Miguel A. Ballicora

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 α-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.

ADP-Glucose Pyrophosphorylase, a Regulatory Enzyme for Bacterial Glycogen Synthesis

SUMMARY The accumulation of α-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 α-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.

Activation of the Potato Tuber ADP-glucose Pyrophosphorylase by Thioredoxin

The potato tuber (Solanum tuberosumL.) ADP-glucose pyrophosphorylase (ADP-GlcPPase) catalyzes the first committed step in starch biosynthesis. The main type of regulation of this enzyme is allosteric, and its activity is controlled by the ratio of activator, 3-phosphoglycerate to inhibitor, Pi. It was reported (Fu, Y., Ballicora, M. A., Leykam, J. F., and Preiss, J. (1998) J. Biol. Chem. 273, 25045–25052) that the enzyme was activated by reduction of the Cys12disulfide linkage present in the catalytic subunits. In this study, both reduced thioredoxin f and m from spinach (Spinacia oleracea) leaves reduced and activated the enzyme at low concentrations (10 μm) of activator (3-phosphoglycerate). Fifty percent activation was at 4.5 and 8.7 μm for reduced thioredoxin f andm, respectively, and 2 orders of magnitude lower than for dithiothreitol. The activation was reversed by oxidized thioredoxin. Cys12 is conserved in the ADP-GlcPPases from plant leaves and other tissues except for the monocot endosperm enzymes. We postulate that in photosynthetic tissues, reduction could play a role in the fine regulation of the ADP-GlcPPase mediated by the ferredoxin-thioredoxin system. This is the first time that a covalent mechanism of regulation is postulated in the synthesis of starch.

Mechanism of Reductive Activation of Potato Tuber ADP-glucose Pyrophosphorylase

The potato tuber (Solanum tuberosumL.) ADP-glucose pyrophosphorylase activity is activated by a incubation with ADP-glucose and dithiothreitol or by ATP, glucose- 1-phosphate, Ca2+, and dithiothreitol. The activation was accompanied by the appearance of new sulfhydryl groups as determined with 5,5′-dithiobis(2-nitrobenzoic acid). By analyzing the activated and nonactivated enzymes on SDS-polyacrylamide gel electrophoresis under nonreducing conditions, it was found that an intermolecular disulfide bridge between the small subunits of the potato tuber enzyme was reduced during the activation. Further experiments showed that the activation was mediated via a slow reduction and subsequent rapid conformational change induced by ADP-glucose. The activation process could be reversed by oxidation with 5,5′-dithiobis(2-nitrobenzoic acid). Incubation with ADP-glucose and dithiothreitol could reactivate the oxidized enzyme. Chemical modification experiments with [14C]iodoacetic acid and 4-vinylpyridine determined that the intermolecular disulfide bridge was located between Cys12 of the small subunits of the potato tuber enzyme. Mutation of Cys12 in the small subunit into either Ala or Ser eliminated the requirement of DTT on the activation and prevented the formation of the intermolecular disulfide of the potato tuber enzyme. The mutants had instantaneous activation rates as the wild-type in the reduced state. A two-step activation model is proposed.

Adenosine 5'-diphosphate-glucose pyrophosphorylase from potato tuber. Significance of the N terminus of the small subunit for catalytic properties and heat stability.

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5[prime]-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081–1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5[prime]-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunits major function is catalysis.

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 α 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 β 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.

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 (α2β2) 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.

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.

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.

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. 397 (2002) 279]. We investigated further the role of the N-terminus by engineering other truncated GBEs and analyzing the branching pattern by high-performance anion-exchange chromatography. The wild type GBE transfers mainly chains with a degree of polymerization (d.p.) of 8-14, the Nd(1-112) enzyme transfers a greater proportion of chains with higher d.p. 15-20, whereas the 63- and 83-amino acid deleted enzymes had an intermediate pattern of transferred chains (d.p. 10-20). These data showed that a progressive shortening of the N-terminus leads to a gradual increase in the length of the transferred chains, suggesting that the N-terminus provides a support for the glucan substrate during the processes of cleavage and transfer of the alpha-(1-4) glucan chains.