RESUMEN

Mitochondrial function is essential for plant growth, but the mechanisms involved in adjusting growth and metabolism to changes in mitochondrial energy production are not fully understood. We studied plants with reduced expression of CYTC-1, one of two genes encoding the respiratory chain component cytochrome c (CYTc) in Arabidopsis, to understand how mitochondria communicate their status to coordinate metabolism and growth. Plants with CYTc deficiency show decreased mitochondrial membrane potential and lower ATP content, even when carbon sources are present. They also exhibit higher free amino acid content, induced autophagy, and increased resistance to nutritional stress caused by prolonged darkness, similar to plants with triggered starvation signals. CYTc deficiency affects target of rapamycin (TOR)-pathway activation, reducing S6 kinase (S6K) and RPS6A phosphorylation, as well as total S6K protein levels due to increased protein degradation via proteasome and autophagy. TOR overexpression restores growth and other parameters affected in cytc-1 mutants, even if mitochondrial membrane potential and ATP levels remain low. We propose that CYTc-deficient plants coordinate their metabolism and energy availability by reducing TOR-pathway activation as a preventive signal to adjust growth in anticipation of energy exhaustion, thus providing a mechanism by which changes in mitochondrial activity are transduced to the rest of the cell.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Citocromos c/genética , Citocromos c/metabolismo , Sirolimus/farmacología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Adenosina Trifosfato/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo

RESUMEN

Plant metabolism is finely orchestrated to allow the occurrence of complementary and sometimes opposite metabolic pathways. In part this is achieved by the allosteric regulation of enzymes, which has been a cornerstone of plant research for many decades. The completion of the Arabidopsis genome and the development of the associated toolkits for Arabidopsis research moved the focus of many researchers to other fields. This is reflected by the increasing number of high-throughput proteomic studies, mainly focused on post-translational modifications. However, follow-up 'classical' biochemical studies to assess the functions and upstream signaling pathways responsible for such modifications have been scarce. In this work, we review the basic concepts of allosteric regulation of enzymes involved in plant carbon metabolism, comprising photosynthesis and photorespiration, starch and sucrose synthesis, glycolysis and gluconeogenesis, the oxidative pentose phosphate pathway and the tricarboxylic acid cycle. Additionally, we revisit the latest results on the allosteric control of the enzymes involved in these pathways. To conclude, we elaborate on the current methods for studying protein-metabolite interactions, which we consider will become crucial for discoveries in the future.

Asunto(s)

Arabidopsis , Carbono , Carbono/metabolismo , Arabidopsis/metabolismo , Proteómica , Fotosíntesis , Vía de Pentosa Fosfato , Procesamiento Proteico-Postraduccional

RESUMEN

Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signal metabolite in plants, linking growth and development to carbon status. Our current understanding of Tre6P metabolism and signaling pathways in plants is based almost entirely on studies performed with Arabidopsis thaliana, a model plant that performs C3 photosynthesis. Conversely, our knowledge on the molecular mechanisms involved in Tre6P regulation of carbon partitioning and metabolism in C4 plants is scarce. This topic is especially relevant due to the agronomic importance of crops performing C4 photosynthesis, such as maize, sorghum and sugarcane. In this review, we focused our attention on recent developments related to Tre6P metabolism in C4 species and raised some open questions that should be addressed in the near future to improve the yield of economically important crops.

Asunto(s)

Arabidopsis , Trehalosa , Trehalosa/metabolismo , Plantas/metabolismo , Arabidopsis/metabolismo , Fotosíntesis , Carbono/metabolismo , Fosfatos/metabolismo

RESUMEN

Sugar-alcohols are major photosynthates in plants from the Rosaceae family. Expression of the gene encoding aldose-6-phosphate reductase (Ald6PRase), the critical enzyme for glucitol synthesis in rosaceous species, is regulated by physiological and environmental cues. Additionally, Ald6PRase is inhibited by small molecules (hexose-phosphates and inorganic orthophosphate) and oxidizing compounds. This work demonstrates that Ald6PRase from peach leaves is phosphorylated in planta at the N-terminus. We also show in vitro phosphorylation of recombinant Ald6PRase by a partially purified kinase extract from peach leaves containing Ca2+-dependent protein kinases (CDPKs). Moreover, phosphorylation of recombinant Ald6PRase was inhibited by hexose-phosphates, phosphoenolpyruvate and pyrophosphate. We further show that phosphorylation of recombinant Ald6PRase was maximal using recombinant CDPKs. Overall, our results suggest that phosphorylation could fine-tune the activity of Ald6PRase.

