Glycogen is generally assumed to serve as a major reserve polysaccharide in bacteria. Adpglucose and starch synthesis in plants is based, in part. The starchsynthesising ability of plants, particularly cereals, may be modified by insertion into their genomes by genetic transformation a dna encoding adp glucose pyrophosphorylase. The question is how the undesirable outcomes of in vivo promiscuity can be prevented. The relationship between nitrate fertilization and tuberbulking inroots from singleleaf explants was examined to clarify the mechanism of tuberformation. A cytosolic adpglucose pyrophosphorylase is a feature of graminaceous endosperms, but not of other. Heat and drought stress responses alter grain characteristics. Adpglucose pyrophosphorylase agpase catalyzes the first committed reaction in the pathway of starch synthesis. Insights into glycogen metabolism in chemolithoautotrophic bacteria from distinctive kinetic and regulatory properties of adp glucose pyrophosphorylase from nitrosomonas europaea.
Unraveling the activation mechanism of the potato tuber adp. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes adpglucose, the precursor molecule of glycogen biosynthesis. Characterization of recombinant udp and adpglucose pyrophosphorylases and glycogen synthase to elucidate glucose1phosphate partitioning into oligo and polysaccharides in streptomyces coelicolor matias d. Regulation adpglucose pyrophosphorylase from chlorella vulgaris. The energy released by hydrolysis of atp is utilised for the forward reaction. In photosynthetic bacteria the synthesis of glycogen follows a similar pathway. This promiscuous behavior has important implications in protein evolution and the acquisition of new functions.
Frontiers mechanism underlying heat stability of the. In this work, glycogen accumulation in the amino acid producer corynebacterium glutamicum was characterized, expression of the c. Here we report that human placenta extracts contain two adp ribose hydrolases, which were characterised after a near fold purification. Ostreococcustauri adpglucosepyrophosphorylasereveals. Characterization of mouse udpglucose pyrophosphatase, a. Experimentalprocedures materialsdu14c glucose 1phosphate glc1p was purchased from ge healthcare. An adenosine diphosphate sugar pyrophosphatase asppase, ec 3. To increase the heat tolerance of the rice enzyme, we engineered two dominant agpase subunits expressed in developing endosperm, the large l2 and small. In plants, the synthesis of starch occurs by utilizing adp glucose as the glucosyl donor for the elongation of alpha1,4glucosidic chains.
Adp glucose pyrophosphorylase, a regulatory enzyme for bacterial glycogen synthesis miguel a. Thus, the two substrates of this enzyme are adpsugar and h 2 o, whereas its two products are amp and alphadaldose 1phosphate. Coexpression of two polypeptides comprising residues 23 and 328431 from the escherichia coli adpglucose pyrophosphorylase yielded an enzyme form as active as the wild type. Pdf adpglucose pyrophosphorylase, a regulatory enzyme. Most of adpglucose linked to starch biosynthesis occurs.
Human placenta hydrolases active on free adpribose. We report the first atomic resolution structure of adp. Computational analysis of adpglucose pyrophosphorylases predicts a fold with two domains. The first committed step in these pathways is the synthesis of adp glucose in a reaction catalyzed by adp glucose. Genes free fulltext a new snp in rice gene encoding. Human adpsugar pyrophosphatasenudt5 antibody mab6414. Adpglucose pyrophosphorylase regulates the synthesis of glycogen in bacteria and of starch in plants. Adpglucose, arising from glucose1phosphate and atp liberating inorganic pyrophosphate ppi6. On the stability of nucleoside diphosphate glucose metabolites. The enzyme was the most stable at ph 7, showing 50% loss of its original activity after 50min heating at 57c.
Cytoplasmic escherichia coli adp sugar pyrophosphatase binds. The enzyme responsible for this activity, referred to as adpg pyrophosphatase agppase, was purified over 1,100fold from barley leaves and subjected to biochemical. The enzyme is hexokinase, which splits atp into adp and the pi is added on to the glucose. Frontiers allosteric control of substrate specificity of. Adpglucose pyrophosphorylase agpase is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. In enzymology, a glucose1phosphate adenylyltransferase ec 2. The dephosphorylation of glucose 6phosphate takes place within the lumen of endoplasmic reticulum. Adpglucose pyrophosphorylase is located in the plastid in. Modulation of allosteric regulation by e38k and g101n mutations in the potato tuber adp glucose pyrophosphorylase shinji wakuta,1 yumi shibata,1 yumiko yoshizaki,1 wataru saburi,1. The nterminal catalytic domain resembles a dinucleotidebinding.
