Alberto A. Iglesias

Higher plant phosphoenolpyruvate carboxylase: Structure and regulation

1. INTRODUCTION Phosphoenolpyruvate (PEP) carboxylase is a key enzyme of photosynthesis in those plant species exhibiting the C4 or CAM pathway for CO2 fixation. It catalyses the&carboxylation of PEP to yield oxaloacetate and Pi. The enzyme is present in plants, algae and bacteria. Its properties are very different depending on the source. In plants it has been described as a cytoplasmic enzyme with a variety of functions ranging from photosynthetic CO2 fixation to nitrogen assimilation [1,2]. After an earlier report by Bandurski and Greiner [3] on the presence of PEP carboxylase in spinach, the enzyme from several sources has been studied and reviewed [1,4]. Moreover, a whole issue of P~ys~ologie Vt?g&ale (~01.21, no.5, 1983) was devoted to the carboxylase. A revision seems worthwhile, since in the last 3 years a lot of new information has been obtained including the elucidation of the primary structure of the enzyme from maize, Anacystis nidulans and ~sc~eric~~a cofi [5- 111, studies on the biosynthesis Correspondence address: C.S.

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.

Control of Starch Composition and Structure through Substrate Supply in the Monocellular Alga Chlamydomonas reinhardtii

In Chlamydomonas, as in higher plants, synthesis of ADP glucose catalyzed by ADP-glucose pyrophosphorylase is rate-limiting for the building of starch in the chloroplast. We have isolated disruptions of the STA1 ADP-glucose pyrophosphorylase structural gene that rendered the enzyme less responsive to the allosteric activator 3-phosphoglycerate. The structure and composition of the residual starch synthesized by all mutants of the STA1 locus is dramatically altered. The residual polysaccharide is shown to be devoid of amylose despite the presence of granule-bound starch synthase, the amylose biosynthetic enzyme. In addition, the fine structure of the mutant amylopectin revealed the presence of an altered chain-length distribution. This distribution mimicks that which is observed during growth and photosynthesis and differs markedly from that observed during storage. We therefore propose that low nucleotide sugar concentrations are either directly or indirectly responsible for the major differences observed in the composition or structure of starch during storage and photosynthesis.

Intrinsic disorder is a key characteristic in partners that bind 14-3-3 proteins.

Proteins named 14‐3‐3 can bind more than 200 different proteins, mostly (but not exclusively) when they are at a phosphorylated state. These partner proteins are involved in different cellular processes, such as cell signaling, transcription factors, cellular morphology, and metabolism; this suggests pleiotropic functionality for 14‐3‐3 proteins. Recent efforts to establish a rational classification of 14‐3‐3 binding partners showed neither structural nor functional relatedness in this group of proteins. Using three natural predictors of disorder in proteins, and the structural available information, we show that >90% of 14‐3‐3 protein partners contain disordered regions. This percentage is significantly high when compared with recent studies on cell signaling and cancer‐related proteins or RNA chaperons. More important, almost all 14‐3‐3‐binding sites are inside disordered regions, this reinforcing the importance of structural disorder in this class of proteins. We also propose that a disorder‐to‐order transition occurs in the binding of 14‐3‐3 proteins with their partners. We discuss the consequences of the latter for consensus binding sequences, specificity, affinity, and thermodynamic control. Proteins 2006.

Characterization of Arabidopsis Lines Deficient in GAPC-1, a Cytosolic NAD-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase

Phosphorylating glyceraldehyde-3-P dehydrogenase (GAPC-1) is a highly conserved cytosolic enzyme that catalyzes the conversion of glyceraldehyde-3-P to 1,3-bis-phosphoglycerate; besides its participation in glycolysis, it is thought to be involved in additional cellular functions. To reach an integrative view on the many roles played by this enzyme, we characterized a homozygous gapc-1 null mutant and an as-GAPC1 line of Arabidopsis (Arabidopsis thaliana). Both mutant plant lines show a delay in growth, morphological alterations in siliques, and low seed number. Embryo development was altered, showing abortions and empty embryonic sacs in basal and apical siliques, respectively. The gapc-1 line shows a decrease in ATP levels and reduced respiratory rate. Furthermore, both lines exhibit a decrease in the expression and activity of aconitase and succinate dehydrogenase and reduced levels of pyruvate and several Krebs cycle intermediates, as well as increased reactive oxygen species levels. Transcriptome analysis of the gapc-1 mutants unveils a differential accumulation of transcripts encoding for enzymes involved in carbon partitioning. According to these studies, some enzymes involved in carbon flux decreased (phosphoenolpyruvate carboxylase, NAD-malic enzyme, glucose-6-P dehydrogenase) or increased (NAD-malate dehydrogenase) their activities compared to the wild-type line. Taken together, our data indicate that a deficiency in the cytosolic GAPC activity results in modifications of carbon flux and mitochondrial dysfunction, leading to an alteration of plant and embryo development with decreased number of seeds, indicating that GAPC-1 is essential for normal fertility in Arabidopsis plants.

