RESUMEN
Many plants accumulate transitory starch reserves in their leaves during the day to buffer their carbohydrate supply against fluctuating light conditions, and to provide carbon and energy for survival at night. It is universally accepted that transitory starch is synthesized from ADP-glucose (ADPG) in the chloroplasts. However, the consensus that ADPG is made in the chloroplasts by ADPG pyrophosphorylase has been challenged by a controversial proposal that ADPG is made primarily in the cytosol, probably by sucrose synthase (SUS), and then imported into the chloroplasts. To resolve this long-standing controversy, we critically re-examined the experimental evidence that appears to conflict with the consensus pathway. We show that when precautions are taken to avoid artefactual changes during leaf sampling, Arabidopsis thaliana mutants that lack SUS activity in mesophyll cells (quadruple sus1234) or have no SUS activity (sextuple sus123456) have wild-type levels of ADPG and starch, while ADPG is 20 times lower in the pgm and adg1 mutants that are blocked in the consensus chloroplastic pathway of starch synthesis. We conclude that the ADPG needed for starch synthesis in leaves is synthesized primarily by ADPG pyrophosphorylase in the chloroplasts.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Adenosina Difosfato Glucosa/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Glucosiltransferasas , Hojas de la Planta/metabolismo , Almidón/metabolismo , Sacarosa/metabolismoRESUMEN
The thermal shift assay (TSA) is a powerful tool used to detect molecular interactions between proteins and ligands. Using temperature as a physical denaturant and an extrinsic fluorescent dye, the TSA tracks protein unfolding. This method precisely determines the midpoint of the unfolding transition (Tm), which can shift upon the addition of a ligand. Though experimental protocols have been well developed, the thermal shift assay data traditionally yielded qualitative results. Quantitative methods for Kd determination relied either on empirical and inaccurate usage of Tm or on isothermal approaches, which do not take full advantage of the melting point precision provided by the TSA. We present a new analysis method based on a model that relies on the equilibrium system between the native and molten globule state of the protein using the van't Hoff equation. We propose the Kd can be determined by plotting Tm values versus the logarithm of ligand concentrations and fitting the data to an equation we derived. After testing this procedure with the monomeric maltose-binding protein and an allosterically regulated homotetrameric enzyme (ADP-glucose pyrophosphorylase), we observed that binding results correlated very well with previously established parameters. We demonstrate how this method could potentially offer a broad applicability to a wide range of protein classes and the ability to detect both active and allosteric site binding compounds.
Asunto(s)
Proteínas/metabolismo , Temperatura , Adenosina Difosfato Glucosa/metabolismo , Escherichia coli/enzimología , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Humanos , Cinética , Ligandos , Maltosa/metabolismo , Proteínas de Unión a Maltosa/metabolismo , Mutagénesis/genética , Desplegamiento Proteico , Trisacáridos/metabolismoRESUMEN
Plant sucrose-phosphate synthase (SPS) contains a glycosyltransferase domain, which specifically catalyzes reactions with the nucleotide sugar uridine diphosphate glucose (UDP-G) as a donor substrate. Unlike plant SPS, bacterial SPS is predicted to bind other nucleotide sugars, such as adenosine diphosphate glucose (ADP-G). This study aimed to identify the UDP-G binding site of sugarcane (Saccharum officinarum) SPS (SoSPS1) and to improve its affinity for ADP-G by site-directed mutagenesis. To achieve targeted mutagenesis, amino acid distribution and comparative modeling studies were performed, followed by site-directed mutagenesis of SoSPS1 in the putative UDP-G binding motif. The N-terminal deletion of SoSPS1 (∆N-SoSPS1) was used for enzymatic analysis. The results showed that mutations in the R-X4-K, E-X7-E, and H-X5-V motifs significantly affect UDP-G and ADP-G binding. Mutations at R496 and K501 severely attenuate the affinity for UDP-G. Additionally, alanine substitutions at E591 and V570 decreased the UDP-G affinity but remarkably increased its ADP-G affinity. The R-X4-K motif plays a crucial role in the UDP-G binding site and catalytic activity of plant SPS; thus, its alteration to other amino acids was not viable. The E-X7-E and H-X5-V motifs may bind to the nucleotide glucose substrate, indicating that these motifs are involved in substrate specificity. These results agree with substrate docking simulations at the mutated residue positions, supporting the experimental results. These results demonstrate that mutation of E591 and V570 severely attenuated the UDP-G affinity, while retaining its activity against ADP-G, offering strategic insights into increasing sucrose synthesis and plant growth.
