Investigation of the Interaction between the Large and Small Subunits of Potato ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield

Structure, function, and evolution of plant ADP-glucose pyrophosphorylase

Key message: This review outlines research performed in the last two decades on the structural, kinetic,regulatory and evolutionary aspects of ADP-glucose pyrophosphorylase, the regulatory enzymefor starch biosynthesis. Abstract: ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the pathway of glycogen and starch synthesis in bacteria and plants, respectively. Plant ADP-Glc PPase is a heterotetramer allosterically regulated by metabolites and post-translational modifications. In this review, we focus on the three-dimensional structure of the plant enzyme, the amino acids that bind the regulatory molecules, and the regions involved in transmitting the allosteric signal to the catalytic site. We provide a model for the evolution of the small and large subunits, which produce heterotetramers with distinct catalytic and regulatory properties. Additionally, we review the various post-translational modifications observed in ADP-Glc PPases from different species and tissues. Finally, we discuss the subcellular localization of the enzyme found in grain endosperm from grasses, such as maize and rice. Overall, this work brings together research performed in the last two decades to better understand the multiple mechanisms involved in the regulation of ADP-Glc PPase. The rational modification of this enzyme could improve the yield and resilience of economically important crops, which is particularly important in the current scenario of climate change and food shortage.Fil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Ballicora, Miguel A.. Loyola University Maryland (lum);Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Production of amylopectin and high-amylose starch in separate potato genotypes

Starch is one of the most important processed products from agriculture. Two main outlets can be identified; starch is either enzymatically processed for the production of sweeteners and as raw material for fermentation or channelled to various applications as dry starch. Native or chemically modified starches are utilized in food as well as non-food applications, where the specific physicochemical properties are main determinants for their respective use. Starch consists of two different molecules, amylose and amylopectin. To be able to take the full benefit of the unique properties of either component it is of interest to divide the production of amylose and amylopectin into separate plant genotypes. In the presented work, potatoes producing either amylopectin or high-amylose starch were achieved using genetic modification. For potato transformation a highly efficient protocol was developed for a herbicide selection gene instead of the commonly used nptII antibiotic selection gene. In order to achieve respective starch qualities, the expression of genes important for amylopectin or amylose synthesis was silenced. Antisense technology as well as the expression of dsRNA was investigated where the expression of dsRNA was determined to be at least ten-fold more efficient for gene silencing. An added benefit of dsRNA expression was that a higher fraction of silenced transgenic lines compared to the use antisense were associated with single copy T-DNA integrations. One amylopectin potato line was furthermore characterized regarding genetic and chemical composition. The T-DNA was found integrated as an inverted repeat with the inverted repeat region extending into potato chromosomal DNA. This transgenic locus was found to be more consistent with integration into a double-stranded chromosomal break than insertion by a mechanism nicking one strand of the locus. The high-amylose trait generally resulted in a higher tuber fresh weight yield, much elevated sugar levels and a decreased starch content. Amylose levels were obtained where very limited amounts of material recognizable as amylopectin could be found. The production of amylopectin and amylose was divided into separate genotypes but additional factors are needed to be able to produce amylose at levels comparable to starch contents of cultivated potatoes

Study of Escherichia Coli ADP-Glucose Pyrophosphorylase Catalysis: Investigating Critical Roles of Conserved Arg32 and Lys42 Residues

