The two AGPase subunits evolve at different rates in angiosperms, yet they are equally sensitive to activity-altering amino acid changes when expressed in bacteria

The rate of protein evolution is generally thought to reflect, at least in part, the proportion of amino acids within the protein that are needed for proper function. In the case of ADP-glucose pyrophosphorylase (AGPase), this premise led to the hypothesis that, because the AGPase small subunit is more conserved compared with the large subunit, a higher proportion of the amino acids of the small subunit are required for enzyme activity compared with the large subunit. Evolutionary analysis indicates that the AGPase small subunit has been subject to more intense purifying selection than the large subunit in the angiosperms. However, random mutagenesis and expression of the maize (Zea mays) endosperm AGPase in bacteria show that the two AGPase subunits are equally predisposed to enzyme activity-altering amino acid changes when expressed in one environment with a single complementary subunit. As an alternative hypothesis, we suggest that the small subunit exhibits more evolutionary constraints in planta than does the large subunit because it is less tissue specific and thus must form functional enzyme complexes with different large subunits. Independent approaches provide data consistent with this alternative hypothesis.     

Phylogenetic Analysis of ADP-Glucose Pyrophosphorylase Subunits Reveals a Role of Subunit Interfaces in the Allosteric Properties of the Enzyme

ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria and plants, respectively. Plant AGPase consists of two large and two small subunits that were derived by gene duplication. AGPase large subunits have functionally diverged, leading to different kinetic and allosteric properties. Amino acid changes that could account for these differences were identified previously by evolutionary analysis. In this study, these large subunit residues were mapped onto a modeled structure of the maize (Zea mays) endosperm enzyme. Surprisingly, of 29 amino acids identified via evolutionary considerations, 17 were located at subunit interfaces. Fourteen of the 29 amino acids were mutagenized in the maize endosperm large subunit (SHRUNKEN-2 [SH2]), and resulting variants were expressed in Escherichia coli with the maize endosperm small subunit (BT2). Comparisons of the amount of glycogen produced in E. coli, and the kinetic and allosteric properties of the variants with wild-type SH2/BT2, indicate that 11 variants differ from the wild type in enzyme properties or in vivo glycogen level. More interestingly, six of nine residues located at subunit interfaces exhibit altered allosteric properties. These results indicate that the interfaces between the large and small subunits are important for the allosteric properties of AGPase, and changes at these interfaces contribute to AGPase functional specialization. Our results also demonstrate that evolutionary analysis can greatly facilitate enzyme structure-function analyses.ADP-glucose pyrophosphorylase (AGPase) catalyzes the conversion of Glc-1-P (G-1-P) and ATP to ADP-Glc and pyrophosphate. This reaction represents a rate-limiting step in starch synthesis (Hannah, 2005). AGPase is an allosteric enzyme whose activity is regulated by small effector molecules. In plants, AGPase is activated by 3-phosphoglyceraldehyde (3-PGA) and deactivated by inorganic phosphate (Pi).Plant AGPase is a heterotetramer consisting of two identical large and two identical small subunits. The large and small subunits of AGPase were generated by a gene duplication. Subsequent sequence divergence has given rise to complementary rather than interchangeable subunits. Indeed, both subunits are needed for AGPase activity (Hannah and Nelson, 1976, Burger et al., 2003). Biochemical studies have indicated that both subunits are important for catalytic and allosteric properties (Hannah and Nelson, 1976; Greene et al., 1996a, 1996b; Ballicora et al., 1998; Laughlin et al., 1998; Frueauf et al., 2001; Kavakli et al., 2001a, 2001b; Cross et al., 2004, 2005; Hwang et al., 2005, 2006, 2007; Kim et al., 2007; Ventriglia et al., 2008). Surprisingly, Georgelis et al. (2007, 2008) showed that, in angiosperms, the small subunit is under greater evolutionary pressure compared with the large subunit. Detailed analyses have shown that the greater constraint on the small subunit is due to its broader tissue expression patterns compared with the large subunit and the fact that the small subunit must interact with multiple large subunits.Large subunits have undergone more duplication events than have small subunits (Georgelis et al., 