Asunto(s)

Prunus persica , Fosforilación , Prunus persica/metabolismo , Fosfatos/metabolismo , Hojas de la Planta/metabolismo , Hexosas/metabolismo

RESUMEN

During germination, seed reserves are mobilised to sustain the metabolic and energetic demands of plant growth. Mitochondrial respiration is presumably required to drive germination in several species, but only recently its role in this process has begun to be elucidated. Using Arabidopsis thaliana lines with changes in the levels of the respiratory chain component cytochrome c (CYTc), we investigated the role of this protein in germination and its relationship with hormonal pathways. Cytochrome c deficiency causes delayed seed germination, which correlates with decreased cyanide-sensitive respiration and ATP production at the onset of germination. In addition, CYTc affects the sensitivity of germination to abscisic acid (ABA), which negatively regulates the expression of CYTC-2, one of two CYTc-encoding genes in Arabidopsis. CYTC-2 acts downstream of the transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4), which binds to a region of the CYTC-2 promoter required for repression by ABA and regulates its expression. The results show that CYTc is a main player during seed germination through its role in respiratory metabolism and energy production. In addition, the direct regulation of CYTC-2 by ABI4 and its effect on ABA-responsive germination establishes a link between mitochondrial and hormonal functions during this process.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Regulación de la Expresión Génica de las Plantas , Germinación/genética , Mitocondrias/metabolismo , Semillas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Sugar alcohols are major photosynthetic products in plant species from the Apiaceae and Plantaginaceae families. Mannose-6-phosphate reductase (Man6PRase) and aldose-6-phosphate reductase (Ald6PRase) are key enzymes for synthesizing mannitol and glucitol in celery (Apium graveolens) and peach (Prunus persica), respectively. In this work, we report the first crystal structures of dimeric plant aldo/keto reductases (AKRs), celery Man6PRase (solved in the presence of mannonic acid and NADP+) and peach Ald6PRase (obtained in the apo form). Both structures displayed the typical TIM barrel folding commonly observed in proteins from the AKR superfamily. Analysis of the Man6PRase holo form showed that residues putatively involved in the catalytic mechanism are located close to the nicotinamide ring of NADP+, where the hydride transfer to the sugar phosphate should take place. Additionally, we found that Lys48 is important for the binding of the sugar phosphate. Interestingly, the Man6PRase K48A mutant had a lower catalytic efficiency with mannose-6-phosphate but a higher catalytic efficiency with mannose than the wild type. Overall, our work sheds light on the structure-function relationships of important enzymes to synthesize sugar alcohols in plants.

Asunto(s)

Fosfatos , Alcoholes del Azúcar , Oxidorreductasas de Alcohol/metabolismo , Aldehído Reductasa/metabolismo , Secuencia de Aminoácidos , Humanos , Manosafosfatos , NADP/metabolismo , Plantas/metabolismo , Azúcares

RESUMEN

KEY MESSAGE: This review outlines research performed in the last two decades on the structural, kinetic, regulatory and evolutionary aspects of ADP-glucose pyrophosphorylase, the regulatory enzyme for starch biosynthesis. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the pathway of glycogen and starch synthesis in bacteria and plants, respectively. Plant ADP-Glc PPase is a heterotetramer allosterically regulated by metabolites and post-translational modifications. In this review, we focus on the three-dimensional structure of the plant enzyme, the amino acids that bind the regulatory molecules, and the regions involved in transmitting the allosteric signal to the catalytic site. We provide a model for the evolution of the small and large subunits, which produce heterotetramers with distinct catalytic and regulatory properties. Additionally, we review the various post-translational modifications observed in ADP-Glc PPases from different species and tissues. Finally, we discuss the subcellular localization of the enzyme found in grain endosperm from grasses, such as maize and rice. Overall, this work brings together research performed in the last two decades to better understand the multiple mechanisms involved in the regulation of ADP-Glc PPase. The rational modification of this enzyme could improve the yield and resilience of economically important crops, which is particularly important in the current scenario of climate change and food shortage.

