B-7405 strain to the synthesis of surface active glycoand aminolipids, respectively.


Assuntos
Proteínas de Bactérias/metabolismo , Glucose/metabolismo , Glicerol/metabolismo , Nocardia/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Di-Hidroxiacetona/metabolismo , Fosfato de Di-Hidroxiacetona/metabolismo , Gluconatos/metabolismo , Gluconeogênese/fisiologia , Glucose 1-Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Desidrogenase de Glutamato (NADP+)/metabolismo , Glicerofosfatos/metabolismo , Glicólise/fisiologia , Ácidos Cetoglutáricos/metabolismo , Cetona Oxirredutases/metabolismo , Via de Pentose Fosfato/fisiologia , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato Carboxilase/metabolismo , Fosfogluconato Desidrogenase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptores Pareados)/metabolismo
9.
Biochem Cell Biol ; 93(3): 236-40, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25707819

RESUMO

Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3(-) complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3 on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.


Assuntos
Inibidores Enzimáticos/farmacologia , Fluoretos/farmacologia , Compostos de Magnésio/farmacologia , Fosfotransferases (Aceptores Pareados)/antagonistas & inibidores , Fosfotransferases (Aceptores Pareados)/metabolismo , Fluoreto de Sódio/farmacologia , Cloreto de Alumínio , Compostos de Alumínio/farmacologia , Cloretos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Fosfotransferases (Aceptores Pareados)/genética , Piruvato Sintase/antagonistas & inibidores , Piruvato Sintase/genética , Piruvato Sintase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Bioresour Technol ; 166: 64-71, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24905044

RESUMO

Shikimate is a key intermediate for the synthesis of the neuraminidase inhibitors. Microbial production of shikimate and related derivatives has the benefit of cost reduction when compared to traditional methods. In this study, an overproducing shikimate Escherichia coli strain was developed by rationally engineering certain metabolic pathways. To achieve this, the shikimate pathway was blocked by deletion of shikimate kinases and quinic acid/shikimate dehydrogenase. EIICB(glc) protein involved in the phosphotransferase system, and acetic acid pathway were also removed to increase the amount of available phosphoenolpyruvate and decrease byproduct formation, respectively. Thereafter, three critical enzymes of mutated 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (encoded by aroG(fbr)), PEP synthase (encoded by ppsA), and transketolase A (encoded by tktA) were modularly overexpressed and the resulting recombinant strain produced 1207 mg/L shikimate in shake flask cultures. Using the fed-batch process, 14.6g/L shikimate with a yield of 0.29 g/g glucose was generated in a 7-L bioreactor.


Assuntos
Reatores Biológicos , Vias Biossintéticas/fisiologia , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/fisiologia , Ácido Chiquímico/metabolismo , 3-Desoxi-7-Fosfo-Heptulonato Sintase , Oxirredutases do Álcool/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptores Pareados) , Ácido Quínico/metabolismo , Transcetolase
11.
Wei Sheng Wu Xue Bao ; 54(1): 24-32, 2014 Jan 04.
Artigo em Chinês | MEDLINE | ID: mdl-24783851