On the Regulation of Phosphoenolpyruvate Carboxylase Activity from Maize Leaves by L-malate. Effect of pH

The effect of L-malate as reversible inhibitor of phosphoenolpyruvate carboxylase purified from maize leaves was studied between pH 7 and 8. The malate concentration required to reach fifty per cent inhibition was pH dependent being higher at pH 8. Complex inhibition kinetics with upwardly curved Dixon plots were obtained. Hill plots showed cooperative effects giving coefficients (n(H)) between 2.0 and 3.9. The K(i) values were 0.8 and 10 mM at pH 7 and 8 respectively. In addition, at lower pH the type of inhibition was mainly competitive with respect to phosphoenolpyruvate while at pH 8 it was non-competitive. The presence of glucose-6-phosphate in the assay medium reduced the competitive inhibition by malate without modifying the non-competitive effect, changing the Hill coefficient values to one at pH 8.0. Moreover, the dissociation constants for phosphoenolpyruvate (plus or minus MgCl(2)) and malate in the presence of glucose-6-phosphate were calculated at pH 7.0, 7.5, and 7.9 from the protection afforded by these compounds against chemical modification of phosphoenolpyruvate carboxylase by phenylglyoxal. The possible existence of competitive as well as non-competitive malate binding sites in phosphoenolpyruvate carboxylase and the physiological meaning of this interaction are discussed.

Characterization of an Arabidopsis thaliana mutant lacking a cytosolic non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase

Non-phosphorylating glyceraldehyde- 3-phosphate dehydrogenase (NP-GAPDH) is a conserved cytosolic protein found in higher plants. In photosynthetic cells, the enzyme is involved in a shuttle transfer mechanism to export NADPH from the chloroplast to the cytosol. To investigate the role of this enzyme in plant tissues, we characterized a mutant from Arabidopsis thaliana having an insertion at the NP-GAPDH gene locus. The homozygous mutant was determined to be null respect to NP-GAPDH, as it exhibited undetectable levels of both transcription of NP-GAPDH mRNA, protein expression and enzyme activity. Transcriptome analysis demonstrated that the insertion mutant plant shows altered expression of several enzymes involved in carbohydrate metabolism. Significantly, cytosolic phosphorylating (NAD-dependent) glyceraldehyde-3-phosphate dehydrogenase mRNA levels are induced in the mutant, which correlates with an increase in enzyme activity. mRNA levels and enzymatic activity of glucose-6-phosphate dehydrogenase were also elevated, correlating with an increase in NADPH concentration. Moreover, increased ROS levels were measured in the mutant plants. Down-regulation of several glycolytic and photosynthetic genes suggests that NP-GAPDH is important for the efficiency of both metabolic processes. The results presented demonstrate that NP-GAPDH has a relevant role in plant growth and development.

Characterization of the Kinetic, Regulatory, and Structural Properties of ADP-Glucose Pyrophosphorylase from Chlamydomonas reinhardtii

ADP-glucose pyrophosphorylase (ADP-Glc PPase) from Chlamydomonas reinhardtii cells was purified over 2000-fold to a specific activity of 81 units/mg protein, and its kinetic and regulatory properties were characterized. Inorganic orthophosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. Rabbit antiserum raised against the spinach leaf ADP-Glc PPase (but not the one raised against the enzyme from Escherichia coli) inhibited the activity of the purified algal enzyme, which migrated as a single protein band in native polyacrylamide gel electrophoresis. Two-dimensional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme from C. reinhardtii is composed of two subunits with molecular masses of 50 and 53 kD, respectively. The molecular mass of the native enzyme is estimated to be 210 kD. Antisera raised against the spinach leaf holoenzyme and against the 51-kD spinach subunit cross-reacted with both subunits of the algal ADP-Glc PPase in immunoblot hybridization, but the cross-reaction was stronger for the 50-kD algal subunit than for the 53-kD subunit. No cross-reaction was observed when antiserum raised against the spinach leaf pyrophosphorylase 54-kD subunit was used. These results suggest that the ADP-Glc PPase from C. reinhardtii is a heterotetrameric protein, since the enzyme from higher plants and its two subunits are structurally more related to the small subunit of the spinach leaf enzyme than to its large subunit. This information is discussed in the context of the possible evolutionary changes leading from the bacterial ADP-Glc PPase to the cyanobacterial and higher plant enzymes.

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacteriumAnabaena sp. strain PCC 7120

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacteriumAnabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of theAnabaena ADPGlc PPase gene and its expression inEscherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48347 Da which is in agreement with the molecular mass determined by SDS-PAGE for theAnabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and theE. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in theAnabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in anE. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the nativeAnabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be theAnabaena enzyme.