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1.
Appl Environ Microbiol ; 85(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30737350

RESUMO

LeLoir glycosyltransferases are important biocatalysts for the production of glycosidic bonds in natural products, chiral building blocks, and pharmaceuticals. Trehalose transferase (TreT) is of particular interest since it catalyzes the stereo- and enantioselective α,α-(1→1) coupling of a nucleotide sugar donor and monosaccharide acceptor for the synthesis of disaccharide derivatives. Heterologously expressed thermophilic trehalose transferases were found to be intrinsically aggregation prone and are mainly expressed as catalytically active inclusion bodies in Escherichia coli To disfavor protein aggregation, the thermostable protein mCherry was explored as a fluorescent protein tag. The fusion of mCherry to trehalose transferase from Pyrobaculum yellowstonensis (PyTreT) demonstrated increased protein solubility. Chaotropic agents like guanidine or the divalent cations Mn(II), Ca(II), and Mg(II) enhanced the enzyme activity of the fusion protein. The thermodynamic equilibrium constant, K eq, for the reversible synthesis of trehalose from glucose and a nucleotide sugar was determined in both the synthesis and hydrolysis directions utilizing UDP-glucose and ADP-glucose, respectively. UDP-glucose was shown to achieve higher conversions than ADP-glucose, highlighting the importance of the choice of nucleotide sugars for LeLoir glycosyltransferases under thermodynamic control.IMPORTANCE The heterologous expression of proteins in Escherichia coli is of great relevance for their functional and structural characterization and applications. However, the formation of insoluble inclusion bodies is observed in approximately 70% of all cases, and the subsequent effects can range from reduced soluble protein yields to a complete failure of the expression system. Here, we present an efficient methodology for the production and analysis of a thermostable, aggregation-prone trehalose transferase (TreT) from Pyrobaculum yellowstonensis via its fusion with mCherry as a thermostable fluorescent protein tag. This fusion strategy allowed for increased enzyme stability and solubility and could be applied to other (thermostable) proteins, allowing rapid visualization and quantification of the mCherry-fused protein of interest. Finally, we have demonstrated that the enzymatic synthesis of trehalose from glucose and a nucleotide sugar is reversible by approaching the thermodynamic equilibrium in both the synthesis and hydrolysis directions. Our results show that uridine establishes an equilibrium constant which is more in favor of the product trehalose than when adenosine is employed as the nucleotide under identical conditions. The influence of different nucleotides on the reaction can be generalized for all LeLoir glycosyltransferases under thermodynamic control as the position of the equilibrium depends solely on the reaction conditions and is not affected by the nature of the catalyst.


Assuntos
Estabilidade Enzimática , Proteínas Recombinantes de Fusão/metabolismo , Transferases/metabolismo , Trealose/metabolismo , Adenosina Difosfato Glucose , Ânions , Catálise , Cátions , Ativação Enzimática , Estabilidade Enzimática/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Glucose/metabolismo , Glicosiltransferases/metabolismo , Cinética , Agregados Proteicos , Pyrobaculum/enzimologia , Pyrobaculum/genética , Proteínas Recombinantes de Fusão/genética , Solubilidade , Transferases/genética , Uridina Difosfato Glucose
2.
Sci Rep ; 8(1): 15509, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30341391

RESUMO

ADP-glucose is the precursor of glycogen biosynthesis in bacteria, and a compound abundant in the starchy plant organs ingested by many mammals. Here we show that the enteric species Escherichia coli is capable of scavenging exogenous ADP-glucose for use as a glycosyl donor in glycogen biosynthesis and feed the adenine nucleotide pool. To unravel the molecular mechanisms involved in this process, we screened the E. coli single-gene deletion mutants of the Keio collection for glycogen content in ADP-glucose-containing culture medium. In comparison to wild-type (WT) cells, individual ∆nupC and ∆nupG mutants lacking the cAMP/CRP responsive inner-membrane nucleoside transporters NupC and NupG displayed reduced glycogen contents and slow ADP-glucose incorporation. In concordance, ∆cya and ∆crp mutants accumulated low levels of glycogen and slowly incorporated ADP-glucose. Two-thirds of the glycogen-excess mutants identified during screening lacked functions that underlie envelope biogenesis and integrity, including the RpoE specific RseA anti-sigma factor. These mutants exhibited higher ADP-glucose uptake than WT cells. The incorporation of either ∆crp, ∆nupG or ∆nupC null alleles sharply reduced the ADP-glucose incorporation and glycogen content initially witnessed in ∆rseA cells. Overall, the data showed that E. coli incorporates extracellular ADP-glucose through a cAMP/CRP-regulated process involving the NupC and NupG nucleoside transporters that is facilitated under envelope stress conditions.