RESUMO

OBJECTIVE: In order to redirect carbon flows into aromatic amino acids biosynthesis pathway and further improve the production of L-tryptophan in Corynebacterium pekinense PD-67, two schemes were implemented. First, the supply of phosphoenolpyruvate (PEP), one of precursors of L-tryptophan biosynthesis, was increased. Second, the feedback inhibition of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DS), a key enzyme in the aromatic amino acids biosynthesis, was relieved and the activity of DS was increased. METHODS: The phosphoenolpyruvate synthase gene (pps) was cloned from C. pekinense PD-67 chromosome by PCR and inserted into expression vector to construct a recombinant plasmid pXPPS; the aroG gene encoding DS isozymes was cloned from Escherichia coli chromosome by PCR and the mutation of Leu175Asp was introduced by site-directed mutagenesis using sequence-overlap extension PCR. The mutated gene named as aroGfbr was cloned to expression vector to construct a recombinant plasmid pXA; and the recombinant plasmid pXAPS co-expressing pps and aroGfbr was constructed. The three recombinant plasmids were transformed into PD-67 to generate the engineering strains PD-67/pXPS, PD-67/pXA and PD-67/pXAPS, respectively. The fermentation characteristics of the three engineering strains were investigated. RESULTS: The expression of pps and aroGfbr was confirmed by enzyme activity assays. The deregulation of feedback inhibition of AroGfbr was confirmed by determining DS activity in the presence of three aromatic amino acids. The overexpression of pps and aroGfbr resulted in an increase of L-tryptophan biosynthesis by 12.1% and 26.8%, respectively, while the co-expression of two genes increased the production of L-tryptophan by 35.9% in the engineering strain PD-67/pXAPS. CONCLUSION: Both of the overexpressions of the pps gene and aroGfbr gene can increase L-tryptophan biosynthesis, while the production was further improved by the co-expression of the two genes.


Assuntos
Corynebacterium/genética , Corynebacterium/metabolismo , Engenharia Genética , Fosfotransferases (Aceptores Pareados)/genética , Triptofano/biossíntese , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Corynebacterium/enzimologia , Expressão Gênica , Vetores Genéticos/genética , Fosfotransferases (Aceptores Pareados)/metabolismo , Análise de Sequência
12.
New Phytol ; 203(2): 495-507, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24697163

RESUMO

Glucan, water dikinase (GWD) is a key enzyme of starch metabolism but the physico-chemical properties of starches isolated from GWD-deficient plants and their implications for starch metabolism have so far not been described. Transgenic Arabidopsis thaliana plants with reduced or no GWD activity were used to investigate the properties of starch granules. In addition, using various in vitro assays, the action of recombinant GWD, ß-amylase, isoamylase and starch synthase 1 on the surface of native starch granules was analysed. The internal structure of granules isolated from GWD mutant plants is unaffected, as thermal stability, allomorph, chain length distribution and density of starch granules were similar to wild-type. However, short glucan chain residues located at the granule surface dominate in starches of transgenic plants and impede GWD activity. A similarly reduced rate of phosphorylation by GWD was also observed in potato tuber starch fractions that differ in the proportion of accessible glucan chain residues at the granule surface. A model is proposed to explain the characteristic morphology of starch granules observed in GWD transgenic plants. The model postulates that the occupancy rate of single glucan chains at the granule surface limits accessibility to starch-related enzymes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Fosfotransferases (Aceptores Pareados)/metabolismo , Amido/química , Amido/metabolismo , Proteínas de Arabidopsis/genética , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Isoamilase/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação , Fosforilação , Fosfotransferases (Aceptores Pareados)/genética , Plantas Geneticamente Modificadas , Solanum tuberosum , Amido/genética , Amido/ultraestrutura , Propriedades de Superfície , beta-Amilase/metabolismo
13.
Plant Signal Behav ; 9(7): e28892, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25763482

RESUMO

Starch phosphorylation mediated by the α-glucan, water dikinase (GWD) is crucial for transitory starch metabolism. The impact of the GWD action on transitory starch metabolism was analyzed in Arabidopsis mutants either lacking or revealing different reduced levels of GWD activity. In these mutants, glucose 6-phosphate (G6P) levels of the transitory leaf starch, the average leaf starch content, as well as alterations in the growth phenotype were determined under different light length conditions, including continuous light. Based on biochemical and growth phenotypical data, we found that the length of the light phase affects the phosphorylation state of the transitory starch and, by this, the average leaf starch content and the resulting growth of the plants. Additionally, we discuss data referring to an involvement of the GWD mediated glucan phosphorylation in starch synthesis, as, e.g., starch phosphorylation occurred even when a dark phase was omitted.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glucanos/metabolismo , Luz , Fosfotransferases (Aceptores Pareados)/metabolismo , Fotoperíodo , Folhas de Planta/metabolismo , Amido/metabolismo , Arabidopsis/crescimento & desenvolvimento , Glucose-6-Fosfato/metabolismo , Fosforilação , Plastídeos/metabolismo , Água
14.
J Biotechnol ; 167(3): 309-15, 2013 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-23871654