Asunto(s)

Evolución Molecular , Glucosa-1-Fosfato Adenililtransferasa/química , Glucosa-1-Fosfato Adenililtransferasa/fisiología , Plantas/enzimología , Regulación Alostérica , Glucosa-1-Fosfato Adenililtransferasa/genética , Modelos Moleculares , Conformación Proteica , Almidón/biosíntesis , Almidón/química

RESUMEN

Until recently, the cyanobacterial phylum only included oxygenic photosynthesizer members. The discovery of Melainabacteria as a group of supposed non-photosynthetic cyanobacteria asked to revisit such scenario. From metagenomic data, we were able to identify sequences encoding putative ADP-glucose pyrophosphorylases (ADP-GlcPPase) from free-living and intestinal Melainabacteria. The respective genes were de novo synthesized and over-expressed in Escherichia coli. The purified recombinant proteins from both Melainabacteria species were active as ADP-GlcPPases, exhibiting Vmax values of 2.3 (free-living) and 7.1 U/mg (intestinal). The enzymes showed similar S0.5 values (∼0.3 mM) for ATP, while the one from the intestinal source exhibited a 6-fold higher affinity toward glucose-1P. Both recombinant ADP-GlcPPases were sensitive to glucose-6P activation (A0.5 ∼0.3 mM) and Pi and ADP inhibition (I0.5 between 0.2 and 3 mM). Interestingly, the enzymes from Melainabacteria were insensitive to 3-phosphoglycerate, which is the principal activator of ADP-GlcPPases from photosynthetic cyanobacteria. As far as we know, this is the first biochemical characterization of an active enzyme from Melainabacteria. This work contributes to a better understanding of the evolution of allosteric regulation in the ADP-GlcPPase family, which is critical for synthesizing the main reserve polysaccharide in prokaryotes (glycogen) and plants (starch). In addition, our results offer further information to discussions regarding the phylogenetic position of Melainabacteria.

Asunto(s)

Proteínas Bacterianas/química , Cianobacterias/enzimología , Glucosa-1-Fosfato Adenililtransferasa/química , Filogenia , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Cianobacterias/genética , Glucosa-1-Fosfato Adenililtransferasa/genética , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

C4 photosynthesis is typically characterized by the spatial compartmentalization of the photosynthetic reactions into mesophyll (M) and bundle sheath (BS) cells. Initial carbon fixation within M cells gives rise to C4 acids, which are transported to the BS cells. There, C4 acids are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle. This work is focused on the study of Setaria viridis, a C4 model plant, closely related to several major feed and bioenergy grasses. First, we performed the heterologous expression and biochemical characterization of Setaria isoforms for chloroplastic NADP-malic enzyme (NADP-ME) and mitochondrial NAD-malic enzyme (NAD-ME). The kinetic parameters obtained agree with a major role for NADP-ME in the decarboxylation of the C4 acid malate in the chloroplasts of BS cells. In addition, mitochondria-located NAD-ME showed regulatory properties that could be important in the context of the operation of the C4 carbon shuttle. Secondly, we compared the proteomes of M and BS compartments and found 825 differentially accumulated proteins that could support different metabolic scenarios. Most interestingly, we found evidence of metabolic strategies to insulate the C4 core avoiding the leakage of intermediates by either up-regulation or down-regulation of chloroplastic, mitochondrial, and peroxisomal proteins. Overall, the results presented in this work provide novel data concerning the complexity of C4 metabolism, uncovering future lines of research that will undoubtedly contribute to the expansion of knowledge on this topic.