Assuntos
Adenosina Difosfato Glucose/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Espaço Extracelular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Nucleotídeos de Adenina/metabolismo , Transporte Biológico , Escherichia coli/genética , Genes Bacterianos , Glicogênio/biossíntese , Glicogênio Sintase/metabolismo , Modelos Biológicos , Estresse Fisiológico
3.
Plant Cell Environ ; 40(9): 1834-1848, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28556250

RESUMO

Nitric oxide (NO) is extensively involved in various growth processes and stress responses in plants; however, the regulatory mechanism of NO-modulated cellular sugar metabolism is still largely unknown. Here, we report that NO significantly inhibited monosaccharide catabolism by modulating sugar metabolic enzymes through S-nitrosylation (mainly by oxidizing dihydrolipoamide, a cofactor of pyruvate dehydrogenase). These S-nitrosylation modifications led to a decrease in cellular glycolysis enzymes and ATP synthase activities as well as declines in the content of acetyl coenzyme A, ATP, ADP-glucose and UDP-glucose, which eventually caused polysaccharide-biosynthesis inhibition and monosaccharide accumulation. Plant developmental defects that were caused by high levels of NO included delayed flowering time, retarded root growth and reduced starch granule formation. These phenotypic defects could be mediated by sucrose supplementation, suggesting an essential role of NO-sugar cross-talks in plant growth and development. Our findings suggest that molecular manipulations could be used to improve fruit and vegetable sweetness.


Assuntos
Arabidopsis/metabolismo , Monossacarídeos/metabolismo , Óxido Nítrico/farmacologia , Complexos de ATP Sintetase/metabolismo , Adenosina Difosfato Glucose/metabolismo , Trifosfato de Adenosina/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Glicólise/efeitos dos fármacos , Mutação/genética , Nitrosação , Oxirredução , Fenótipo , Desenvolvimento Vegetal/efeitos dos fármacos , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Solubilidade , Amido/metabolismo , Sacarose/farmacologia , Ácido Tióctico/análogos & derivados , Ácido Tióctico/metabolismo , Uridina Difosfato Glucose/metabolismo
4.
Sci Rep ; 7: 40124, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-28054650

RESUMO

Starch is the main storage carbohydrate in higher plants. Although several enzymes and regulators for starch biosynthesis have been characterized, a complete regulatory network for starch synthesis in cereal seeds remains elusive. Here, we report the identification and characterization of the rice Brittle1 (OsBT1) gene, which is expressed specifically in the developing endosperm. The osbt1 mutant showed a white-core endosperm and a significantly lower grain weight than the wild-type. The formation and development of compound starch granules in osbt1 was obviously defective: the amyloplast was disintegrated at early developmental stages and the starch granules were disperse and not compound in the endosperm cells in the centre region of osbt1 seeds. The total starch content and amylose content was decreased and the physicochemical properties of starch were altered. Moreover, the degree of polymerization (DP) of amylopectin in osbt1 was remarkably different from that of wild-type. Map-based cloning of OsBT1 indicated that it encodes a putatively ADP-glucose transporter. OsBT1 coded protein localizes in the amyloplast envelope membrane. Furthermore, the expression of starch synthesis related genes was also altered in the osbt1 mutant. These findings indicate that OsBT1 plays an important role in starch synthesis and the formation of compound starch granules.


