Glycogen: Biosynthesis and Regulation.

Glycogen accumulation occurs in Escherichia coli and Salmonella enterica serovar Typhimurium as well as in many other bacteria. Glycogen will be formed when there is an excess of carbon under conditions in which growth is limited because of the lack of a growth nutrient, e.g.

Characterization of the AGPase large subunit isoforms from tomato indicates that the recombinant L3 subunit is active as a monomer.

The enzyme AGPase [ADP-Glc (glucose) pyrophosphorylase] catalyses a rate-limiting step in starch synthesis in tomato (Solanum lycopersicon) fruit, which undergoes a transient period of starch accumulation. It has been a generally accepted paradigm in starch metabolism that the enzyme naturally functions primarily as a heterotetramer comprised of two large subunits (L) and two small subunits (S). The tomato genome harbours a single gene encoding S and three genes for L proteins, which are expressed in both a tissue- and time-specific manner.

Oligosaccharide binding in Escherichia coli glycogen synthase.

Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure.

Glycogen: Biosynthesis and Regulation.

The accumulation of glycogen occurs in Escherichia coli and Salmonella enterica serovar Typhimurium as well as in many other bacteria. Glycogen will be formed when there is an excess of carbon under conditions in which growth is limited due to the lack of a growth nutrient, e.g.

The crystal structures of the open and catalytically competent closed conformation of Escherichia coli glycogen synthase.

Escherichia coli glycogen synthase (EcGS, EC 2.4.1.

Two Arabidopsis ADP-glucose pyrophosphorylase large subunits (APL1 and APL2) are catalytic.

ADP-glucose (Glc) pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (alpha(2)beta(2)) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants result from the synergy of both types of subunits.

Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function 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. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene.

Regulatory properties of potato-Arabidopsis hybrid ADP-glucose pyrophosphorylase.

In higher plants, ADP-glucose pyrophosphorylase (ADPGlc-PPase) is a heterotetrameric enzyme comprised of two small and two large subunits. Potato-Arabidopsis hybrid ADPGlc-PPases were generated and their regulatory properties analyzed. We show that ADPGlc-PPase subunits from two different species can interact, producing active enzymes with new regulatory properties.

Molecular architecture of the glucose 1-phosphate site in ADP-glucose pyrophosphorylases.

ADP-Glc pyrophosphorylase (PPase), a key regulatory enzyme in the biosynthetic pathway of starch and bacterial glycogen, catalyzes the synthesis of ADP-Glc from Glc-1-P and ATP. A homology model of the three-dimensional structure of the Escherichia coli enzyme complexed with ADP-Glc has been generated to study the substrate-binding site in detail. A set of amino acids in the model has been identified to be in close proximity to the glucose moiety of the ADP-Glc ligand.

The ADP-glucose binding site of the Escherichia coli glycogen synthase.

Bacterial glycogen/starch synthases are retaining GT-B glycosyltransferases that transfer glucosyl units from ADP-Glc to the non-reducing end of glycogen or starch. We modeled the Escherichia coli glycogen synthase based on the coordinates of the inactive form of the Agrobacterium tumefaciens glycogen synthase and the active form of the maltodextrin phosphorylase, a retaining GT-B glycosyltransferase belonging to a different family. In this model, we identified a set of conserved residues surrounding the sugar nucleotide substrate, and we replaced them with different amino acids by means of site-directed mutagenesis.

Domain swapping between a cyanobacterial and a plant subunit ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the regulatory step in the pathway for synthesis of bacterial glycogen and starch in plants. ADP-Glc PPases from cyanobacteria (homotetramer) and from potato (Solanum tuberosum) tuber (heterotetramer) are activated by 3-phosphoglycerate and inhibited by inorganic orthophosphate. To study the function of two putative domains, chimeric enzymes were constructed.

ADP-Glucose Pyrophosphorylase: A Regulatory Enzyme for Plant Starch Synthesis.

In plants, the synthesis of starch occurs by utilizing ADP-glucose as the glucosyl donor for the elongation of alpha-1,4-glucosidic chains. In photosynthetic bacteria the synthesis of glycogen follows a similar pathway. The first committed step in these pathways is the synthesis of ADP-glucose in a reaction catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase).

Crystal structure of potato tuber ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase catalyzes the first committed and rate-limiting step in starch biosynthesis in plants and glycogen biosynthesis in bacteria. It is the enzymatic site for regulation of storage polysaccharide accumulation in plants and bacteria, being allosterically activated or inhibited by metabolites of energy flux. We report the first atomic resolution structure of ADP-glucose pyrophosphorylase.

