Allosteric regulation of the higher plant ADP-glucose pyrophosphorylase is a product of synergy between the two subunits. (49/172)

The higher plant ADP-glucose pyrophosphorylase (AGPase) is a heterotetramer consisting of two regulatory large subunits (LSs) and two catalytic small subunits (SSs). To further characterize the roles of these subunits in determining enzyme function, different combinations of wildtype LS (LWT) and variant forms (LUpReg1, LM345) were co-expressed with wildtype SS (SWT) and variant forms (STG-15 and Sdevo330) and their enzyme properties compared to those measured for the heterotetrameric wildtype enzyme and SS homotetrameric enzymes. Analysis of the allosteric regulatory properties of the various enzymes indicates that although the LS is required for optimal activation by 3-phosphoglyceric acid and resistance to Pi, the overall allosteric regulatory and kinetic properties are specified by both subunits. Our results show that the regulatory and kinetic properties of AGPase are not simply due to the LS modulating the properties of the SS but, instead, are a product of synergistic interaction between the two subunits.  (+info)

QTLs for enzyme activities and soluble carbohydrates involved in starch accumulation during grain filling in maize. (50/172)

ADPglucose, the essential substrate for starch synthesis, is synthesized in maize by a pathway involving at least invertases, sucrose synthase, and ADPglucose pyrophosphorylase, as shown by the starch-deficient mutants, mn1, sh1, and bt2 or sh2, respectively. To improve understanding of the relationship between early grain-filling traits and carbohydrate composition in mature grain, QTLs linked to soluble invertase, sucrose synthase, and ADPglucose pyrophosphorylase activities and to starch, sucrose, fructose, and glucose concentrations were investigated. In order to take into account the specific time-course of each enzyme activity during grain filling, sampling was carried out at three periods (15, 25, and 35 d after pollination) on 100 lines from a recombinant inbred family, grown in the field. The MQTL method associated with QTL interaction analysis revealed numerous QTLs for all traits, but only one QTL was consistently observed at the three sampling periods. Some chromosome zones were heavily labelled, forming clusters of QTLs. Numerous possible candidate genes of the starch synthetic pathway co-located with QTLs. Four QTLs were found close to the locus Sh1 (bin 9.01) coding for the sucrose synthase. In order to confirm the importance of this locus, the CAPS polymorphism of the Sh1 gene was analysed in 45 genetically unrelated maize lines from various geographical origins. The DNA polymorphism was significantly associated with phenotypic traits related to grain filling (starch and amylose content, grain matter, and ADPglucose pyrophosphorylase activity at 35 DAP). Thus, the Sh1 locus could provide a physiologically pertinent marker for maize selection.  (+info)

Identification and characterization of a novel plastidic adenine nucleotide uniporter from Solanum tuberosum. (51/172)

Homologs of BT1 (the Brittle1 protein) are found to be phylogenetically related to the mitochondrial carrier family and appear to occur in both mono- and dicotyledonous plants. Whereas BT1 from cereals is probably involved in the transport of ADP-glucose, which is essential for starch metabolism in endosperm plastids, BT1 from a noncereal plant, Solanum tuberosum (StBT1), catalyzes an adenine nucleotide uniport when functionally integrated into the bacterial cytoplasmic membrane. Import studies into intact Escherichia coli cells harboring StBT1 revealed a narrow substrate spectrum with similar affinities for AMP, ADP, and ATP of about 300-400 mum. Transiently expressed StBT1-green fluorescent protein fusion protein in tobacco leaf protoplasts showed a plastidic localization of the StBT1. In vitro synthesized radioactively labeled StBT1 was targeted to the envelope membranes of isolated spinach chloroplasts. Furthermore, we showed by real time reverse transcription-PCR a ubiquitous expression pattern of the StBT1 in autotrophic and heterotrophic potato tissues. We therefore propose that StBT1 is a plastidic adenine nucleotide uniporter used to provide the cytosol and other compartments with adenine nucleotides exclusively synthesized inside plastids.  (+info)

Purification and characterization of adenosine diphosphate glucose pyrophosphorylase from maize/potato mosaics. (52/172)

Adenosine diphosphate glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in starch biosynthesis. The reaction produces ADP-glucose and pyrophosphate from glucose-1-P and ATP. Investigations from a number of laboratories have shown that alterations in allosteric properties as well as heat stability of this enzyme have dramatic positive effects on starch synthesis in the potato (Solanum tuberosum) tuber and seeds of important cereals. Here, we report the characterization of purified recombinant mosaic AGPases derived from protein motifs normally expressed in the maize (Zea mays) endosperm and the potato tuber. These exhibit properties that should be advantageous when expressed in plants. We also present an in-depth characterization of the kinetic and allosteric properties of these purified recombinant AGPases. These data point to previously unrecognized roles for known allosteric effectors.  (+info)

Trehalose 6-phosphate regulates starch synthesis via posttranslational redox activation of ADP-glucose pyrophosphorylase. (53/172)