Assuntos
Adenosina Difosfato Glucose/metabolismo , Endosperma/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Oryza/enzimologia , Plastídeos/enzimologia , Amido/biossíntese , Amilopectina/metabolismo , Deleção de Genes , Proteínas de Membrana Transportadoras/genética , Oryza/genética , Oryza/metabolismo
5.
Plant Physiol ; 170(3): 1271-83, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26754668

RESUMO

Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose (ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter (OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093 were due to their poor uptake of ADP-[(14)C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when these plant lines were grown under elevated CO2. Overall, our results indicate that rice lines with enhanced ADPglc synthesis and import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.


Assuntos
Adenosina Difosfato Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Amido/metabolismo , Genes de Plantas , Proteínas Facilitadoras de Transporte de Glucose/genética , Mutação , Oryza/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plastídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sementes/metabolismo , Zea mays/enzimologia , Zea mays/genética
6.
Plant Physiol ; 167(4): 1321-31, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25717036

RESUMO

A unique CO2-Responsive CONSTANS, CONSTANS-like, and Time of Chlorophyll a/b Binding Protein1 (CCT) Protein (CRCT) containing a CCT domain but not a zinc finger motif is described, which is up-regulated under elevated CO2 in rice (Oryza sativa). The expression of CRCT showed diurnal oscillation peaked at the end of the light period and was also increased by sugars such as glucose and sucrose. Promoter ß-glucuronidase analysis showed that CRCT was highly expressed in the phloem of various tissues such as leaf blade and leaf sheath. Overexpression or RNA interference knockdown of CRCT had no appreciable effect on plant growth and photosynthesis except that tiller angle was significantly increased by the overexpression. More importantly, starch content in leaf sheath, which serves as a temporary storage organ for photoassimilates, was markedly increased in overexpression lines and decreased in knockdown lines. The expressions of several genes related to starch synthesis, such as ADP-glucose pyrophospholylase and α-glucan phospholylase, were significantly changed in transgenic lines and positively correlated with the expression levels of CRCT. Given these observations, we suggest that CRCT is a positive regulator of starch accumulation in vegetative tissues, regulating coordinated expression of starch synthesis genes in response to the levels of photoassimilates.


Assuntos
Dióxido de Carbono/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Amido/metabolismo , Adenosina Difosfato Glucose/metabolismo , Metabolismo dos Carboidratos , Clorofila/metabolismo , Clorofila A , Expressão Gênica , Técnicas de Silenciamento de Genes , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Oryza/citologia , Oryza/genética , Floema/citologia , Floema/genética , Floema/metabolismo , Fosforilases/genética , Fosforilases/metabolismo , Fotossíntese , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética
7.
J Bacteriol ; 197(8): 1394-407, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25666133

RESUMO

UNLABELLED: α-Glucan phosphorylases contribute to degradation of glycogen and maltodextrins formed in the course of maltose metabolism in bacteria. Accordingly, bacterial α-glucan phosphorylases are classified as either glycogen or maltodextrin phosphorylase, GlgP or MalP, respectively. GlgP and MalP enzymes follow the same catalytic mechanism, and thus their substrate spectra overlap; however, they differ in their regulation: GlgP genes are constitutively expressed and the enzymes are controlled on the activity level, whereas expression of MalP genes are transcriptionally controlled in response to the carbon source used for cultivation. We characterize here the modes of control of the α-glucan phosphorylase MalP of the Gram-positive Corynebacterium glutamicum. In accordance to the proposed function of the malP gene product as MalP, we found transcription of malP to be regulated in response to the carbon source. Moreover, malP transcription is shown to depend on the growth phase and to occur independently of the cell glycogen content. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. Since the latter is considered a typical feature of GlgPs, we propose that C. glutamicum MalP acts as both maltodextrin and glycogen phosphorylase and, based on these findings, we question the current system for classification of bacterial α-glucan phosphorylases. IMPORTANCE: Bacterial α-glucan phosphorylases have been classified conferring to their purpose as either glycogen or maltodextrin phosphorylases. We found transcription of malP in C. glutamicum to be regulated in response to the carbon source, which is recognized as typical for maltodextrin phosphorylases. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. The latter is considered a typical feature of GlgPs. These findings, taken together, suggest that C. glutamicum MalP is the first α-glucan phosphorylase that does not fit into the current system for classification of bacterial α-glucan phosphorylases and exemplifies the complex mechanisms underlying the control of glycogen content and maltose metabolism in this model organism.