Asunto(s)
Adenosina Difosfato Glucosa/química , Glucosiltransferasas/química , Saccharum/enzimología , Saccharum/genética , Uridina Difosfato Glucosa/química , Adenosina Difosfato Glucosa/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Escherichia coli/metabolismo , Expresión Génica , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Cinética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , N-Glicosil Hidrolasas/metabolismo , Proteínas Recombinantes , Saccharum/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Uridina Difosfato Glucosa/metabolismoRESUMEN
The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK1110, annotated as ADP-dependent glucokinase. The encoding gene was expressed in Escherichia coli and the gene product, TK-GLK, was produced in soluble and active form. The recombinant enzyme was extremely thermostable. Thermostability was increased significantly in the presence of ammonium sulfate. ADP was the preferred co-factor for TK-GLK, which could be replaced with CDP but with a 60% activity. TK-GLK was a metal ion-dependent enzyme which exhibited glucokinase, glucosamine kinase and glucose 6-phosphatase activities. It catalyzed the phosphorylation of both glucose and glucosamine with nearly the same rate and affinity. The apparent Km values for glucose and glucosamine were 0.48 ± 0.03 and 0.47 ± 0.09 mM, respectively. The catalytic efficiency (kcat/Km) values against these two substrates were 6.2 × 105 ± 0.25 and 5.8 × 105 ± 0.75 M-1 s-1. The apparent Km value for dephosphorylation of glucose 6-phosphate was ~14-fold higher than that of glucose phosphorylation. Similarly, catalytic efficiency (kcat/Km) for phosphatase reaction was ~19-fold lower than that for the kinase reaction. To the best of our knowledge, this is the first report that describes the reversible nature of a euryarchaeal ADP-dependent glucokinase.
Asunto(s)
Adenosina Difosfato Glucosa/química , Adenosina Difosfato/química , Proteínas Arqueales/química , Glucoquinasa/química , Glucosamina/química , Glucosa/química , Thermococcus/enzimología , Adenosina Difosfato/metabolismo , Adenosina Difosfato Glucosa/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Sitios de Unión , Biocatálisis , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glucoquinasa/genética , Glucoquinasa/metabolismo , Glucosamina/metabolismo , Glucosa/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Thermococcus/química , TermodinámicaRESUMEN
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.
Asunto(s)
Adenosina Difosfato Glucosa/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Espacio Extracelular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Nucleótidos de Adenina/metabolismo , Transporte Biológico , Escherichia coli/genética , Genes Bacterianos , Glucógeno/biosíntesis , Glucógeno Sintasa/metabolismo , Modelos Biológicos , Estrés FisiológicoRESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , Monosacáridos/metabolismo , Óxido Nítrico/farmacología , Complejos de ATP Sintetasa/metabolismo , Adenosina Difosfato Glucosa/metabolismo , Adenosina Trifosfato/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Glucólisis/efectos de los fármacos , Mutación/genética , Nitrosación , Oxidación-Reducción , Fenotipo , Desarrollo de la Planta/efectos de los fármacos , Raíces de Plantas/anatomía & histología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Solubilidad , Almidón/metabolismo , Sacarosa/farmacología , Ácido Tióctico/análogos & derivados , Ácido Tióctico/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Difosfato Glucosa/metabolismo , Endospermo/enzimología , Proteínas de Transporte de Membrana/metabolismo , Oryza/enzimología , Plastidios/enzimología , Almidón/biosíntesis , Amilopectina/metabolismo , Eliminación de Gen , Proteínas de Transporte de Membrana/genética , Oryza/genética , Oryza/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Difosfato Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Almidón/metabolismo , Genes de Plantas , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Mutación , Oryza/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Plastidios/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Semillas/metabolismo , Zea mays/enzimología , Zea mays/genéticaRESUMEN
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.
Asunto(s)
Dióxido de Carbono/metabolismo , Regulación de la Expresión Génica de las Plantas , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Almidón/metabolismo , Adenosina Difosfato Glucosa/metabolismo , Metabolismo de los Hidratos de Carbono , Clorofila/metabolismo , Clorofila A , Expresión Génica , Técnicas de Silenciamiento del Gen , Glucosa-1-Fosfato Adenililtransferasa/genética , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Glucuronidasa/genética , Glucuronidasa/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos , Oryza/citología , Oryza/genética , Floema/citología , Floema/genética , Floema/metabolismo , Fosforilasas/genética , Fosforilasas/metabolismo , Fotosíntesis , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genéticaRESUMEN
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.
Asunto(s)
Adenosina Difosfato Glucosa/metabolismo , Corynebacterium glutamicum/enzimología , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Fosforilasas/metabolismo , Transcripción Genética/fisiología , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Fosforilasas/genéticaRESUMEN
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.
