In enzymology, a glucose 1phosphate adenylyltransferase ec 2. An adenosine diphosphate sugar pyrophosphatase asppase, ec 3. The starchsynthesising ability of plants, particularly cereals, may be modified by insertion into their genomes by genetic transformation a dna encoding adp glucose pyrophosphorylase. Although phosphofructokinase typically uses atp as a phosphoryl donor, we found only pyrophosphate ppilinked activity. Group i consists of bacterial types of adp ribose pyrophosphatase, such as adprase in e. Smith john innes centre, colney lane, norwich nr4 7uh, united kingdom d. Identification of regions critically affecting kinetics. Adenosine diphosphate sugar pyrophosphatase prevents.
Reduction of agpase activity led to an inhibition of starch synthesis in oilseed rape and delayed oil synthesis, although. The consequences for sucrosestarch interconversions have now been studied in growing potato solanum tuberosum l. Overexpression of inorganic pyrophosphatase ppase from escherichia coli in the cytosol of plants ppa1 plants leads to a decrease of inorganic pyrophosphate ppi. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed. Rice plastidial nglycosylated nucleotide pyrophosphatase phosphodiesterase is transported from the ergolgi to the chloroplast through the secretory pathway w yohei nanjo,a hiromasa oka,a noriko ikarashi,a kentaro kaneko,a aya kitajima,a toshiaki mitsui,a,1. One of those residues, asp142 in the adpglucose pyrophosphorylase fromescherichia coli, was predicted to be near the substrate. Pdf aspartate residue 142 is important for catalysis by adp. Read characterization of mouse udp glucose pyrophosphatase, a nudix hydrolase encoded by the nudt14 gene, biochemical and biophysical research communications on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at. M and may be nonspecific, as the same preparation hydrolyzed adp glucose, nadh and ap2a. In oxygenic photosynthetic organisms, adp glc ppase is mainly activated by 3phosphoglycerate 3pga and to a lesser extent by other metabolites. In contrast, the pyrophosphatase activity of the microsomal fraction table iii was much greater at ph 5. Adp sugar pyrophosphatase nudt5 is a member of the nudix superfamily of enzymes. To perform a structurefunction relationship study of the escherichia coli adp glc ppase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model.
Human placenta hydrolases active on free adp ribose. Group ii consists of eukaryotic types of adp sugar pyrophosphatase, such as nudt5 in humans and ysa1 in saccharomyces cerevisiae, which hydrolyze adp sugars, including adp ribose, adp mannose, and adp glucose. Adp glucose pyrophosphorylase agpase is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. Constitutive release of atp and evidence for major. Cytoplasmic escherichia coli adp sugar pyrophosphatase binds. Leacha, adepartment of bioagricultural sciences and pest management and program in plant molecular biology, colorado state university, fort collins, co 805231177, usa b usdaars, national center for genetic resources preservation, fort collins, co, usa. Constitutive release of atp and evidence for major contribution of ectonucleotide pyrophosphatase and nucleoside diphosphokinase to extracellular nucleotide concentrations eduardo r.
Adpglucose pyrophosphorylase is located in the plastid in. Adp glucose pyrophosphorylase agpase catalyzes the first committed reaction in the pathway of starch synthesis. Gluconeogenesis glycogen metabolism masarykova univerzita. Ministry of health and longterm care assistive devices program 7th floor, 5700 yonge street toronto, on m2m 4k5.
The gene encoding the enzyme adpglucose pyrophosphorylase has been isolated from wheat leaf and wheat endosperm tissue. Cuttings of sweet potato leaves were grown on a medium containing 1,10, 30, and 50 mm nitrate and analyzed with regard tomorphologicaland biochemical traits. Human adpsugar pyrophosphatasenudt5 antibody mab6414. Hexokinase is the key glycolytic enzyme and the reaction is irreversible. Both tnk1 and tnk2 have been shown to influence glut4 mediated glucose transport in an adp ribosylationdependent manner 99,104. Adp glucose pyrophosphorylase agpase is a ratelimiting enzyme associated with starch biosynthesis in plants. Activity and expression of adpglucose pyrophosphorylase during. Addition of 30 mm suc to isolated chloroplasts did not lead to monomerization of agpb in the dark over a 15min period data not shown. According to this view, the plastidial adp glucose adpg pyrophosphorylase agp is the sole enzyme catalyzing the synthesis of the starch precursor molecule adpg.
One of two different adpglucose pyrophosphorylase genes from. The question is how the undesirable outcomes of in vivo promiscuity can be prevented. However, starch synthesis in the glgctm line c3 was unaltered or lower following an unexpected extension of the night fig. Jun 19, 2017 the substrate specificity of enzymes is crucial to control the fate of metabolites to different pathways. Here we show that this mutation is accompanied by a decrease in storage protein accumulation and seed size, which indicates that alteration of a single enzymatic step can change the network of storage metabolism as a whole. In enzymology, a glucose1phosphate adenylyltransferase ec 2. The dephosphorylation of glucose 6phosphate takes place within the lumen of endoplasmic reticulum. Abstractnucleotide pyrophosphatasephosphodiesterase npp is a widely distributed enzymatic activity. Systematic characterization of the adpribose pyrophosphatase.