Assuntos
Adenosina Difosfato Glucose/metabolismo , Corynebacterium glutamicum/enzimologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Fosforilases/metabolismo , Transcrição Genética/fisiologia , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Fosforilases/genética
8.
PLoS One ; 9(8): e104997, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25133777

RESUMO

In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low.


Assuntos
Adenosina Difosfato Glucose/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatografia Líquida de Alta Pressão , Glucose-1-Fosfato Adenililtransferase/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Sintase do Amido/metabolismo , Espectrometria de Massas em Tandem , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-6-Fosfato Isomerase , Plastídeos/enzimologia , Plastídeos/metabolismo , Sintase do Amido/genética
9.
Plant Biotechnol J ; 12(9): 1297-307, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25052102

RESUMO

The rice Waxy (Wx) gene encodes granule-bound starch synthase 1 (EC 2.4.1.242), OsGBSS1, which is responsible for amylose synthesis in rice seed endosperm. In this study, we determined the functional contribution of eight amino acids on the activity of OsGBSS1 by introducing site-directed mutated Wx gene constructs into the wx mutant glutinous rice. The eight amino acid residues are suspected to play roles in OsGBSS1 structure maintenance or function based on homologous enzyme sequence alignment and homology modelling. Both OsGBSS1 activity and amylose content were analysed in homozygous transgenic lines carrying the mutated OsGBSS1 (Wx) genes. Our results indicate that mutations at diverse sites in OsGBSS1 reduces its activity by affecting its starch-binding capacity, its ADP-glucose-binding capability or its protein stability. Our results shed new light on the structural basis of OsGBSS1 activity and the mechanisms of OsGBSS1 activity on amylose synthesis in vivo. This study also demonstrates that it is feasible to finely modulate amylose content in rice grains by modifying the OsGBSS1 activity.


Assuntos
Amilose/metabolismo , Oryza/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Sintase do Amido/química , Sintase do Amido/genética , Adenosina Difosfato Glucose/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Cruzamentos Genéticos , Regulação da Expressão Gênica de Plantas , Homozigoto , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Oryza/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Plasmídeos/metabolismo , Ligação Proteica , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Sintase do Amido/metabolismo
10.
Biochimie ; 101: 215-20, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24508535

RESUMO

Trehalose-6-phosphate synthase plays an important role in trehalose metabolism. It catalyzes the transfer of glucose from UDP-glucose (UDPG) to glucose 6-phosphate to produce trehalose-6-phosphate. Herein we describe the characterization of a trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum. The dimeric enzyme could utilize UDPG, ADP-Glucose (ADPG) and GDP-Glucose (GDPG) as glycosyl donors and various phosphorylated monosaccharides as glycosyl acceptors. The optimal temperature and pH were found to be 60 °C and pH 6, and the enzyme exhibited notable pH and thermal stability. The enzymatic activity could be stimulated by divalent metal ions and polyanions heparin and chondroitin sulfate. Moreover, the protein was considerably resistant to additives ethanol, EDTA, urea, DTT, SDS, ß-mercaptoethanol, methanol, isopropanol and n-butanol. Molecular modeling and mutagenesis analysis revealed that the N-loop region was important for the catalytic efficiency of the enzyme, indicating different roles of N-loop sequences in different trehalose-6-phosphate synthases.