2008). This has led to the creation of five groups of large subunits that differ in their patterns of tissue of expression (Akihiro et al., 2005; Crevillen et al., 2005; Ohdan et al., 2005). Crevillen et al. (2003) studied the biochemical properties of four Arabidopsis (Arabidopsis thaliana) AGPases consisting of the four different large subunits and the only functional small subunit in Arabidopsis. The different AGPases had different kinetic and allosteric properties. More specifically, the AGPases differed in their affinity for the allosteric regulator 3-PGA and the substrates G-1-P and ATP. This possibly reflects the different 3-PGA, G-1-P, and ATP levels in the various tissues. This evidence indicates that not only did the different large subunit groups subfunctionalize in terms of expression, but also these groups may have specialized in terms of protein function. While the study of Crevillen et al. (2003) pointed to functional specialization of the large subunit, the identity of the amino acid sites in the large subunit that account for these kinetic and allosteric differences was not pursued.Georgelis et al. (2008) presented supporting evidence for AGPase large subunit specialization by identifying positively selected amino acid sites in the phylogenetic branches following gene duplication events. We also identified amino acid residues that were conserved in one large subunit group but not conserved in another large subunit group (type I functional divergence; Gu, 1999) and amino acid residues that are conserved within large subunit groups but are variable among large subunit groups (type II functional divergence; Gu, 2006). Positively selected type I and type II sites could have contributed to specialization of the different large subunit groups. Indeed, positively selected type II sites in several proteins have been proven via site-directed mutagenesis (Bishop, 2005; Norrgård et al., 2006; Cavatorta et al., 2008; Courville et al., 2008) to be important for protein function and functional specialization. Additionally, several positively selected type I and type II amino acid sites in the large AGPase subunit identified in our previous evolutionary analysis (Georgelis et al., 2008) have been implicated in the kinetic and allosteric properties and heat stability of AGPase. The role of these sites was demonstrated by site-directed mutagenesis experiments of large subunits from Arabidopsis, maize endosperm, and potato (Solanum tuberosum) tuber (Ballicora et al., 1998, 2005; Kavakli et al., 2001a; Jin et al., 2005; Linebarger et al., 2005; Ventriglia et al., 2008). These analyses indicate that the rest of the amino acid sites identified as positive type I and type II sites in our previous evolutionary analysis (Georgelis et al., 2008) represent promising candidate targets for mutagenesis.To identify large subunit amino acids that are possibly important in controlling enzyme properties and that may have contributed to large subunit specialization, we conducted site-directed mutagenesis of the maize endosperm large subunit encoded by Shrunken-2 (Sh2). We specifically identified amino acids of SH2 that correspond to amino acid sites that were detected as positive type I and type II sites during the large subunit evolution (Georgelis et al., 2008). We then replaced the SH2 residues with amino acids of a group different from the SH2 family. Several amino acid sites important for the kinetic and allosteric properties and heat stability of AGPase were identified. Our results indicate that the subunit interfaces between the large and small subunits are important for the allosteric properties of AGPase. They also indicate that amino acid changes at subunit interfaces have been important for AGPase specialization in terms of allosteric properties. These experiments also support the idea that the majority of positively selected sites as detected by codon substitution models (Nielsen and Yang, 1998; Yang et al., 2000) and type II sites are not false positives. Site-directed mutagenesis of such sites can greatly facilitate enzyme structure-function analyses.     

马铃薯AGPase大小亚基功能研究

马铃薯 1,6 二磷酸腺苷葡萄糖焦磷酸化酶 (AGPase)是淀粉合成的限速酶 ,该酶有大、小两个亚基形成异源四聚体。总结了迄今为止已克隆的马铃薯AGPase大、小亚基编码基因、小亚基和底物结合位点的识别、以及大亚基异构调控因子结合位点识别的研究结果 ,提出了大小亚基非自然重组是深入研究AGPase的途径 ,建立体内条件下高效可靠代谢调控研究手段是AGPase研究所必需的。     

A low-starch barley mutant,risø 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase,reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit

To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Ris? 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Ris? 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A- and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene.     