Trehalose is the most widespread disaccharide in nature, occurring in bacteria, fungi, insects, and plants. Its precursor, trehalose 6-phosphate (T6P), is also indispensable for the regulation of sugar utilization and growth, but the sites of action are largely unresolved. Here we use genetic and biochemical approaches to investigate whether T6P acts to regulate starch synthesis in plastids of higher plants. Feeding of trehalose to Arabidopsis leaves led to stimulation of starch synthesis within 30 min, accompanied by activation of ADP-glucose pyrophosphorylase (AGPase) via posttranslational redox modification. The response resembled sucrose but not glucose feeding and depended on the expression of SNF1-related kinase. We also analyzed transgenic Arabidopsis plants with T6P levels increased by expression of T6P synthase or decreased by expression of T6P phosphatase (TPP) in the cytosol. Compared with wild type, leaves of T6P synthase-expressing plants had increased redox activation of AGPase and increased starch, whereas TPP-expressing plants showed the opposite. Moreover, TPP expression prevented the increase in AGPase activation in response to sucrose or trehalose feeding. Incubation of intact isolated chloroplasts with 100 muM T6P significantly and specifically increased reductive activation of AGPase within 15 min. Results provide evidence that T6P is synthesized in the cytosol and acts on plastidial metabolism by promoting thioredoxin-mediated redox transfer to AGPase in response to cytosolic sugar levels, thereby allowing starch synthesis to be regulated independently of light. The discovery informs about the evolution of plant metabolism and how chloroplasts of prokaryotic origin use an intermediate of the ancient trehalose pathway to report the metabolic status of the cytosol.  (+info)

Expression profiling of genes involved in starch synthesis in sink and source organs of rice. (54/172)

A comprehensive analysis of the transcript levels of genes which encode starch-synthesis enzymes is fundamental for the assessment of the function of each enzyme and the regulatory mechanism for starch biosynthesis in source and sink organs. Using quantitative real-time RT-PCR, an examination was made of the expression profiles of 27 rice genes encoding six classes of enzymes, i.e. ADPglucose pyrophosphorylase (AGPase), starch synthase, starch branching enzyme, starch debranching enzyme, starch phosphorylase, and disproportionating enzyme in developing seeds and leaves. The modes of gene expression were tissue- and developmental stage-specific. Four patterns of expression in the seed were identified: group 1 genes, which are expressed very early in grain formation and are presumed to be involved in the construction of fundamental cell machineries, de novo synthesis of glucan primers, and initiation of starch granules; group 2 genes, which are highly expressed throughout endosperm development; group 3 genes, which have transcripts that are low at the onset but which rise steeply at the start of starch synthesis in the endosperm and are thought to play essential roles in endosperm starch synthesis; and group 4 genes, which are expressed scantly, mainly at the onset of grain development, and might be involved in synthesis of starch in the pericarp. The methodology also revealed that the defect in the cytosolic AGPase small subunit2b (AGPS2b) transcription from the AGPS2 gene in endosperm sharply enhanced the expressions of endosperm and leaf plastidial AGPS1, the endosperm cytosolic AGPase large subunit2 (AGPL2), and the leaf plastidial AGPL1.  (+info)

Heat stability of maize endosperm ADP-glucose pyrophosphorylase is enhanced by insertion of a cysteine in the N terminus of the small subunit. (55/172)

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme in starch biosynthesis. However, plant AGPases differ in several parameters, including spatial and temporal expression, allosteric regulation, and heat stability. AGPases of cereal endosperms are heat labile, while those in other tissues, such as the potato (Solanum tuberosum) tuber, are heat stable. Sequence comparisons of heat-stable and heat-labile AGPases identified an N-terminal motif unique to heat-stable enzymes. Insertion of this motif into recombinant maize (Zea mays) endosperm AGPase increased the half-life at 58 degrees C more than 70-fold. Km values for physiological substrates were unaffected, although Kcat was doubled. A cysteine within the inserted motif gives rise to small subunit homodimers not found in the wild-type maize enzyme. Placement of this N-terminal motif into a mosaic small subunit containing the N terminus from maize endosperm and the C terminus from potato tuber AGPase increases heat stability more than 300-fold.  (+info)

Biosynthesis of bacterial glycogen. Mutagenesis of a catalytic site residue of ADP-glucose pyrophosphorylase from Escherichia coli. (56/172)

Site-directed mutagenesis was used to explore the role of Lys-195 in ADP-glucose pyrophosphorylase from Escherichia coli. This residue, which is conserved in every bacterial and plant source sequenced to date, was originally identified as a potential catalytic site residue by covalent modification studies. Mutation of Lys-195 to glutamine produces an enzyme whose Km for glucose 1-phosphate is 600-fold greater than that measured for the wild-type enzyme. The effect on glucose 1-phosphate is very specific since kinetic constants measured for ATP, Mg2+, and the allosteric activator, fructose 1,6-bisphosphate, are unchanged relative to those measured for the wild-type enzyme. Furthermore, the catalytic rate constant, Kcat, for the glutamine mutant is similar to that of the wild-type enzyme. Taken together, the results suggest a role for Lys-195 in binding of glucose 1-phosphate and exclude its role as a participant in the rate-determining step(s) in the catalytic reaction mechanism. To further study the effect of charge, shape, size, and hydrophobicity of the amino acid residue at position 195, a series of mutants were prepared including arginine, histidine, isoleucine, and glutamic acid. In every case, the kinetic constants measured for ATP, Mg2+, and fructose 1,6-bisphosphate were similar to wild-type constants, reinforcing the notion that this residue is responsible for a highly localized effect at the glucose 1-phosphate-binding site and also suggesting that the protein can accommodate a wide range of substitutions at this position without losing its global folding properties. Thermal stability measurements corroborate this finding. The mutations did, however, produce a range of glucose 1-phosphate Km values from 100- to 10,000-fold greater than wild-type, which indicate that both size and charge properties of lysine are essential for proper binding of glucose 1-phosphate at the catalytic site. AMP binding was also affected by the nature of the mutation at position 195. A model for glucose 1-phosphate, ATP, and AMP binding is presented.  (+info)