Assuntos
Proteínas Arqueais/química , Glucosiltransferases/química , Thermoplasma/enzimologia , Adenosina Difosfato Glucose/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas Arqueais/genética , Domínio Catalítico , Estabilidade Enzimática , Glucosiltransferases/genética , Glicosilação , Açúcares de Guanosina Difosfato/química , Concentração de Íons de Hidrogênio , Magnésio/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Estrutura Quaternária de Proteína , Especificidade por Substrato , Uridina Difosfato Glucose/química , Zinco/química
11.
J Biosci Bioeng ; 117(5): 531-8, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24231376

RESUMO

Exopolysaccharides produced by photosynthetic cyanobacteria have received considerable attention in recent years for their potential applications in the production of renewable biofuels. Particularly, cyanobacterial cellulose is one of the most promising products because it is extracellularly secreted as a non-crystalline form, which can be easily harvested from the media and converted into glucose units. In cyanobacteria, the production of UDP-glucose, the cellulose precursor, is a key step in the cellulose synthesis pathway. UDP-glucose is synthesized from UTP and glucose-1-phosphate (Glc-1P) by UDP-glucose pyrophosphorylase (UGPase), but this pathway in cyanobacteria has not been well characterized. Therefore, to elucidate the overall cellulose biosynthesis pathway in cyanobacteria, we studied the putative UGPase All3274 and seven other putative NDP-sugar pyrophosphorylases (NSPases), All4645, Alr2825, Alr4491, Alr0188, Alr3400, Alr2361, and Alr3921 of Anabaena sp. PCC 7120. Assays using the purified recombinant proteins revealed that All3274 exhibited UGPase activity, All4645, Alr2825, Alr4491, Alr0188, and Alr3921 exhibited pyrophosphorylase activities on ADP-glucose, CDP-glucose, dTDP-glucose, GDP-mannose, and UDP-N-acetylglucosamine, respectively. Further characterization of All3274 revealed that the kcat for UDP-glucose formation was one or two orders lower than those of other known UGPases. The activity and dimerization tendency of All3274 increased at higher enzyme concentrations, implying catalytic activation by dimerization. However, most interestingly, All3274 dimerization was inhibited by UTP and Glc-1P, but not by UDP-glucose. This study presents the first in vitro characterization of a cyanobacterial UGPase, and provides insights into biotechnological attempts to utilize the photosynthetic production of cellulose from cyanobacteria.


Assuntos
Anabaena/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/isolamento & purificação , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Adenosina Difosfato Glucose/metabolismo , Sequência de Aminoácidos , Celulose/metabolismo , Clonagem Molecular , Reagentes para Ligações Cruzadas , Glucose/análogos & derivados , Glucose/metabolismo , Cinética , Dados de Sequência Molecular , Açúcares de Nucleosídeo Difosfato/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Especificidade por Substrato , Nucleotídeos de Timina/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/biossíntese , UTP-Glucose-1-Fosfato Uridililtransferase/química
12.
Plant Cell Physiol ; 55(2): 320-32, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24092883

RESUMO

Nucleotide pyrophosphatase/phosphodiesterase (NPP) is a widely distributed enzymatic activity occurring in both plants and mammals that catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides. Unlike mammalian NPPs, the physiological function of plant NPPs remains largely unknown. Using a complete rice NPP1-encoding cDNA as a probe, in this work we have screened a rice shoot cDNA library and obtained complete cDNAs corresponding to six NPP genes (NPP1-NPP6). As a first step to clarify the role of NPPs, recombinant NPP1, NPP2 and NPP6 were purified from transgenic rice cells constitutively expressing NPP1, NPP2 and NPP6, respectively, and their enzymatic properties were characterized. NPP1 and NPP6 exhibited hydrolytic activities toward ATP, UDP-glucose and the starch precursor molecule, ADP-glucose, whereas NPP2 did not recognize nucleotide sugars as substrates, but hydrolyzed UDP, ADP and adenosine 5'-phosphosulfate. To gain insight into the physiological function of rice NPP1, an npp1 knockout mutant was characterized. The ADP-glucose hydrolytic activities in shoots of npp1 rice seedlings were 8% of those of the wild type (WT), thus indicating that NPP1 is a major determinant of ADP-glucose hydrolytic activity in rice shoots. Importantly, when seedlings were cultured at 160 Pa CO2 under a 28°C/23°C (12 h light/12 h dark) regime, npp1 shoots and roots were larger than those of wild-type (WT) seedlings. Furthermore, the starch content in the npp1 shoots was higher than that of WT shoots. Growth and starch accumulation were also enhanced under an atmospheric CO2 concentration (40 Pa) when plants were cultured under a 33°C/28°C regime. The overall data strongly indicate that NPP1 exerts a negative effect on plant growth and starch accumulation in shoots, especially under high CO2 concentration and high temperature conditions.