Kinetic and regulatory properties of plant ADP-glucose pyrophosphorylase genetically modified by heterologous expression of potato<Emphasis Type="Italic"> upreg</Emphasis> mutants in vitro and in vivo

In this study, the uses of the mutated genes, upreg1 and upreg2, encoding upregulated ADP-glucose pyrophosphorylase (AGPase) large subunits with increased enzymatic activity, to improve crop yield productivity was evaluated in vitro and in planta. For in vitro examination, wild type and upregs were co-expressed with three different AGPase small subunit genes from potato and perilla to produce nine AGPase isoforms. In kinetic experiments, 3-Phosphoglycerate increased the V _max and decreased the K _M for the recombinant AGPase. Regardless of the specific small subunit, Upreg-type AGPases had much larger increases in enzymatic activity with concomitant decreases in values as compared to the wild type enzyme. Transformation of lettuce with the upreg1 gene altered the regulatory properties of leaf AGPase. AGPases from transgenic lettuce showed greater 3-PGA activation and lower Pi inhibition than was observed for wild type AGPase. Fresh weights of the aerial parts of transgenic plants were larger than non-transgenic controls. Based on these results, upreg mutant genes could be used for the genetic improvement of plant AGPases other than potato and effectively increase crop yield productivity.     

Transcription profiles of hydrogenases related genes in the cyanobacterium Lyngbya majuscula CCAP 1446/4

Background  

Lyngbya majuscula CCAP 1446/4 is a N₂-fixing filamentous nonheterocystous strain that contains two NiFe-hydrogenases: an uptake (encoded by hupSL) and a bidirectional enzyme (encoded by hoxEFUYH). The biosynthesis/maturation of NiFe-hydrogenases is a complex process requiring several accessory proteins for e.g. for the incorporation of metals and ligands in the active center (large subunit), and the insertion of the FeS clusters (small subunit). The last step in the maturation of the large subunit is the cleavage of a C-terminal peptide from its precursor by a specific endopeptidase. Subsequently, the mature large and small subunits can assemble forming a functional enzyme.     

Probing Allosteric Binding Sites of the Maize Endosperm ADP-Glucose Pyrophosphorylase

Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA). Several arginine (Arg) side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine-scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady-state kinetic and regulatory parameters were measured for each mutant. All of the Arg mutants examined—in both the small and large subunits—bound 3-PGA more weakly than the wild type (A₅₀ increased by 3.5- to 20-fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA. In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the Arg mutants. Phosphate binding was also affected by Arg substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and inorganic phosphate compete directly.ADP-Glc pyrophosphorylase (AGPase), a key enzyme in starch biosynthesis, catalyzes the formation of ADP-Glc from ATP and Glc-1-P (G-1-P). Maize (Zea mays) AGPase, like nearly all higher plant homologs, is a highly regulated heterotetramer containing two small and two large subunits. By contrast, virtually all bacterial forms of the enzyme are homotetramers. Evidence from eight independent plant transgenic or genetic experiments (L.C. Hannah and T.W. Greene, unpublished data; Stark et al., 1992; Giroux et al., 1996; Smidansky et al., 2002, 2003; Sakulsingharoj et al., 2004; Obana et al., 2006; Wang et al., 2007) has shown that altering the allosteric properties and/or heat stability of AGPase can significantly increase starch content and starch turnover and, in turn, seed yield. Increased seed number giving rise to enhanced starch content occurs in some cases. Such observations have inspired efforts to understand AGPase regulation at a molecular level.Virtually all known AGPases are subject to allosteric activation and inhibition by various metabolites associated with the specific carbon utilization pathway of the organism. For example, the bacterial AGPase from Agrobacterium tumefaciens is activated by Fru-6-P (F-6-P) and inhibited by inorganic phosphate (P_i), whereas the Escherichia coli AGPase is activated by Fru-1,6-bisP but inhibited by AMP. Rhodospirillum rubrum AGPase is activated by both Fru-1,6-bisP and F-6-P, and inhibited by P_i, while Anabaena AGPase mimics plant AGPases in its activation by 3-phosphoglycerate (3-PGA) and inhibition by P_i. Using both chemical modification and site-directed mutagenesis, several Arg and Lys residues participating in allosteric regulation have been mapped to the C-terminal segments of the Anabaena and potato (Solanum tuberosum) tuber enzymes (Charng et al., 1994; Sheng et al., 1996; Ballicora et al., 1998, 2002).Unfortunately, only limited atomic-level structural data are available for AGPases. The three-dimensional structure of a bacterial homotetrameric enzyme from A. tumefaciens has recently been solved (Cupp-Vickery et al., 2008). Only one crystal structure is available for a higher plant AGPase: a nonnative, low-activity form of the enzyme from potato tuber (small subunit homotetramer; Jin et al., 2005). Although both structures reflect inactive conformations due to high concentrations of ammonium sulfate in the crystallization buffer, important information about potential substrate-binding sites was predicted by molecular modeling based on the known structures of thymidilyltransferases. While this class of enzymes likely binds sugar phosphates in the same manner as AGPases, thymidilyltransferases are not regulated allosterically. Both AGPase crystal structures suggest that the enzyme functions as a dimer of dimers, similar to the mechanism proposed for the Escherichia coli enzyme on the basis of ligand-binding studies (Haugen and Preiss, 1979). All available evidence leads to the conclusion that tetramers are required for AGPase catalytic activity.Both available AGPase crystal structures show two domains in each subunit: an N-terminal catalytic domain, which resembles previously reported pyrophosphorylase structures (Jin et al., 2005; Cupp-Vickery et al., 2008) and a C-terminal domain that makes strong hydrophobic interactions with the catalytic domain. In the potato small subunit homotetramer, two of the three bound sulfate ions (per monomer) are located in a crevice between the N- and C-terminal domains, separated by 7.24 Å. We have arbitrarily labeled these sites as sulfate 1 and sulfate 2, respectively. The third sulfate ion (in site 3) binds between two protein-adjacent monomers. When ATP is included in the crystallization buffer, two substrate molecules are bound in two of the four presumptive active sites, consistent with the notion that the protein functions as a dimer of dimers. On the other hand, one of the sulfate ions originally found in site 3 is lost when ATP is bound, despite the large distance between their respective binding sites. The A. tumefaciens AGPase homotetramer binds a single sulfate ion (per monomer) with 100% occupancy (Cupp-Vickery et al., 2008).All known allosteric regulators of higher plant AGPases contain one or more phosphate moieties. Because of their structural similarity, it is likely that the sulfate ions found in AGPase crystal structures bind in sites normally occupied by P_i or anionic, phosphorylated ligands such as F-6-P, G-6-P, and 3-PGA. Several studies suggest that all AGPase activators and inhibitors compete for binding to the same or closely adjacent sites within a subunit (Morell et al., 1988; Boehlein et al., 2008). Like P_i, sulfate reverses 3-PGA-mediated activation for the potato, A. tumefaciens, and maize enzymes (I_0.5 = 2.8 mm in the presence of 6 mm 3-PGA, potato tuber AGPase; I_0.5 = 20 mm in the presence of 2.5 mm 3-PGA, maize endosperm AGPase; Jin et al., 2005; S.K. Boehlein, unpublished data). In addition, both sulfate and P_i significantly affect maize AGPase thermal stability. For these reasons, we analyzed sulfate ion binding to the potato small subunit homotetramer to guide Ala-scanning mutagenesis studies on the analogous anion-binding sites within the heterotetrameric maize endosperm AGPase. Replacements were made in both the small and the large subunits of the maize endosperm AGPase. More conservative changes (Gln or Lys) were employed when Ala mutants displayed no catalytic activity. We chose not to create homology models of the maize subunits to help understand the behavior of Arg mutants. While computational models often predict core structures accurately, small details such as ligand-binding sites and subunit-subunit contacts are less reliable. This is particularly important for sulfate ion-binding site 3, which is located at the interface between two subunits. The problems are compounded by the lack of experimental data for an AGPase large subunit.Our studies revealed that altering any Arg residue that participates in a sulfate ion binding—either in the small or the large subunits of maize AGPase—drastically altered the enzyme''s overall allosteric properties. This indicates that effector-binding sites in both subunits function in concert in the native heterotetramer, reminiscent of their synergistic participation in catalysis. It also directly supports the notion that sulfate ion-binding sites are also involved in binding allosteric effectors. On the other hand, while mutations at all sulfate ion-binding sites affected allostery, substantial variation was observed for the different Arg side chains. Finally we note that while the various AGPases of plant and bacterial origin exhibit vastly different allosteric properties, presumably due to differing selection pressures over evolutionary time, single amino acid changes of the maize endosperm enzyme can create allosteric properties that mimic those exhibited by bacterial and other AGPases.     