RESUMO

Pseudomonas fluorescens invoked a metabolic reconfiguration that resulted in enhanced production of pyruvate under the challenge of hydrogen peroxide (H2O2). Although this stress led to a sharp reduction in the activities of numerous tricarboxylic acid (TCA) cycle enzymes, there was a marked increase in the activities of catalase and various NADPH-generating enzymes to counter the oxidative burden. The upregulation of phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) coupled with the reduction of pyruvate dehydrogenase (PDH) in the H2O2-challenged cells appear to be important contributors to the elevated levels of pyruvate found in these bacteria. Increased pyruvate synthesis was evident in the presence of a variety of carbon sources including d-glucose. Intact cells rapidly consumed d-glucose with the concomitant formation of this monocarboxylic acid. At least a 12-fold increase in pyruvate production within 1h was observed in the stressed cells. These findings may be exploited in the development of technologies aimed at the conversion of carbohydrates into pyruvate.


Assuntos
Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Pseudomonas fluorescens/efeitos dos fármacos , Piruvatos/metabolismo , Redes e Vias Metabólicas , Fosfotransferases (Aceptores Pareados)/metabolismo , Pseudomonas fluorescens/metabolismo , Piruvato Quinase/metabolismo , Regulação para Cima/efeitos dos fármacos
15.
Nucleic Acids Res ; 41(18): 8546-58, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23892291

RESUMO

Organisms across all three domains of life use gene regulatory networks (GRNs) to integrate varied stimuli into coherent transcriptional responses to environmental pressures. However, inferring GRN topology and regulatory causality remains a central challenge in systems biology. Previous work characterized TrmB as a global metabolic transcription factor in archaeal extremophiles. However, it remains unclear how TrmB dynamically regulates its ∼100 metabolic enzyme-coding gene targets. Using a dynamic perturbation approach, we elucidate the topology of the TrmB metabolic GRN in the model archaeon Halobacterium salinarum. Clustering of dynamic gene expression patterns reveals that TrmB functions alone to regulate central metabolic enzyme-coding genes but cooperates with various regulators to control peripheral metabolic pathways. Using a dynamical model, we predict gene expression patterns for some TrmB-dependent promoters and infer secondary regulators for others. Our data suggest feed-forward gene regulatory topology for cobalamin biosynthesis. In contrast, purine biosynthesis appears to require TrmB-independent regulators. We conclude that TrmB is an important component for mediating metabolic modularity, integrating nutrient status and regulating gene expression dynamics alone and in concert with secondary regulators.


Assuntos
Proteínas Arqueais/metabolismo , Regulação da Expressão Gênica em Archaea , Redes Reguladoras de Genes , Halobacterium salinarum/genética , Fatores de Transcrição/metabolismo , Glucose/metabolismo , Halobacterium salinarum/metabolismo , Fosfotransferases (Aceptores Pareados)/genética , Regiões Promotoras Genéticas , Transcrição Genética
16.
J Biotechnol ; 167(2): 111-22, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23792782