All the reaction steps take place in the cytoplasm. The ancestral activation promiscuity of adpglucose. Addition of extra copies of the gene to a plant by transformation enhances starch production, increasing the food value of the crop. Impact of heterologous expression of escherichia coli udp. Structurefunction relationships of cyanobacterial adpglucose. Synthesis of this activated form of glucose, used by starch or glycogen synthases, is catalyzed by adpglucose pyrophosphorylase ec 2. Enzyme that can either act as an adp sugar pyrophosphatase in absence of diphosphate or catalyze the synthesis of atp in presence of diphosphate by similarity. This promiscuous behavior has important implications in protein evolution and the acquisition of new functions.
The enzyme responsible for this activity, referred to as adpg pyrophosphatase agppase, was purified over 1,100fold from barley leaves and subjected to biochemical. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes adp glucose, the precursor molecule of glycogen biosynthesis. Overexpression of pyrophosphatase leads to increased. The substrate specificity of enzymes is crucial to control the fate of metabolites to different pathways. The gene encoding the enzyme adp glucose pyrophosphorylase has been isolated from wheat leaf and wheat endosperm tissue.
An alternative explanation for the increase in glycogen content during induced galu expression is the interference by udp glucose with putative adp glucose futile cycling. Adenosine diphosphate sugar pyrophosphatase prevents glycogen. Atypical glycolysis in clostridium thermocellum applied. Unless otherwise indicated, all steps were carried out at 4c.
Most of adpglucose linked to starch biosynthesis occurs. According to this view, the plastidial adpglucose adpg pyrophosphorylase agp is the sole enzyme catalyzing the synthesis of the starch precursor molecule adpg. The soluble fraction was thus free of that pyrophosphatase with which we are concerned. Unraveling the activation mechanism of the potato tuber adp. Thus, the two substrates of this enzyme are atp and alphad glucose 1phosphate, whereas its two products are diphosphate and adp glucose. Thus, the two substrates of this enzyme are atp and alphadglucose 1phosphate, whereas its two products are diphosphate and adpglucose. However, there is growing evidence that many enzymes can catalyze alternative reactions. Conversely, insertion of a gene encoding mrna antisense to the mrna encoded. The enzyme was the most stable at ph 7, showing 50% loss of its original activity after 50min heating at 57c. Barley grains, deficient in cytosolic small subunit of adp.
Characterization of adpglucose transport across the. In this work, we analyzed the activation promiscuity of adp glc ppase subunits from the. We report the first atomic resolution structure of adp. The enzyme is hexokinase, which splits atp into adp and the pi is added on to the glucose. Aug 31, 2004 sucrose and starch are end products of two segregated gluconeogenic pathways, and their production takes place in the cytosol and chloroplast of green leaves, respectively. Adpglucose pyrophosphorylase in wheat grains nature. Acceleration of potato tuber sprouting by the expression of a.
The presence of an adenosine diphosphate sugar pyrophosphatase aspp in e. Seedspecific suppression of adpglucose pyrophosphorylase. Further evidence that the expression of the inorganic pyrophosphatase transgene has no major effect on the flux through glycolysis comes from the measurements of atp and adp. Dec 25, 2009 read characterization of mouse udpglucose pyrophosphatase, a nudix hydrolase encoded by the nudt14 gene, biochemical and biophysical research communications on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Adpglucose pyrophosphorylase regulates the synthesis of glycogen in bacteria and of starch in plants. Taking also into account the fact that udp glucose concentrations, in the range 0. Human placenta hydrolases active on free adpribose. Sugar pyrophosphatase nudt5, nucleoside diphosphatelinked moiety x motif 5.
Characterization of mouse udpglucose pyrophosphatase, a. In absence of diphosphate, hydrolyzes with similar activities various modified nucleoside diphosphates such as adp ribose, adp mannose, adp glucose, 8oxogdp and 8oxodgdp pubmed. Adpglucose pyrophosphorylase, a regulatory enzyme for. The first committed step in these pathways is the synthesis of adpglucose in a reaction catalyzed by adpglucose pyrophosphorylase adpglc ppase. While neb develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications. Sdspage and subsequent staining of the purified rice adpg hydrolytic npp revealed a. Frontiers allosteric control of substrate specificity of. The relationship between nitrate fertilization and tuberbulking inroots from singleleaf explants was examined to clarify the mechanism of tuberformation.
Read overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrosestarch interconversions, and increased levels of nucleotides in growing potato tubers, planta on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. A diverse protein family important for crop improvement rebecca m. Adpglucose pyrophosphorylase potato starch synthesis sucrose induction. In photosynthetic bacteria the synthesis of glycogen follows a similar pathway.