Asunto(s)

Setaria (Planta) , Cloroplastos/metabolismo , Malato Deshidrogenasa/metabolismo , Fotosíntesis , Hojas de la Planta/metabolismo , Plantas/metabolismo , Setaria (Planta)/metabolismo

RESUMEN

This review commemorates the 50th anniversary of the Nobel Prize in Chemistry awarded to Luis F. Leloir 'for his discovery of sugar-nucleotides and their role in the biosynthesis of carbohydrates'. He and his co-workers discovered that activated forms of simple sugars, such as UDP-glucose and UDP-galactose, are essential intermediates in the interconversion of sugars. They elucidated the biosynthetic pathways for sucrose and starch, which are the major end-products of photosynthesis, and for trehalose. Trehalose 6-phosphate, the intermediate of trehalose biosynthesis that they discovered, is now a molecule of great interest due to its function as a sugar signalling metabolite that regulates many aspects of plant metabolism and development. The work of the Leloir group also opened the doors to an understanding of the biosynthesis of cellulose and other structural cell wall polysaccharides (hemicelluloses and pectins), and ascorbic acid (vitamin C). Nucleotide-sugars also serve as sugar donors for a myriad of glycosyltransferases that conjugate sugars to other molecules, including lipids, phytohormones, secondary metabolites, and proteins, thereby modifying their biological activity. In this review, we highlight the diversity of nucleotide-sugars and their functions in plants, in recognition of Leloir's rich and enduring legacy to plant science.

Asunto(s)

Pared Celular , Plantas , Metabolismo de los Hidratos de Carbono , Pared Celular/metabolismo , Glicosiltransferasas/metabolismo , Uridina Difosfato Glucosa/metabolismo

RESUMEN

Phosphoenolpyruvate carboxykinase (PEPCK) plays a crucial role in gluconeogenesis. In this work, we analyze the proteolysis of Arabidopsis thaliana PEPCK1 (AthPEPCK1) in germinating seedlings. We found that the amount of AthPEPCK1 protein peaks at 24-48 h post-imbibition. Concomitantly, we observed shorter versions of AthPEPCK1, putatively generated by metacaspase-9 (AthMC9). To study the impact of AthMC9 cleavage on the kinetic and regulatory properties of AthPEPCK1, we produced truncated mutants based on the reported AthMC9 cleavage sites. The Δ19 and Δ101 truncated mutants of AthPEPCK1 showed similar kinetic parameters and the same quaternary structure as the wild type. However, activation by malate and inhibition by glucose 6-phosphate were abolished in the Δ101 mutant. We propose that proteolysis of AthPEPCK1 in germinating seedlings operates as a mechanism to adapt the sensitivity to allosteric regulation during the sink-to-source transition.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Fosfoenolpiruvato Carboxiquinasa (ATP) , Regulación Alostérica , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Gluconeogénesis , Fosfoenolpiruvato , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Proteolisis

RESUMEN

Raffinose (Raf) protects plant cells during seed desiccation and under different abiotic stress conditions. The biosynthesis of Raf starts with the production of UDP-galactose by UDP-sugar pyrophosphorylase (USPPase) and continues with the synthesis of galactinol by galactinol synthase (GolSase). Galactinol is then used by Raf synthase to produce Raf. In this work, we report the biochemical characterization of USPPase (BdiUSPPase) and GolSase 1 (BdiGolSase1) from Brachypodium distachyon. The catalytic efficiency of BdiUSPPase was similar with galactose 1-phosphate and glucose 1-phosphate, but 5- to 17-fold lower with other sugar 1-phosphates. The catalytic efficiency of BdiGolSase1 with UDP-galactose was three orders of magnitude higher than with UDP-glucose. A structural model of BdiGolSase1 allowed us to determine the residues putatively involved in the binding of substrates. Among these, we found that Cys261 lies within the putative catalytic pocket. BdiGolSase1 was inactivated by oxidation with diamide and H2O2. The activity of the diamide-oxidized enzyme was recovered by reduction with dithiothreitol or E. coli thioredoxin, suggesting that BdiGolSase1 is redox-regulated.