RESUMO

Xanthomonas campestris pv. campestris (Xcc) synthesizes huge amounts of the exopolysaccharide xanthan and is a plant pathogen affecting Brassicaceae, among them the model plant Arabidopsis thaliana. Xanthan is produced as a thickening agent at industrial scale by fermentation of Xcc. In an approach based on 2D gel electrophoresis, protein samples from different growth phases were characterized to initialize analysis of the Xanthomonas phosphoproteome. The 2D gels were stained with Pro-Q Diamond phosphoprotein stain to identify putatively phosphorylated proteins. Spots of putatively phosphorylated proteins were excised from the gel and analyzed by mass spectrometry. Three proteins were confirmed to be phosphorylated, the phosphoglucomutase/phosphomannomutase XanA that is important for xanthan and lipopolysaccharide biosynthesis, the phosphoenolpyruvate synthase PspA that is involved in gluconeogenesis, and an anti-sigma factor antagonist RsbR that was so far uncharacterized in xanthomonads. The growth phase in which the samples were collected had an influence on protein phosphorylation in Xcc, particular distinct in case of RsbR, which was phosphorylated during the transition from the late exponential growth phase to the stationary phase.


Assuntos
Proteínas de Bactérias/metabolismo , Proteômica/métodos , Xanthomonas campestris/metabolismo , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional/métodos , Gluconeogênese/fisiologia , Lipopolissacarídeos/metabolismo , Dados de Sequência Molecular , Fosfoglucomutase/metabolismo , Fosforilação , Fosfotransferases (Aceptores Pareados)/metabolismo , Fosfotransferases (Fosfomutases)/metabolismo , Polissacarídeos Bacterianos/metabolismo , Xanthomonas campestris/crescimento & desenvolvimento
17.
Nat Chem Biol ; 9(7): 416-21, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23708076

RESUMO

Despite its importance in central metabolism and bacterial cell signaling, protein histidine phosphorylation has remained elusive with respect to its extent and functional roles in biological systems because of the lack of adequate research tools. We report the development of the first pan-phosphohistidine (pHis) antibody using a stable pHis mimetic as the hapten. This antibody was successfully used in ELISA, western blotting, dot blot assays and immunoprecipitation and in detection and identification of histidine-phosphorylated proteins from native cell lysates when coupled with MS analysis. We also observed that the amount of protein pHis in Escherichia coli lysates depends on carbon source and nitrogen availability in the growth medium. In particular, we found that the amount of pHis on phosphoenolpyruvate synthase (PpsA) is sensitive to nitrogen availability in vivo and that α-ketoglutarate inhibits phosphotransfer from phosphorylated PpsA to pyruvate. We expect this antibody to open opportunities for investigating other pHis proteins and their functions.


Assuntos
Anticorpos/química , Histidina/química , Proteínas/química , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Histidina/análogos & derivados , Concentração de Íons de Hidrogênio , Íons , Ácidos Cetoglutáricos/metabolismo , Espectrometria de Massas , Fosforilação , Fosfotransferases (Aceptores Pareados)/metabolismo , Piruvato Sintase/química , Proteínas Recombinantes/química
18.
Mikrobiol Z ; 75(1): 3-13, 2013.
Artigo em Russo | MEDLINE | ID: mdl-23516834

RESUMO

Synthesis of biosurfactants (surface-active substances, SAS) was investigated under the conditions of growth of Rhodococcus erythropolis IMV Ac-5017 and Acinetobacter calcoaceticus IMV B-7241 on hydrophobic (n-hexadecane, liquid paraffins, sunflower oil) and hydrophilic (ethanol) substrates depending on concentration (0.01-0.5 mM) and time of copper cations introduction in the medium. It is established that Cu2+ addition in the exponential phase of growth of the strains IMV B-7241 and IMV Ac-5017 on all studied substrates was accompanied by the increase of conventional concentration of SAS by 25-140% as compared with the indices in the medium without copper cations. Maximum synthesis intensification of SAS of A. calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 was observed in the case of Cu2+ introduction in the medium with hydrocarbons. The increase of SAS synthesis in the presence of copper cations is determined by their activating effect on activity of alkane hydroxylase of the both strains, as well as 4-nitroso-N,N-dimethylaniline-dependent alcohol dehydrogenase and enzymes of biosynthesis of surface active glyco-(phosphoenolpyruvate-synthetase) and aminolipids (NADP(+)-dependent glutamate dehydrogenase) in A. calcoaceticus IMV B-7241.