Adp sugar pyrophosphatase nudt5 is a member of the nudix superfamily of enzymes. In plants, glucose is stored as starch, a polysaccharide chemically similar to glycogen, but in this case the glucosyl donor is adp glucose 16. Adp glc pyrophosphorylase ppase, a key regulatory enzyme in the biosynthetic pathway of starch and bacterial glycogen, catalyzes the synthesis of adp glc from glc1p and atp. The full text of this article hosted at is unavailable due to technical difficulties. 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. Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. 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. Human adpsugar pyrophosphatasenudt5 antibody af6414. Proteomics analysis reveals noncontrolled activation of. Coupled chemoenzymatic reactions in continuous flow. A homology model of the threedimensional structure of the escherichia coli enzyme complexed with adp glc has been generated to study the substratebinding site in detail.
Thus, whereas npp1 and npp6 catalyze the hydrolytic breakdown of nucleotide sugars such as adp glucose, udp glucose and adp ribose, npp2 did not recognize these compounds as substrates, but favored the hydrolysis of udp k cat k m of 0. Jul 03, 2001 an adenosine diphosphate sugar pyrophosphatase asppase, ec 3. Glc1p, atp, adp glucose adp glc, 3phosphoglycerate 3pga, nadp, glucose 6phosphate dehydrogenase from leuconostoc mesenteroides, phosphoglucomutase from rabbit muscle, and inorganic pyrophosphatase were purchased. The only difference from the wild type was a slightly modified affinity for allosteric effectors. Dec 25, 2009 in escherichia coli, an adp glucose pyrophosphatase, encoded by the nudf aspp gene, has been reported to regulate adp glucose levels linked to bacterial glycogen synthesis. A cytosolic adpglucose pyrophosphorylase is a feature of. 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. Thus, the two substrates of this enzyme are atp and alphadglucose 1phosphate, whereas its two products are diphosphate and adpglucose. Adpsugar pyrophosphatasenudt5 antibody h00011164a01. Plastid adpglucose pyrophosphorylase small subunit agps2a and agps2b and soluble starch synthase ssiib and ssiic also exhibited enhanced expression in the flo45 mutant.
Plant agpase is a heterotetramer formed by two small subunits and two large subunits. The presence of an adenosine diphosphate sugar pyrophosphatase aspp in e. Adpgucose starch synthase tarc chloroplast phospho glucomutase glucose1 phosphate glucose6 hexose adp glucos pyrophos atp phorylase phosphate phosphate isomerase fructose6phosphate fructosei. Realtime rtpcr experiments revealed that ppdk was expressed at considerably higher levels in the flo45 mutant than in the wild type during the grain filling stage. It comprises two types of subunits, but despite its importance little is known about their roles. Pyruvate pyr and fructose 6phosphate fru6p activate the enzyme from agrobacterium tumefaciens by increasing v max about 10 and 20fold, respectively. The starchsynthesising ability of plants, particularly cereals, may be modified by insertion into their genomes by genetic transformation a dna encoding adpglucose pyrophosphorylase. The enzyme preparation contained noactivity ofpglucomutase, glucose lphosphatase, adp glucose pyrophosphatase, aglucan phosphorylase, starch synthase, a branching enzyme, or a debranching enzyme. A plastidial phosphodiesterase that prevents starch biosynthesis. Cytoplasmic escherichia coli adp sugar pyrophosphatase. Directed molecular evolution of adpglucose pyrophosphorylase. Adenosine diphosphate sugar pyrophosphatase prevents. Adp glucose 1p glucose 6p glycolysis glucose shipped out glycogen phosphorylase udp glucose pyrophosphatase glycogen synthase massaction ratios are the ratio of products over reactants for any given reaction phosphoglucomutase q phosphoglucomutase glucose 6p glucose 1p for instance knowing whether this ratio is or adp gucose starch synthase tarc chloroplast phospho glucomutase glucose 1 phosphate glucose 6 hexose adp glucos pyrophos atp phorylase phosphate phosphate isomerase fructose6phosphate fructosei. As a first step in characterizing the role of mammalian ugppase, we cloned and analyzed recombinant mouse ugppase.
Adpglucose pyrophosphorylase, a regulatory enzyme for. Pdf the accumulation of alpha1,4polyglucans is an important strategy to cope with transient starvation conditions in the environment. Recent studies have identified an adp glucose pyrophosphatase that, analogously to ugppase, has been proposed to control adp glucose levels, and hence starch accumulation, in some tissues 17, 18. 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. Npp1 is a major determinant of adpglucose pyrophosphatase agppase activity and can reduce the plastidic pool of adpglucose required for starch accumulation in rice 14. Crystal structure of potato tuber adpglucose pyrophosphorylase xiangshu jin1,2,3, miguel a ballicora2, jack preiss2, and james h geiger1, 1department of chemistry, michigan state university, east lansing, mi, usa and 2department of biochemistry and molecular biology, michigan state university, east lansing, mi, usa adpglucose pyrophosphorylase catalyzes the.