Asunto(s)

Brachypodium/enzimología , Galactosiltransferasas/metabolismo , Nucleotidiltransferasas/metabolismo , Rafinosa/biosíntesis , Peróxido de Hidrógeno , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo

RESUMEN

ATP-dependent phosphoenolpyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana: AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. AthPEPCK1 and AthPEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had no effect on AthPEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fine allosteric regulation.

Asunto(s)

Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Fosfoenolpiruvato Carboxiquinasa (ATP)/química , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Ácido Cítrico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fluorometría/métodos , Fumaratos/metabolismo , Cinética , Malatos/metabolismo , Manganeso/metabolismo , Ácido Oxaloacético/metabolismo , Fotosíntesis , Unión Proteica , Proteínas Recombinantes/metabolismo , Ácido Succínico/metabolismo , Temperatura de Transición

RESUMEN

Most oxidoreductases that use NAD+ or NADP+ to transfer electrons in redox reactions display a strong preference for the cofactor. The catalytic efficiency of peach glucitol dehydrogenase (GolDHase) for NAD+ is 1800-fold higher than that for NADP+. Herein, we combined structural and kinetic data to reverse the cofactor specificity of this enzyme. Using site-saturation mutagenesis, we obtained the D216A mutant, which uses both NAD+ and NADP+, although with different catalytic efficiencies (1000 ± 200 and 170 ± 30 M-1 s-1, respectively). This mutant was used as a template to introduce further mutations by site-directed mutagenesis, using information from the fruit fly NADP-dependent GolDHase. The D216A/V217R/D218S triple mutant displayed a 2-fold higher catalytic efficiency with NADP+ than with NAD+. Overall, our results indicate that the triple mutant has the potential to be used for metabolic and cellular engineering and for cofactor recycling in industrial processes.

Asunto(s)

Coenzimas/metabolismo , L-Iditol 2-Deshidrogenasa/metabolismo , NADP/metabolismo , Proteínas de Plantas/metabolismo , Prunus persica/enzimología , Cinética , L-Iditol 2-Deshidrogenasa/química , L-Iditol 2-Deshidrogenasa/genética , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética

RESUMEN

Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.

Asunto(s)

Aldehído Reductasa/metabolismo , Hojas de la Planta/enzimología , Proteínas de Plantas/metabolismo , Prunus domestica/enzimología , Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/genética , Fructosadifosfatos/metabolismo , Fructosadifosfatos/farmacología , Fructosafosfatos/metabolismo , Fructosafosfatos/farmacología , Glucofosfatos/metabolismo , Glucofosfatos/farmacología , Disulfuro de Glutatión/metabolismo , Hexosafosfatos/metabolismo , Hexosafosfatos/farmacología , Immunoblotting , Cinética , Modelos Biológicos , NADP/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacología , Fosfatos/metabolismo , Fosfatos/farmacología , Filogenia , Hojas de la Planta/genética , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Prunus domestica/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Compuestos de Sulfhidrilo/metabolismo

RESUMEN

ADP-glucose pyrophosphorylase regulates the synthesis of glycogen in bacteria and of starch in plants. The enzyme from plants is mainly activated by 3-phosphoglycerate and is a heterotetramer comprising two small and two large subunits. Here, we found that two highly conserved residues are critical for triggering the activation of the potato tuber ADP-glucose pyrophosphorylase, as shown by site-directed mutagenesis. Mutations in the small subunit, which bears the catalytic function in this potato tuber form, had a more dramatic effect on disrupting the allosteric activation than those introduced in the large subunit, which is mainly modulatory. Our results strongly agree with a model where the modified residues are located in loops responsible for triggering the allosteric activation signal for this enzyme, and the sensitivity to this activation correlates with the dynamics of these loops. In addition, previous biochemical data indicates that the triggering mechanism is widespread in the enzyme family, even though the activator and the quaternary structure are not conserved.

