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. In the present study the allosteric contributions of the different L subunits were compared by expressing each one in Escherichia coli, in conjunction with S and individually, and characterizing the resulting enzyme activity. Our results indicate different kinetic characteristics of the tomato L1/S and L3/S heterotetramers. Surprisingly, the recombinant L3 protein was also active when expressed alone and size-exclusion and immunoblotting showed that it functioned as a monomer. Subunit interaction modelling pointed to two amino acids potentially affecting subunit interactions. However, directed mutations did not have an impact on subunit tetramerization. These results indicate a hitherto unknown active role for the L subunit in the synthesis of ADP-Glc.

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. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

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). Most of the ADP-Glc PPases are allosterically regulated by intermediates of the major carbon assimilatory pathway in the organism. Based on specificity for activator and inhibitor, classification of ADP-Glc PPases has been expanded into nine distinctive classes. According to predictions of the secondary structure of the ADP-Glc PPases, they seem to have a folding pattern common to other sugar nucleotide pyrophosphorylases. All the ADP-Glc PPases as well as other sugar nucleotide pyrophosphorylases appear to have evolved from a common ancestor, and later, ADP-Glc PPases developed specific regulatory properties, probably by addition of extra domains. Studies of different domains by construction of chimeric ADP-Glc PPases support this hypothesis. In addition to previous chemical modification experiments, the latest random and site-directed mutagenesis experiments with conserved amino acids revealed residues important for catalysis and regulation.

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 two polypeptides could not be separated by chromatographic procedures. Separate expression of these polypeptides produced inactive unstable forms. All these results indicated that the ADP-glucose pyrophosphorylase comprises two domains with a strong interaction between them. That interaction is important for allosteric properties and structural stability.

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). Generally, this enzyme is allosterically regulated by intermediates of the major carbon assimilatory pathway in the respective organism. In oxygenic photosynthesizers, ADPGlc PPase is mainly regulated by 3-phosphoglycerate (activator) and inorganic orthophosphate (inhibitor), interacting in four different patterns. Recent reports have shown that in higher plants, some of the enzymes could also be redox regulated. In eukaryotes, the enzyme is a heterotetramer comprised of two distinct subunits, a catalytic and a modulatory subunit. The latter has been proposed as related to variations in regulation of the enzyme in different plant tissues. Random and site-directed mutagenesis experiments of conserved amino acids revealed important residues for catalysis and regulation. Prediction of the ADPGlc PPase secondary structure suggests that it shares a common folding pattern to other sugar-nucleotide pyrophosphorylases, and they evolved from a common ancestor.

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., a nitrogen source. This review describes the enzymatic reactions involved in glycogen synthesis and the allosteric regulation of the first enzyme, ADP-glucose pyrophosphorylase. The properties of the enzymes involved in glycogen synthesis, ADP-glucose pyrophosphorylase, glycogen synthase, and branching enzyme are also characterized. The data describing the genetic regulation of the glycogen synthesis are also presented. An alternate pathway for glycogen synthesis in mycobacteria is also described.

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., a nitrogen source. The structural genes of the glycogen biosynthetic enzymes of E. coli and S. serovar Typhimurium have been cloned previously, and that has provided insights in the genetic regulation of glycogen synthesis. An important aspect of the regulation of glycogen synthesis is the allosteric regulation of the ADP-Glc PPase. The current information, views, and concepts regarding the regulation of enzyme activity and the expression of the glycogen biosynthetic enzymes are presented in this review. The recent information on the amino acid residues critical for the activity of both glycogen synthase and branching enzyme (BE) is also presented. The residue involved in catalysis in the E. coli ADP-Glc PPase was determined by comparing a predicted structure of the enzyme with the known three-dimensional structures of sugar-nucleotide PPase domains. The molecular cloning of the E. coliglg K-12 structural genes greatly facilitated the subsequent study of the genetic regulation of bacterial glycogen biosynthesis. Results from studies of glycogen excess E. coli B mutants SG3 and AC70R1, which exhibit enhanced levels of the enzymes in the glycogen synthesis pathway (i.e., they are derepressed mutants), suggested that glycogen synthesis is under negative genetic regulation.