In plants, the synthesis of starch occurs by utilizing adpglucose as the glucosyl donor for the elongation of. Pdf adenosine diphosphate sugar pyrophosphatase prevents. Enzyme that can either act as an adp sugar pyrophosphatase in absence of diphosphate or catalyze the synthesis of atp in presence of diphosphate pubmed. Substrate binding properties of potato tuber adpglucose. Constitutive release of atp and evidence for major contribution of ectonucleotide pyrophosphatase and. This product is covered by one or more patents, trademarks andor s owned or controlled by new england biolabs, inc neb. Download a pdf containing pricing for our full product list. Adpglucose pyrophosphorylase adpglc ppase from higher. Biochimica et biophysica acta bba general subjects. This article is from plant and cell physiology, volume 55. The first committed step in these pathways is the synthesis of adp glucose in a reaction catalyzed by adp glucose. M and may be nonspecific, as the same preparation hydrolyzed adpglucose, nadh and ap2a. Starch was determined by using a glucose oxidasebased test kit boehringer mannheim.
Nudc is a nudix pyrophosphatase that efficiently hydrolyzes nad. Pyrophosphatase 54 glucose 6 phosphate fructose6phosphate h 20 fructosei, 6. While some glucose transporters, such as glut1, constitutively localize to the cell membrane, glut4 translocates to the plasma membrane in response to insulin stimulation. Dec 25, 2009 taking also into account the fact that udpglucose concentrations, in the range 0. It was recently shown that potato solanum tuberosum tuber agpase is subject to redoxdependent posttranslational regulation, involving formation of an intermolecular cys bridge between the two catalytic subunits agpb of the heterotetrameric holoenzyme a. Adpribaseii showed similar properties, except that the km for adpribose was 50. Intracellular glucose was phosphorylated by glucokinase using gtp rather than atp. Frontiers on the kinetic and allosteric regulatory.
Starch synthesis is initiated by the activation of glucose by converting glc1p to adp glc through an adp glucose pyrophosphorylase agpase. Adpribaseii showed similar properties, except that the km for adp ribose was 50. Adp ribose, adp glucose and metabolic cofactors such as fad, coenzyme a and acetyl coa at varying efficiency 3 and unpublished results. Thus, the two substrates of this enzyme are adp sugar and h 2 o, whereas its two products are amp and alphadaldose 1phosphate. The enzyme is essential to the biosynthesis of starch in plants. Adpglucose pyrophosphorylase is activated by posttranslational. Glc pyrophosphorylase, and results in decreased adp. Adp glucose pyrophosphorylase adp glc ppase is the enzyme responsible for the regulation of bacterial glycogen synthesis. Extra copies of the dna enhance and antisense variants thereof reduce the starchsynthesising ability of the plants by enhancing or inhibiting expression of the enzyme.
As illustrated in figure 1, sdspage and subsequent staining of the two barley adpg hydrolytic npp isoforms revealed a single band of. A novel dual allosteric activation mechanism of escherichia. Acceleration of potato tuber sprouting by the expression. In this work, we analyzed the activation promiscuity of adp glc ppase subunits. Regulatory properties of adp glucose pyrophosphorylase are. Frontiers mechanism underlying heat stability of the. Adp glucose pyrophosphorylase adp glc ppase catalyzes the first committed step in the synthesis of glycogen in bacteria and starch in algae and plants. Nucleotide pyrophosphatasephosphodiesterase 1 exerts a. The aim of this work was to discover whether both of the alternative transcripts produced by the gene encoding a small subunit ssu of adp glucose pyrophosphorylase agpase in barley produce proteins with significant roles in starch synthesis in the plant. Assistive devices program health care professionals mohltc. Adp glucose pyrophosphorylase is located in the plastid in developing tomato fruit1 diane m. Human adpsugar pyrophosphatasenudt5 antibody af6414. Rice plastidial nglycosylated nucleotide pyrophosphatase.
Adpribasei fulfils the requirements of a specific turnover pathway consistent with a cellular role for free adpribose. There are also biochemical data that support the model 19. In plants, the synthesis of starch occurs by utilizing adp glucose as the glucosyl donor for the elongation of alpha1,4glucosidic chains. Cofactor specificities of glycolytic enzymes in clostridium thermocellum were studied with cellobiosegrown cells from batch cultures. Plastids prepared from crude homogenates of columella and pericarp, the starchaccumulating tissues of developing fruit, contained. For smallscale extractions, plant tissues were homogenized with 3fold of extraction buffer 50 mm mes, ph 6. Changes in the activities of sucrose synthase susy, adp glucose pyrophosphorylase agpase, udp glucose pyrophosphorylase ugpase, alkaline inorganic pyrophosphatase, 3phosphoglycerate 3pga phosphatase and amylases were monitored in relation to accumulation of starch in developing pods of mung bean vigna radiata l. Studies in other species have led to the identification of only a. Atp adp glucose phosphoglucose isomerase atp adp 1,3bisphosphoglycerate fructose 1,6bisphosphate fructose 6phosphat glucose 6phosphat glucose 6phosphatase p i p i endoplasmic reticulum cytosol p i. Members of this family are pyrophosphohydrolases that act upon substrates with the general structure of a nucleoside nu diphosphate di linked to another moiety, x ndpx to yield nmp plus px 1. The recombinant enzyme was purified to electrophoretic homogeneity to accurately characterize its oligomeric, kinetic, and regulatory properties. The energy released by hydrolysis of atp is utilised for the forward reaction. Role of enzymes of sucrosestarch conversion in seed sink.
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