Asunto(s)

Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Tubérculos de la Planta/enzimología , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Activación Enzimática , Glucosa-1-Fosfato Adenililtransferasa/química , Ácidos Glicéricos/metabolismo , Cinética , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Triptófano/genética

RESUMEN

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/metabolismo

RESUMEN

ADP-glucose pyrophosphorylase is the enzyme responsible for the regulation of glycogen synthesis in bacteria. The enzyme N-terminal domain has a Rossmann-like fold with three neighbor loops facing the substrate ATP. In the Escherichia coli enzyme, one of those loops also faces the regulatory site containing Lys(39), a residue involved in binding of the allosteric activator fructose-1,6-bisphosphate and its analog pyridoxal-phosphate. The other two loops contain Trp(113) and Gln(74), respectively, which are highly conserved among all the ADP-glucose pyrophosphorylases. Molecular modeling of the E. coli enzyme showed that binding of ATP correlates with conformational changes of the latter two loops, going from an open to a closed (substrate-bound) form. Alanine mutants of Trp(113) or Gln(74) did not change apparent affinities for the substrates, but they became insensitive to activation by fructose-1,6-bisphosphate. By capillary electrophoresis we found that the mutant enzymes still bind fructose-1,6-bisphosphate, with similar affinity as the wild type enzyme. Since the mutations did not alter binding of the activator, they must have disrupted the communication between the regulatory and the substrate sites. This agrees with a regulatory mechanism where the interaction with the allosteric activator triggers conformational changes at the level of loops containing residues Trp(113) and Gln(74).

Asunto(s)

Escherichia coli/enzimología , Fructosadifosfatos/metabolismo , Glucosa-1-Fosfato Adenililtransferasa/química , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Regulación Alostérica , Electroforesis Capilar , Glucosa-1-Fosfato Adenililtransferasa/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida

RESUMEN

Phosphoenolpyruvate (PEP) carboxylase (PEPCase) from orange fruit juice sacs has been cloned and heterogously expressed in high yield. The purified recombinant enzyme displays properties typical of plant PEPCase, including activation by sugar phosphates and inhibition by malate and citrate. Malate inhibition is weak in the physiological pH range, and the enzyme is also poorly affected by Glu and Asp, known inhibitors of C(3) plants PEPCases. However, it is strongly inhibited by citrate. Orange fruit PEPCase phosphorylation by mammalian protein kinase A decreased inhibition by malate. The enzyme presents an unusual high molecular mass in the absence of PEP, while in its presence it displays a more common tetrameric arrangement. The overall properties of the enzyme suggest that it is suited for organic acid synthesis and NADH reoxidation in the mature fruit. The present study provides the first analysis of a recombinant fruit PEPCase.

RESUMEN

Aldose-6-phosphate reductase (A6PRase) is a key enzyme for glucitol biosynthesis in plants from the Rosaceae family. To gain on molecular tools for enzymological studies, we developed an accurate system for the heterologous expression of A6PRase from apple leaves. The recombinant enzyme was expressed with a His-tag alternatively placed in the N- or C-terminus, thus allowing the one-step protein purification by immobilized metal affinity chromatography. Both, the N- and the C-term tagged enzymes exhibited similar affinity toward substrates, although the k(cat) of the latter enzyme was 80-fold lower than that having the His-tag in the N-term. Gel filtration chromatography showed different oligomeric structures arranged by the N- (dimer) and the C-term (monomer) tagged enzymes. These results, reinforced by homology modeling studies, point out the relevance of the C-term domain in the structure of A6PRase to conform an enzyme having optimal specific activity and the proper quaternary structure.

Asunto(s)

Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Malus/enzimología , Hojas de la Planta/enzimología , Estructura Cuaternaria de Proteína , Oxidorreductasas de Alcohol/biosíntesis , Oxidorreductasas de Alcohol/aislamiento & purificación , Clonación Molecular , Escherichia coli/genética , Cinética , Modelos Moleculares , Homología de Secuencia de Aminoácido , Especificidad por Sustrato