Carbohydrates have been most notable as energy sources for mammals, bacteria (glycogen) and plants (starch) – and in many other species. As such the biosynthesis of carbohydrates is essential to the sustainability of many forms of life, on earth. Adenosine-‘5-diphosphate glucose pyrophosphate (ADP-Glc pyrophosphorylase; ADP-Glc PPase) is the allosterically controlled “first committed step” in both the biosynthetic pathways of starch (~25% amylose and ~75% amylopectin, in plants and algae) and glycogen (in bacteria), preceding the starch/glycogen synthase reaction. By catalyzing the following reaction, ATP + α –D-Glc-1P ADP-Glc + PPi , ADP-Glc PPase functions as the primary enzyme in the reaction that provides the glycosyl precursor for the elongation of α-1,4-polyglucans. ADP-Glc PPase is a tetrameric allosterically regulated enzyme. The structure of this enzyme is homotetrameric in enteric bacteria (Escherichia coli) – α4, and heterotetrameric in plants and other photosynthetic eukaryotes – consisting of two small subunits (50-52 kDa) and two large subunits (~60kDa): (α2β2). The plant ADP-Glc PPases are allosterically regulated: mainly by 3-phosphoglycerate (3PGA) as an activator and inhibited in the presence of inorganic phosphate (Pi). There is an allosteric disparity in the bacterial enzyme’s counterpart. For instance, the E. coli ADP-Glc PPases enzyme is activated by fructose-1,6-bisphosphate (FBP) and inhibited by adenosine-‘5-monophosphate (AMP). All ADP-Glc PPases, to date, are noted to have a divalent metal ion cofactor (Mg+2). Comparative modeling of the E. coli ADP-Glc PPase with ATP bound in the active site predicted critical interactions for Lys42. In the model, this residue interacts with the catalytic Asp142 and the β-phosphate of the ATP substrate, which comprises the reaction “leaving group”. Lys42 is highly conserved in the ADP-Glc PPases known to be catalytic, but absent in plant subunits that are catalytically deficient. It is also conserved in other homologues of the sugar-nucleotide pyrophosphorylase superfamily. To investigate the role of Lys42 in E. coli ADP-Glc PPase, we performed site-directed mutagenesis. As a result, we observed a markedly decreased kcat (\u3e3 orders of magnitude lower than the wild type (WT)). We analyzed another conserved residue that interacts with the phosphates of ATP (Arg32). In mutating the Arg32 residue, we observed the S0.5 of the ATP substrate for the mutants was only two to three times higher than that of the WT. But more significant was the marked decrease in specific activity (and kcat) for the Arg32 mutants (1 -3 orders of magnitude). Our results indicate that the interaction of Arg32 guanidinium moeity and structural length of the Arg32 side chain is critical for overall catalysis. Modeling of the E. coli enzyme WT and Arg32 mutants suggest that two nitrogen atoms of the Arg32 guanidinium side chain may interact with the γ-phosphate of the ATP substrate making the PPi product a more stable leaving group. These results show that both residues in the E. coli ADP-Glc PPase are catalytic (Lys42), important for orientation and positioning of the ATP substrate (Arg32) and overall essential for the production of ADP-glucose

Genome sequence and analysis of the tuber crop potato

Potato (Solanum tuberosum L.) is the world’s most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital cro

Mechanism Underlying Heat Stability of the Rice Endosperm Cytosolic ADP-Glucose Pyrophosphorylase

Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme’s regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature

On the ancestral UDP-glucose pyrophosphorylase activity of GalF from Escherichia coli

In bacteria, UDP-glucose is a central intermediate in carbohydrate metabolism. The enzyme responsible for its synthesis is encoded by the galU gene and its deletion generates cells unable to ferment galactose. In some bacteria, there is a second gene, galF, encoding for a protein with high sequence identity to GalU. However, the role of GalF has been contradictory regarding its catalytic capability and not well understood. In this work we show that GalF derives from a catalytic (UDP-glucose pyrophosphorylase) ancestor, but its activity is very low compared to GalU. We demonstrated that GalF has some residual UDP-glucose pyrophosphorylase activity by in vitro and in vivo experiments in which the phenotype of a galU- strain was reverted by the over-expression of GalF and its mutant. To demonstrate its evolutionary path of "enzyme inactivation" we enhanced the catalysis by mutagenesis and showed the importance of the quaternary structure. This study provides important information to understand the structural and functional evolutionary origin of the protein GalF in enteric bacteria.Fil: Ebrecht, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Loyola University; Estados UnidosFil: Orlof, Agnieszka M.. Loyola University; Estados UnidosFil: Sasoni, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Ballicora, Miguel A.. Loyola University; Estados Unido