Assuntos
Dióxido de Carbono/metabolismo , Oryza/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Amido/metabolismo , Adenosina Difosfato Glucose/metabolismo , Sequência de Bases , Dióxido de Carbono/farmacologia , Células Cultivadas , DNA Complementar/genética , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Dados de Sequência Molecular , Mutação , Oryza/efeitos dos fármacos , Oryza/genética , Oryza/fisiologia , Diester Fosfórico Hidrolases/genética , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/enzimologia , Brotos de Planta/genética , Brotos de Planta/fisiologia , Pirofosfatases/genética , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/genética , Plântula/fisiologia , Análise de Sequência de DNA , Temperatura
13.
New Phytol ; 200(4): 1064-75, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23952675

RESUMO

Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants. Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume. We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape. SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules.


Assuntos
Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Cloroplastos/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/crescimento & desenvolvimento , Sintase do Amido/metabolismo , Amido/biossíntese , Adenosina Difosfato Glucose/metabolismo , Agrobacterium/enzimologia , Proteínas de Arabidopsis , Glucanos/metabolismo , Glicogênio Sintase/metabolismo , Heterozigoto , Isoenzimas/metabolismo , Metaboloma , Mutação/genética , Tamanho das Organelas , Interferência de RNA , Solubilidade
14.
Plant Physiol ; 163(1): 75-85, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23872660

RESUMO

STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants.


Assuntos
Adenosina Difosfato Glucose/metabolismo , Arabidopsis/crescimento & desenvolvimento , Amido/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Mutação , Estresse Oxidativo , Fosforilação , Fotossíntese , Sintase do Amido/genética , Sintase do Amido/metabolismo
15.
J Biotechnol ; 166(3): 65-75, 2013 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-23608552

RESUMO

ADP-glucose pyrophosphorylase, encoded by glgC, catalyzes the first step of glycogen and glucosylglycer(ol/ate) biosynthesis. Here we report the construction of the first glgC null mutant of a marine cyanobacterium (Synechococcus sp. PCC 7002) and investigate its impact on dark anoxic metabolism (autofermentation). The glgC mutant had 98% lower ADP-glucose, synthesized no glycogen and produced appreciably more soluble sugars (mainly sucrose) than wild type (WT). Some glucosylglycerol was still observed, which suggests that the mutant has another, inefficient ADP-glucose synthesis pathway. In contrast, hypersaline conditions (1M NaCl) were lethal to the mutant strain, indicating that, unlike other strains, the elevated sucrose does not compensate for the reduced GG as osmolyte. In contrast to WT, nitrate limitation did not cause bleaching of N-containing pigments or carbohydrate accumulation in the glgC mutant, indicating impaired recycling of nitrogen stores. Despite the 2-fold increase in osmolytes, both the respiration and autofermentation rates of the glgC mutant were appreciably slower (2-4-fold) and correlated quantitatively with the lower fraction of insoluble carbohydrates relative to WT (85% vs. 12%). However, the remaining insoluble carbohydrates still accounted for a high fraction of the carbohydrate catabolized (38%), indicating that insoluble carbohydrates rather than osmolytes were the preferred substrate for autofermentation.