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

Escherichia coli glycogen synthase (EcGS, EC 2.4.1.21) is a retaining glycosyltransferase (GT) that transfers glucose from adenosine diphosphate glucose to a glucan chain acceptor with retention of configuration at the anomeric carbon. EcGS belongs to the GT-B structural superfamily. Here we report several EcGS x-ray structures that together shed considerable light on the structure and function of these enzymes. The structure of the wild-type enzyme bound to ADP and glucose revealed a 15.2 degrees overall domain-domain closure and provided for the first time the structure of the catalytically active, closed conformation of a glycogen synthase. The main chain carbonyl group of His-161, Arg-300, and Lys-305 are suggested by the structure to act as critical catalytic residues in the transglycosylation. Glu-377, previously thought to be catalytic is found on the alpha-face of the glucose and plays an electrostatic role in the active site and as a glucose ring locator. This is also consistent with the structure of the EcGS(E377A)-ADP-HEPPSO complex where the glucose moiety is either absent or disordered in the active site.

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. In Arabidopsis (Arabidopsis thaliana), two genes encode small subunits (APS1 and APS2) and four large subunits (APL1-APL4). Here, we show that in Arabidopsis, APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a noncatalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-phosphoglycerate). Mutation of the Glc-1-P binding site of Arabidopsis and potato (Solanum tuberosum) isoforms confirmed these observations. To determine the relevance of these activities in planta, a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacks ADP-Glc PPase activity, APS1 mRNA, and APS1 protein, and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild-type phenotype, indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glc synthesis in planta.

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. Depending on the subunit combinations, hybrid heterotetramers showed responses to allosteric effectors [3-phosphoglycerate (3-PGA) and Pi] in the micromolar or millimolar range. While hybrid potato small subunit (PSS) and the Arabidopsis large subunit APL1 showed an extremely sensitive response to 3-PGA and Pi, hybrid PSS/Arabidopsis APL2 was very insensitive to them. Intermediate responses were determined for other subunit combinations.

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. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu(102) and Pro(103) was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains.

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. Kinetic analysis of the mutants revealed the involvement of these residues in ADP-Glc binding. Replacement of Asp21, Asn246 or Tyr355 for Ala decreased the apparent affinity for ADP-Glc 18-, 45-, and 31-fold, respectively. Comparison with other crystallized retaining GT-B glycosyltransferases confirmed the striking similarities among this group of enzymes even though they use different substrates.

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 role of these amino acids (Glu(194), Ser(212), Tyr(216), Asp(239), Phe(240), Trp(274), and Asp(276)) was studied by site-directed mutagenesis through the characterization of the kinetic properties and thermal stability of the designed mutants. All purified alanine mutants had 1 or 2 orders of magnitude lower apparent affinity for Glc-1-P compared with the wild type, indicating that the selected set of amino acids plays an important role in their interaction with the substrate. These amino acids, which are conserved within the ADP-Glc PPase family, were replaced with other residues to investigate the effect of size, hydrophobicity, polarity, aromaticity, or charge on the affinity for Glc-1-P. In this study, the architecture of the Glc-1-P-binding site is characterized. The model overlaps with the Glc-1-P site of other PPases such as Pseudomonas aeruginosa dTDP-Glc PPase and Salmonella typhi CDP-Glc PPase. Therefore, the data reported here may have implications for other members of the nucleotide-diphosphoglucose PPase family.

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. PSSANA contained the N-terminus (292 amino acids) of the potato tuber ADP-Glc PPase small subunit (PSS) and the C-terminus (159 residues) of the Anabaena PCC 7120 enzyme. ANAPSS was the inverse chimera. These constructs were expressed separately or together with the large subunit of the potato tuber ADP-Glc PPase (PLS), to obtain homo- and heterotetrameric chimeric proteins. Characterization of these forms showed that the N-terminus determines stability and regulatory redox-dependent properties. The chimeric forms exhibited intermediate 3-phosphoglycerate activation properties with respect to the wild-type homotetrameric enzymes, indicating that the interaction between the putative N- and C-domains determines the affinity for the activator. Characterization of the chimeric heterotetramers showed the functionality of the large subunit, mainly in modulating regulation of the enzyme by the coordinate action of 3-phosphoglycerate and inorganic orthophosphate.

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. Crystals of potato tuber ADP-glucose pyrophosphorylase alpha subunit were grown in high concentrations of sulfate, resulting in the sulfate-bound, allosterically inhibited form of the enzyme. The N-terminal catalytic domain resembles a dinucleotide-binding Rossmann fold and the C-terminal domain adopts a left-handed parallel beta helix that is involved in cooperative allosteric regulation and a unique oligomerization. We also report structures of the enzyme in complex with ATP and ADP-glucose. Communication between the regulator-binding sites and the active site is both subtle and complex and involves several distinct regions of the enzyme including the N-terminus, the glucose-1-phosphate-binding site, and the ATP-binding site. These structures provide insights into the mechanism for catalysis and allosteric regulation of the enzyme.