Resurrecting the ancestral enzymatic role of a modulatory subunit.

In the post-genomic era, functional prediction of genes is largely based on sequence similarity searches, but sometimes the homologues bear different roles because of evolutionary adaptations. For instance, the existence of enzyme and non-enzyme homologues poses a difficult case for function prediction and the extent of this phenomenon is just starting to be surveyed. Different evolutionary paths are theoretically possible for the loss or acquisition of enzyme function.

The ADP-glucose pyrophosphorylase from Escherichia coli comprises two tightly bound distinct domains.

Computational analysis of ADP-glucose pyrophosphorylases predicts a fold with two domains. Co-expression of two polypeptides comprising residues 1-323 and 328-431 from the Escherichia coli ADP-glucose pyrophosphorylase yielded an enzyme form as active as the wild type. The only difference from the wild type was a slightly modified affinity for allosteric effectors.

The active site of the Escherichia coli glycogen synthase is similar to the active site of retaining GT-B glycosyltransferases.

Bacterial glycogen synthases transfer a glucosyl unit, retaining the anomeric configuration, from ADP-glucose to the non-reducing end of glycogen. We modeled the Escherichia coli glycogen synthase based on three glycosyltransferases with a GT-B fold. Comparison between the model and the structure of the active site of crystallized retaining GT-B glycosyltransferases identified conserved residues with the same topology.

Identification and characterization of a critical region in the glycogen synthase from Escherichia coli.

The cysteine-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) inactivates the Escherichia coli glycogen synthase (Holmes, E., and Preiss, J. (1982) Arch.

An assay for adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase.

Adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5'-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer.

Characterization of the branching patterns of glycogen branching enzyme truncated on the N-terminus.

Truncation of 112 amino acids at the N-terminus (Nd(1-112)) changes the chain transfer pattern of the Escherichia coli glycogen branching enzyme (GBE) [Arch. Biochem. Biophys.

ADP-glucose pyrophosphorylase, a regulatory enzyme for bacterial glycogen synthesis.

The accumulation of alpha-1,4-polyglucans is an important strategy to cope with transient starvation conditions in the environment. In bacteria and plants, the synthesis of glycogen and starch occurs by utilizing ADP-glucose as the glucosyl donor for elongation of the alpha-1,4-glucosidic chain. The main regulatory step takes place at the level of ADP-glucose synthesis, a reaction catalyzed by ADP-Glc pyrophosphorylase (PPase).

The different large subunit isoforms of Arabidopsis thaliana ADP-glucose pyrophosphorylase confer distinct kinetic and regulatory properties to the heterotetrameric enzyme.

ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis and is allosterically regulated by the levels of 3-phosphoglycerate and phosphate in plants. ADP-glucose pyrophosphorylases from plants are heterotetramers composed of two types of subunits (small and large). In this study, the six Arabidopsis thaliana genes coding for ADP-glucose pyrophosphorylase isoforms (two small and four large subunits) have been cloned and expressed in an Escherichia coli mutant deficient in ADP-glucose pyrophosphorylase activity.

ADP-glucose pyrophosphorylase from potato tuber: site-directed mutagenesis of homologous aspartic acid residues in the small and large subunits.

Asp142 in the homotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase) enzyme from Escherichia coli was demonstrated to be involved in catalysis of this enzyme [Frueauf, J.B., Ballicora, M.

The X-ray crystallographic structure of Escherichia coli branching enzyme.

Branching enzyme catalyzes the formation of alpha-1,6 branch points in either glycogen or starch. We report the 2.3-A crystal structure of glycogen branching enzyme from Escherichia coli.

Characterization of chimeric ADPglucose pyrophosphorylases of Escherichia coli and Agrobacterium tumefaciens. Importance of the C-terminus on the selectivity for allosteric regulators.

ADPglucose pyrophosphorylase catalyzes the regulatory step in the pathway for bacterial glycogen synthesis. The enzymes from different organisms exhibit distinctive regulatory properties related to the main carbon metabolic pathway. Escherichia coli ADPglucose pyrophosphorylase is mainly activated by fructose 1,6-bisphosphate (FBP), whereas the Agrobacterium tumefaciens enzyme is activated by fructose 6-phosphate (F6P) and pyruvate.

Alteration of inhibitor selectivity by site-directed mutagenesis of Arg(294) in the ADP-glucose pyrophosphorylase from Anabaena PCC 7120.

Previous alanine scanning mutagenesis of ADP-glucose pyrophosphorylase from Anabaena PCC 7120 indicated that Arg(294) plays a role in inhibition by orthophosphate [J. Sheng, J. Preiss, Biochemistry 36 (1997) 13077].