Assuntos
Adenosina Difosfato Glucose/metabolismo , Metabolismo Energético , Glicogênio/metabolismo , Synechococcus/metabolismo , Proteínas de Bactérias/metabolismo , Metabolismo dos Carboidratos , Fermentação , Técnicas de Inativação de Genes , Glucose/metabolismo , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glucosídeos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicogênio/biossíntese , Salinidade , Sacarose/metabolismo
16.
Plant Cell Physiol ; 54(2): 282-94, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23292602

RESUMO

Sucrose synthase (SuSy) is a highly regulated cytosolic enzyme that catalyzes the conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate glucose and fructose. In cereal endosperms, it is widely assumed that the stepwise reactions of SuSy, UDPglucose pyrophosphorylase and ADPglucose (ADPG) pyrophosphorylase (AGP) take place in the cytosol to convert sucrose into ADPG necessary for starch biosynthesis, although it has also been suggested that SuSy may participate in the direct conversion of sucrose into ADPG. In this study, the levels of the major primary carbon metabolites, and the activities of starch metabolism-related enzymes were assessed in endosperms of transgenic maize plants ectopically expressing StSUS4, which encodes a potato SuSy isoform. A total of 29 fertile lines transformed with StSUS4 were obtained, five of them containing a single copy of the transgene that was still functional after five generations. The number of seeds per ear of the five transgenic lines containing a single StSUS4 copy was comparable with that of wild-type (WT) control seeds. However, transgenic seeds accumulated 10-15% more starch at the mature stage, and contained a higher amylose/amylopectin balance than WT seeds. Endosperms of developing StSUS4-expressing seeds exhibited a significant increase in SuSy activity, and in starch and ADPG contents when compared with WT endosperms. No significant changes could be detected in the transgenic seeds in the content of soluble sugars, and in activities of starch metabolism-related enzymes when compared with WT seeds. A suggested metabolic model is presented wherein both AGP and SuSy are involved in the production of ADPG linked to starch biosynthesis in maize endosperm cells.


Assuntos
Adenosina Difosfato Glucose/metabolismo , Amilose/metabolismo , Endosperma/enzimologia , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/metabolismo , Zea mays/enzimologia , Amilopectina/metabolismo , Endosperma/genética , Ativação Enzimática , Ensaios Enzimáticos , Regulação Enzimológica da Expressão Gênica , Modelos Biológicos , Oxirredução , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Solubilidade , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Zea mays/genética
17.
FEBS Lett ; 587(2): 165-9, 2013 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-23196182

RESUMO

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. In filamentous cyanobacteria, the sucrose cleavage direction plays a key physiological function in carbon metabolism, nitrogen fixation, and stress tolerance. In unicellular strains, the function of sucrose synthase has not been elucidated. We report a detailed biochemical characterization of sucrose synthase from Thermosynechococcus elongatus after the gene was artificially synthesized for optimal expression in Escherichia coli. The homogeneous recombinant sucrose synthase was highly specific for ADP as substrate, constituting the first one with this unique characteristic, and strongly suggesting an interaction between sucrose and glycogen metabolism.


Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/enzimologia , Glucosiltransferases/metabolismo , Adenosina Difosfato Glucose/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cianobactérias/genética , Estabilidade Enzimática , Genes Bacterianos , Glucosiltransferases/química , Glucosiltransferases/genética , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura , Difosfato de Uridina/metabolismo , Uridina Difosfato Glucose/metabolismo
18.
Plant J ; 70(2): 231-42, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22098298

RESUMO

Many plants, including Arabidopsis thaliana, retain a substantial portion of their photosynthate in leaves in the form of starch, which is remobilized to support metabolism and growth at night. ADP-glucose pyrophosphorylase (AGPase) catalyses the first committed step in the pathway of starch synthesis, the production of ADP-glucose. The enzyme is redox-activated in the light and in response to sucrose accumulation, via reversible breakage of an intermolecular cysteine bridge between the two small (APS1) subunits. The biological function of this regulatory mechanism was investigated by complementing an aps1 null mutant (adg1) with a series of constructs containing a full-length APS1 gene encoding either the wild-type APS1 protein or mutated forms in which one of the five cysteine residues was replaced by serine. Substitution of Cys81 by serine prevented APS1 dimerization, whereas mutation of the other cysteines had no effect. Thus, Cys81 is both necessary and sufficient for dimerization of APS1. Compared to control plants, the adg1/APS1(C81S) lines had higher levels of ADP-glucose and maltose, and either increased rates of starch synthesis or a starch-excess phenotype, depending on the daylength. APS1 protein levels were five- to tenfold lower in adg1/APS1(C81S) lines than in control plants. These results show that redox modulation of AGPase contributes to the diurnal regulation of starch turnover, with inappropriate regulation of the enzyme having an unexpected impact on starch breakdown, and that Cys81 may play an important role in the regulation of AGPase turnover.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cisteína/genética , Glucose-1-Fosfato Adenililtransferase/genética , Folhas de Planta/genética , Amido/metabolismo , Adenosina Difosfato Glucose/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Ritmo Circadiano , Cisteína/metabolismo , Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/metabolismo , Immunoblotting , Maltose/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Oxirredução , Fotoperíodo , Folhas de Planta/metabolismo , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
19.
Mol Biol Rep ; 39(1): 585-91, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21584701