Asunto(s)
Adenosina Difosfato Glucosa/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatografía Líquida de Alta Presión , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Hojas de la Planta/enzimología , Hojas de la Planta/metabolismo , Almidón Sintasa/metabolismo , Espectrometría de Masas en Tándem , Arabidopsis/enzimología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Glucosa-1-Fosfato Adenililtransferasa/genética , Glucosa-6-Fosfato Isomerasa , Plastidios/enzimología , Plastidios/metabolismo , Almidón Sintasa/genéticaRESUMEN
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.
Asunto(s)
Amilosa/metabolismo , Oryza/enzimología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Almidón Sintasa/química , Almidón Sintasa/genética , Adenosina Difosfato Glucosa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Cruzamientos Genéticos , Regulación de la Expresión Génica de las Plantas , Homocigoto , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Oryza/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Plásmidos/metabolismo , Unión Proteica , Estabilidad Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Almidón Sintasa/metabolismoRESUMEN
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.
Asunto(s)
Anabaena/enzimología , UTP-Glucosa-1-Fosfato Uridililtransferasa/aislamiento & purificación , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Adenosina Difosfato Glucosa/metabolismo , Secuencia de Aminoácidos , Celulosa/metabolismo , Clonación Molecular , Reactivos de Enlaces Cruzados , Glucosa/análogos & derivados , Glucosa/metabolismo , Cinética , Datos de Secuencia Molecular , Azúcares de Nucleósido Difosfato/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Especificidad por Sustrato , Nucleótidos de Timina/metabolismo , UTP-Glucosa-1-Fosfato Uridililtransferasa/biosíntesis , UTP-Glucosa-1-Fosfato Uridililtransferasa/químicaRESUMEN
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.
Asunto(s)
Dióxido de Carbono/metabolismo , Oryza/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/metabolismo , Almidón/metabolismo , Adenosina Difosfato Glucosa/metabolismo , Secuencia de Bases , Dióxido de Carbono/farmacología , Células Cultivadas , ADN Complementario/genética , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Datos de Secuencia Molecular , Mutación , Oryza/efectos de los fármacos , Oryza/genética , Oryza/fisiología , Hidrolasas Diéster Fosfóricas/genética , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/enzimología , Brotes de la Planta/genética , Brotes de la Planta/fisiología , Pirofosfatasas/genética , Plantones/efectos de los fármacos , Plantones/enzimología , Plantones/genética , Plantones/fisiología , Análisis de Secuencia de ADN , TemperaturaRESUMEN
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.
Asunto(s)
Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Cloroplastos/metabolismo , Hojas de la Planta/enzimología , Hojas de la Planta/crecimiento & desarrollo , Almidón Sintasa/metabolismo , Almidón/biosíntesis , Adenosina Difosfato Glucosa/metabolismo , Agrobacterium/enzimología , Proteínas de Arabidopsis , Glucanos/metabolismo , Glucógeno Sintasa/metabolismo , Heterocigoto , Isoenzimas/metabolismo , Metaboloma , Mutación/genética , Tamaño de los Orgánulos , Interferencia de ARN , SolubilidadRESUMEN
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.
Asunto(s)
Adenosina Difosfato Glucosa/metabolismo , Arabidopsis/crecimiento & desarrollo , Almidón/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Mutación , Estrés Oxidativo , Fosforilación , Fotosíntesis , Almidón Sintasa/genética , Almidón Sintasa/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Difosfato Glucosa/metabolismo , Metabolismo Energético , Glucógeno/metabolismo , Synechococcus/metabolismo , Proteínas Bacterianas/metabolismo , Metabolismo de los Hidratos de Carbono , Fermentación , Técnicas de Inactivación de Genes , Glucosa/metabolismo , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Glucósidos/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Glucógeno/biosíntesis , Salinidad , Sacarosa/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Difosfato Glucosa/metabolismo , Amilosa/metabolismo , Endospermo/enzimología , Regulación de la Expresión Génica de las Plantas , Glucosiltransferasas/metabolismo , Zea mays/enzimología , Amilopectina/metabolismo , Endospermo/genética , Activación Enzimática , Pruebas de Enzimas , Regulación Enzimológica de la Expresión Génica , Modelos Biológicos , Oxidación-Reducción , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Solubilidad , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Zea mays/genéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cianobacterias/enzimología , Glucosiltransferasas/metabolismo , Adenosina Difosfato Glucosa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cianobacterias/genética , Estabilidad de Enzimas , Genes Bacterianos , Glucosiltransferasas/química , Glucosiltransferasas/genética , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Temperatura , Uridina Difosfato/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
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.