Assuntos
Acinetobacter calcoaceticus/metabolismo , Cobre/metabolismo , Rhodococcus/metabolismo , Tensoativos/metabolismo , Acinetobacter calcoaceticus/efeitos dos fármacos , Álcool Desidrogenase/metabolismo , Alcanos/metabolismo , Cátions Bivalentes , Cobre/farmacologia , Meios de Cultura , Citocromo P-450 CYP4A/metabolismo , Etanol/metabolismo , Desidrogenase de Glutamato (NADP+) , Óleo Mineral/metabolismo , Fosfotransferases (Aceptores Pareados) , Óleos Vegetais/metabolismo , Rhodococcus/efeitos dos fármacos , Óleo de Girassol
19.
Mikrobiol Z ; 75(5): 18-26, 2013.
Artigo em Russo | MEDLINE | ID: mdl-24479309

RESUMO

The effect of yeast autolysate and microelements on synthesis of surface-active substances (SAS, biosurfactants) was investigated under cultivation of Acinetobacter calcoaceticus IMV B-7241 on various carbon substrates (n-hexadecane, ethanol, glycerol). The authors have shown a possibility to substitute the yeast autolysate and microelement mixture in the composition of ethanol- and n-hexadecane-containing media by copper sulfate (0.16 micromol/l) and iron sulfate (3.6 micromol/l), and in the medium with glycerol by 0.21 mmol/l of KCl, 38 micromol/l of zinc sulfate and 0.16 micromol/l of copper sulfate. Under such conditions of cultivation of the strain IMV B-7241 the SAS concentration exceeded that on the initial media, which contained the yeast autolysate and microelements, 1.2-1.6 times. The authors have also established the activating effect of low (0.01 mM) concentrations of Fe2+ on activity of the enzymes of biosynthesis of surface-active amino- (NADP-dependent glutamate dehydrogenase) and glycolipids (phosphoenolpyruvate(PhEP)-synthetase, PhEP-carboxykinase), as well as of anaplerotic reaction(PhEP-carboxylase). A necessity to introduce zinc cations into glycerol-containing medium is determined by their stimulating effect on activity of 4-dinitroso-N,N-dimethylaniline-dependent alcohol dehydrogenase--one of the enzymes of this substrate catabolism in A. calcoaceticus IMV B-7241.


Assuntos
Acinetobacter calcoaceticus/metabolismo , Alcanos/metabolismo , Proteínas de Bactérias/metabolismo , Misturas Complexas/farmacologia , Etanol/metabolismo , Glicerol/metabolismo , Tensoativos/metabolismo , Álcool Desidrogenase/metabolismo , Misturas Complexas/química , Misturas Complexas/metabolismo , Sulfato de Cobre/metabolismo , Sulfato de Cobre/farmacologia , Compostos Ferrosos/metabolismo , Compostos Ferrosos/farmacologia , Desidrogenase de Glutamato (NADP+)/metabolismo , Glicolipídeos/biossíntese , Fosfoenolpiruvato Carboxilase/metabolismo , Fosfotransferases (Aceptores Pareados)/metabolismo , Leveduras/química , Sulfato de Zinco/metabolismo , Sulfato de Zinco/farmacologia
20.
Genetika ; 48(5): 608-16, 2012 May.
Artigo em Russo | MEDLINE | ID: mdl-22830256

RESUMO

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.


Assuntos
Acil-Butirolactonas/metabolismo , Burkholderia cenocepacia/genética , Regulação Bacteriana da Expressão Gênica , Fosfotransferases (Aceptores Pareados)/genética , Protease La/genética , Percepção de Quorum/genética , Animais , Biofilmes , Burkholderia cenocepacia/crescimento & desenvolvimento , Burkholderia cenocepacia/patogenicidade , Masculino , Camundongos , Mutação , Fosfotransferases (Aceptores Pareados)/metabolismo , Protease La/metabolismo , Virulência/genética
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