RESUMO

The metabolic pathways leading to the synthesis of bacterial glycogen involve the action of several enzymes, among which glycogen synthase (GS) catalyzes the elongation of the α-1,4-glucan. GS from Agrobacterium tumefaciens uses preferentially ADPGlc, although UDPGlc can also be used as glycosyl donor with less efficiency. We present here a continuous spectrophotometric assay for the determination of GS activity using ADP- or UDPGlc. When ADPGlc was used as the substrate, the production of ADP is coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH). With UDPGlc as substrate, UDP was converted to ADP via adenylate kinase and subsequent coupling to PK and LDH reactions. Using this assay, we determined the kinetic parameters of GS and compared them with those obtained with the classical radiochemical method. For this purpose, we improved the expression procedure of A. tumefaciens GS using Escherichia coli BL21(DE3)-RIL cells. This assay allows the continuous monitoring of glycosyltransferase activity using ADPGlc or UDPGlc as sugar-nucleotide donors.


Assuntos
Agrobacterium tumefaciens/enzimologia , Glicogênio Sintase/isolamento & purificação , Glicogênio Sintase/metabolismo , Glicogênio/biossíntese , Espectrofotometria/métodos , Adenosina Difosfato Glucose/metabolismo , Clonagem Molecular , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Cinética , Uridina Difosfato Glucose/metabolismo
20.
Proc Natl Acad Sci U S A ; 109(1): 321-6, 2012 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-22184213

RESUMO

Sucrose synthase (SUS) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate-glucose and fructose. In Arabidopsis, a multigene family encodes six SUS (SUS1-6) isoforms. The involvement of SUS in the synthesis of UDP-glucose and ADP-glucose linked to Arabidopsis cellulose and starch biosynthesis, respectively, has been questioned by Barratt et al. [(2009) Proc Natl Acad Sci USA 106:13124-13129], who showed that (i) SUS activity in wild type (WT) leaves is too low to account for normal rate of starch accumulation in Arabidopsis, and (ii) different organs of the sus1/sus2/sus3/sus4 SUS mutant impaired in SUS activity accumulate WT levels of ADP-glucose, UDP-glucose, cellulose and starch. However, these authors assayed SUS activity under unfavorable pH conditions for the reaction. By using favorable pH conditions for assaying SUS activity, in this work we show that SUS activity in the cleavage direction is sufficient to support normal rate of starch accumulation in WT leaves. We also demonstrate that sus1/sus2/sus3/sus4 leaves display WT SUS5 and SUS6 expression levels, whereas leaves of the sus5/sus6 mutant display WT SUS1-4 expression levels. Furthermore, we show that SUS activity in leaves and stems of the sus1/sus2/sus3/sus4 and sus5/sus6 plants is ∼85% of that of WT leaves, which can support normal cellulose and starch biosynthesis. The overall data disprove Barratt et al. (2009) claims, and are consistent with the possible involvement of SUS in cellulose and starch biosynthesis in Arabidopsis.


Assuntos
Arabidopsis/enzimologia , Celulose/biossíntese , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Mutação/genética , Amido/biossíntese , Adenosina Difosfato Glucose/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Cinética , Luz , Cloreto de Magnésio/farmacologia , Extratos Vegetais/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/efeitos da radiação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Uridina Difosfato Glucose/metabolismo
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