Posttranslational redox modification of adpglucose. The subcellular location of activity and protein of adpglucose pyrophosphorylase agpase in developing tomato lycopersicon esculentum fruit was determined following a report that the enzyme might be present inside and outside the plastids in this organ. Glycogen formation in corynebacterium glutamicum and role. The aim of this work was to discover whether both of the alternative transcripts produced by the gene encoding a small subunit ssu of adpglucose pyrophosphorylase agpase in barley produce proteins with significant roles in starch synthesis in the plant. Hexokinase is the key glycolytic enzyme and the reaction is irreversible. Smith john innes centre, colney lane, norwich nr4 7uh, united kingdom d. Substrate binding properties of potato tuber adpglucose. Frontiers on the roles of wheat endosperm adpglucose. Moleculararchitectureoftheglucose1phosphatesitein adp. Herein, we report the molecular cloning and recombinant expression of both genes separately or together followed by the characterization of the respective enzymes.
Theenzymicactivitywasstable for at least 2 months in a freezer. Jojournalurnal crystal structure of potato tuber adpglucose. Adpglucose pyrophosphorylase from escherichia coli, was predicted to be near the substrate site. Download fulltext pdf identification of the adpglucose pyrophosphorylase isoforms essential for starch synthesis in the leaf and seed endosperm of rice oryza sativa l. Plastids prepared from crude homogenates of columella and pericarp, the starchaccumulating tissues of developing fruit, contained 8% to 18. A plastidial phosphodiesterase that prevents starch biosynthesis milagros rodrguezlo. Starch synthesis in potato tubers is regulated by post.
Insights into glycogen metabolism in chemolithoautotrophic. Both genes were upregulated in maturing pollen, a stage when. According to this view, the plastidial adpglucose adpg pyrophosphorylase agp is the sole enzyme catalyzing the synthesis of the starch precursor molecule adpg. The adpglucose pyrophosphorylase from escherichia coli.
Bacterial adpglucose pyrophosphorylases are allosterically regulated by metabolites that are key intermediates of central pathways in the respective microorganism. The major form of adpglucose pyrophosphorylase in maize. Alanine scanning mutagenesis of adpglucose pyrophosphorylase from anabaena sp. Jojournalurnal crystal structure of potato tuber adp. Adpglucose pyrophosphorylase, a regulatory enzyme for bacterial glycogen synthesis miguel a. Pdf adenosine diphosphate sugar pyrophosphatase prevents. In plants, the synthesis of starch occurs by utilizing adpglucose as the glucosyl donor for the elongation of. Modulation of allosteric regulation by e38k and g101n. In bacteria and plants, the synthesis of glycogen and starch occurs by utilizing adp glucose as the glucosyl donor for elongation of the alpha1,4glucosidic chain.
Adp sugar pyrophosphatase aspp is a member of the nudix nucleoside diphosphate linked to some other moiety x hydrolase family of enzymes that catalyzes the hydrolytic breakdown of adpglucose adpg linked to glycogen biosynthesis. 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. Starch was determined by using a glucose oxidasebased test kit boehringer mannheim. Adp glucose1p glucose6p glycolysis glucose shipped out glycogen phosphorylase udpglucose pyrophosphatase glycogen synthase massaction ratios are the ratio of products over reactants for any given reaction phosphoglucomutase q phosphoglucomutase glucose6p glucose1p for instance knowing whether this ratio is or pyrophosphatasenudt5 antibody. Regulation adpglucose pyrophosphorylase from chlorella. 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. Transcriptional and allosteric regulation of adpglc pyrophosphorylase agpase plays a major role in. 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.
Pdf adpglucose pyrophosphorylase, a regulatory enzyme for. Sugar pyrophosphatase nudt5, nucleoside diphosphatelinked moiety x motif 5. Pdf identification of the adpglucose pyrophosphorylase. To elucidate the function that asp142 might play in the e. Characterization of recombinant udp and adpglucose. Adpsugar pyrophosphatase nudt5 is a member of the nudix superfamily of enzymes. The enzyme preparation contained noactivity ofpglucomutase, glucoselphosphatase, adpglucose pyrophosphatase, aglucan phosphorylase, starch synthase, a branching enzyme, or a debranching enzyme.
Higher concentration of ppi in grain amyloplast inhibits starch biosynthesis by inhibiting agpase, and hence there is another important enzyme involved in regulation of starch synthesis is alkaline inorganic pyrophosphatase ppi. Adp glucose pyrophosphorylase is located in the plastid in developing tomato fruit1 diane m. Adp glucose, arising from glucose 1phosphate and atp liberating inorganic pyrophosphate ppi6. Nucleotide pyrophosphatasephosphodiesterase 1 exerts a. Crystals of potato tuber adpglucose pyrophosphorylase a subunit were grown in high concentrations of sulfate, resulting in the sulfatebound, allosterically inhibited form of the enzyme.