Accelerated evolution and coevolution drove the evolutionary history of AGPase sub-units during angiosperm radiation

Background and Aims

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme of starch biosynthesis. In the green plant lineage, it is composed of two large (LSU) and two small (SSU) sub-units encoded by paralogous genes, as a consequence of several rounds of duplication. First, our aim was to detect specific patterns of molecular evolution following duplication events and the divergence between monocotyledons and dicotyledons. Secondly, we investigated coevolution between amino acids both within and between sub-units.

Methods

A phylogeny of each AGPase sub-unit was built using all gymnosperm and angiosperm sequences available in databases. Accelerated evolution along specific branches was tested using the ratio of the non-synonymous to the synonymous substitution rate. Coevolution between amino acids was investigated taking into account compensatory changes between co-substitutions.

Key Results

We showed that SSU paralogues evolved under high functional constraints during angiosperm radiation, with a significant level of coevolution between amino acids that participate in SSU major functions. In contrast, in the LSU paralogues, we identified residues under positive selection (1) following the first LSU duplication that gave rise to two paralogues mainly expressed in angiosperm source and sink tissues, respectively; and (2) following the emergence of grass-specific paralogues expressed in the endosperm. Finally, we found coevolution between residues that belong to the interaction domains of both sub-units.

Conclusions

Our results support the view that coevolution among amino acid residues, especially those lying in the interaction domain of each sub-unit, played an important role in AGPase evolution. First, within SSU, coevolution allowed compensating mutations in a highly constrained context. Secondly, the LSU paralogues probably acquired tissue-specific expression and regulatory properties via the coevolution between sub-unit interacting domains. Finally, the pattern we observed during LSU evolution is consistent with repeated sub-functionalization under ‘Escape from Adaptive Conflict’, a model rarely illustrated in the literature.     

Isolation and characterization of cDNA clones encoding ADP-glucose pyrophosphorylase (AGPase) large and small subunits from chickpea (Cicer arietinum L.)

Four cDNA clones encoding two large subunits and two small subunits of the starch regulatory enzyme ADP-glucose pyrophosphorylase (AGPase) were isolated from a chickpea (Cicer arietinum L.) stem cDNA library. DNA sequence and Southern blot analyses of these clones, designated CagpL1, CagpL2 (large subunits) and CagpS1 and CagpS2 (small subunits), revealed that these isoforms represented different AGPase large and small subunits. RNA expression analysis indicated that CagpL1 was expressed strongly in leaves with reduced expression in the stem. No detectable expression was observed in seeds and roots. CagpL2 was expressed moderately in seeds followed by weak expression in leaves, stems and roots. Similar analysis showed that CagpS1 and CagpS2 displayed a spatial expression pattern similar to that observed for CagpL2 with the exception that CagpS1 showed a much higher expression in seeds than CagpS2. The spatial expression patterns of these different AGPase subunit sequences indicate that different AGPase isoforms are used to control starch biosynthesis in different organs during chickpea development.     

A family of putative Kir potassium channels in prokaryotes

Background  

Prior to this report, members of the inward rectifier family, or Kir, have been found only in eukaryotes. Like most K⁺ channels, the pore-forming part of the protein is formed by four identical, or closely related, subunits. Each subunit contains a transmembrane M1-P-M2 motif that is followed by a relatively large C-terminus region unique to Kir's.     