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. Here we studied the ancestral role of a model non-catalytic modulatory subunit. With a rational approach, we "resurrected" enzymatic activity from that subunit to experimentally prove that it derived from a catalytic ancestor. We show that this protein (L subunit ADP-glucose pyrophosphorylase) evolved to have a regulatory role, losing catalytic residues more than 130 million years ago, but preserving, possibly as a by-product, the substrate site architecture. Inactivation of catalytic subunits could be the consequence of a general evolutionary strategy to explore new regulatory roles in hetero-oligomers.

Truncation of the amino terminus of branching enzyme changes its chain transfer pattern.

Previous work has reported the production of an Escherichia coli branching enzyme with a 112-residue deletion at the amino terminal by limited proteolysis. Here, we study the chain transfer pattern of this enzyme. Gel-permeation chromatography of in vitro branched amylose shows that the truncated branching enzyme transfers fewer short chains (degree of polymerization [d.p.] <20) and a greater proportion of intermediate size chains (d.p. 30-90) than the native enzyme. High-performance anion-exchange chromatography (HPAEC) of the branching limited alpha-glucan product indicates that the truncated branching enzyme transfers a smaller proportion of chains with d.p. 4-11 and more chains longer than d.p. 12. Also, the genes encoding native or truncated branching enzyme were individually expressed in a branching enzyme-deficient mutant, AC71 (glgB(-)). By HPAEC analysis of the purified alpha-glucans we find that truncated branching enzyme transfers fewer chains of d.p. 5-11 and more chains longer than d.p. 12 relative to the full-length enzyme. These observations allow us to conclude that truncation of the amino-terminal domain has altered the branching pattern of the enzyme. Our results are consistent with the construction of hybrid branching enzymes from the maize isoforms.

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]. In this study, analysis of several site-directed mutants in the presence of different metabolic effectors showed that the primary inhibitor for two of the mutant proteins, R294A and R294Q, was no longer orthophosphate but rather NADPH, which was a reversal in the pattern of inhibitor selectivity from the wild-type. Despite the differences in charge and size, analysis of the purified R294K, R294E, and R294Q mutant enzymes demonstrated similar decreases in orthophosphate affinity as the R294A mutant, while most of the other kinetic values were similar to those reported for the wild-type. All these results suggest that the positive charge of Arg(294) is not specifically involved in orthophosphate binding and that it is important in determining inhibitor selectivity.

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.A. and Preiss J. (2001) J. Biol. Chem., 276, 46319-46325]. The residue is highly conserved throughout the family of ADP-Glc PPases, as well as throughout the super-family of sugar-nucleotide pyrophosphorylases. In the heterotetrameric ADP-Glc PPase from potato (Solanum tuberosum L.) tuber, the homologous residue is present in both the small (Asp145) and the large (Asp160) subunits. It has been proposed that the small subunit of plant ADP-Glc PPases is catalytic, while the large subunit is modulatory; however, no catalytic residues have been identified. To investigate the function of these conserved Asp residues in the ADP-Glc PPase from potato tuber, we used site-directed mutagenesis to introduce either an Asn or a Glu. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of ADP-Glc showed a significant decrease (more than four orders of magnitude) in the specific activity of the SD145NLwt, SD145NLD160N, and SD145NLD160E mutants, while the effect was smaller (approximately two orders of magnitude) with the SD145ELwt, SD145ELD160N, and SD145ELD160E mutants. By contrast, mutation of the large subunit alone did not affect the specific activity but did alter the apparent affinity for the activator 3-phosphoglycerate, showing two types of apparent roles for this residue in the different subunits. These results show that mutation of Asp160 of the large subunit does not affect catalysis, thus the large subunit is not catalytic, and that the negative charge of Asp145 in the small subunit is necessary for enzyme catalysis.

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. To confirm the importance of these residues predicted by the model, we studied them in E. coli glycogen synthase by site-directed mutagenesis. Mutations D137A, R300A, K305A, and H161A decreased the specific activity 8100-, 2600-, 1200-, and 710-fold, respectively. None of these mutations increased the Km for glycogen and only H161A and R300A had a higher Km for ADP-Glc of 11- and 8-fold, respectively. These residues were essential, validating the model that shows a strong similarity between the active site of E. coli glycogen synthase and the other retaining GT-B glycosyltransferases known to date.