Contrasted patterns of selection since maize domestication on duplicated genes encoding a starch pathway enzyme

Maize domestication from teosinte (Zea mays ssp. parviglumis) was accompanied by an increase of kernel size in landraces. Subsequent breeding has led to a diversification of kernel size and starch content among major groups of inbred lines. We aim at investigating the effect of domestication on duplicated genes encoding a key enzyme of the starch pathway, the ADP-glucose pyrophosphorylase (AGPase). Three pairs of paralogs encode the AGPase small (SSU) and large (LSU) subunits mainly expressed in the endosperm, the embryo and the leaf. We first validated the putative sequence of LSU_leaf through a comparative expression assay of the six genes. Second, we investigated the patterns of molecular evolution on a 2 kb coding region homologous among the six genes in three panels: teosintes, landraces, and inbred lines. We corrected for demographic effects by relying on empirical distributions built from 580 previously sequenced ESTs. We found contrasted patterns of selection among duplicates: three genes exhibit patterns of directional selection during domestication (SSU_end, LSU_emb) or breeding (LSU_leaf), two exhibit patterns consistent with diversifying (SSU_leaf) and balancing selection (SSU_emb) accompanying maize breeding. While patterns of linkage disequilibrium did not reveal sign of coevolution between genes expressed in the same organ, we detected an excess of non-synonymous substitutions in the small subunit functional domains highlighting their role in AGPase evolution. Our results offer a different picture on AGPase evolution than the one depicted at the Angiosperm level and reveal how genetic redundancy can provide flexibility in the response to selection.     

Both subunits of ADP-glucose pyrophosphorylase are regulatory

The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits.     

Comparative computational analysis of ADP Glucose Pyrophosphorylase in plants

ADP-glucose pyrophosphorylase (AGPase), a key enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Ample evidence has shown that the AGPase catalyzes the rate limiting step in starch biosynthesis in higher plants. In this study, we compiled detailed comparative information about ADP glucose pyrophosphorylase in selected plants by analyzing their structural features e.g. amino acid content, physico-chemical properties, secondary structural features and phylogenetic classification. Functional analysis of these proteins includes identification of important 10 to 20 amino acids long motifs arise because specific residues and regions proved to be important for the biological function of a group of proteins, which are conserved in both structure and sequence during evolution. Phylogenetic analysis depicts two main clusters. Cluster I encompasses large subunits (LS) while cluster II contains small subunits (SS).     

Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

Background  

Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish).     

The major form of ADP-glucose pyrophosphorylase in maize endosperm is extra-plastidial.

Preparations enriched in plastids were used to investigate the location of ADP-glucose pyrophosphorylase (AGPase) in the developing endosperm of maize (Zea mays L.). These preparations contained more than 25% of the total activity of the plastid marker enzymes alkaline pyrophosphatase and soluble starch synthase, less than 2% of the cytosolic marker enzymes alcohol dehydrogenase and pyrophosphate, fructose 6-phosphate 1-phosphotransferase, and approximately 3% of the AGPase activity. Comparison with the marker enzyme distribution suggests that more than 95% of the activity of AGPase in maize endosperm is extra-plastidial. Two proteins were recognized by antibodies to the small subunit of AGPase from maize endosperm Brittle-2 (Bt2). The larger of the two proteins was the major small subunit in homogenates of maize endosperm, and the smaller, less abundant of the two proteins was enriched in preparations containing plastids. These results suggest that there are distinct plastidial and cytosolic forms of AGPase, which are composed of different subunits. Consistent with this was the finding that the bt2 mutation specifically eliminated the extraplastidial AGPase activity and the larger of the two proteins recognized by the antibody to the Bt2 subunit.     

Investigation of subunit function in ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key regulatory enzyme in higher plant starch biosynthesis, is composed of a pair of large and small subunits (alpha(2)beta(2)). Current evidence suggests that the large subunit has primarily a regulatory function, while the small subunit has both regulatory and catalytic roles. To define the structure-function relationship of the large subunit (LS), the LS of potato AGPase was subjected to chemical mutagenesis and coexpressed with the wild-type (WT) small subunit (SS) cDNA in an AGPase defective Escherichia coli strain. An LS mutant (M143) was isolated, which accumulated very low levels of glycogen compared to the WT recombinant AGPase, but maintained normal catalytic activity when assayed under saturating conditions. Sequence analysis revealed that M143 has a single amino acid change, V463I, which lies adjacent to the C-terminus. This single mutation had no effect on the Km for ATP and Mg(2+), which were similar to the WT enzyme. The K(m) for glucose 1-P, however, was sixfold higher than the WT enzyme. These results suggest that the LS plays a role in binding glucose 1-P through its interaction with the SS.