Relative turnover numbers of maize endosperm and potato tuber ADP-glucose pyrophosphorylases in the absence and presence of 3-phosphoglyceric acid

Adenosine diphosphate glucose pyrophosphorylase (AGPase; EC 2.7.7.27) synthesizes the starch precursor, ADP-glucose. It is a rate-limiting enzyme in starch biosynthesis and its activation by 3-phosphoglyceric acid (3PGA) and/or inhibition by inorganic phosphate (Pi) are believed to be physiologically important. Leaf, tuber and cereal embryo AGPases are highly sensitive to these effectors, whereas endosperm AGPases are much less responsive. Two hypotheses can explain the 3PGA activation differences. Compared to leaf AGPases, endosperm AGPases (i) lack the marked ability to be activated by 3PGA or (ii) they are less dependent on 3PGA for activity. The absence of purified preparations has heretofore negated answering this question. To resolve this issue, heterotetrameric maize ( Zea mays L.) endosperm and potato ( Solanum tuberosum L.) tuber AGPases expressed in Escherichia coli were isolated and the relative amounts of enzyme protein were measured by reaction to antibodies against a motif resident in both small subunits. Resulting reaction rates of both AGPases are comparable in the presence but not in the absence of 3PGA when expressed on an active-protein basis. We also placed the potato tuber UpReg1 mutation into the maize AGPase. This mutation greatly enhances 3PGA sensitivity of the potato AGPase but it has little effect on the maize AGPase. Thirdly, lysines known to bind 3PGA in potato tuber AGPase, but missing from the maize endosperm AGPase, were introduced into the maize enzyme. These had minimal effect on maize endosperm activity. In conclusion, the maize endosperm AGPase is not nearly as dependent on 3PGA for activity as is the potato tuber AGPase.     

Isolation of a heat-stable maize endosperm ADP-glucose pyrophosphorylase variant

Heat stress reduces maize yield and several lines of evidence suggest that the heat lability of maize endosperm ADP-glucose pyrophosphorylase (AGPase) contributes to this yield loss. AGPase catalyzes a rate-limiting step in starch synthesis. Herein, we present a novel maize endosperm AGPase small subunit variant, termed BT2-TI that harbors a single amino acid change of residue 462 from threonine to isoleucine. The mutant was isolated by random mutagenesis and heterologous expression in a bacterial system. BT2-TI exhibits enhanced heat stability compared to wildtype maize endosperm AGPase.The TI mutation was placed into another heat-stable small subunit variant, MP. MP is composed of sequences from the maize endosperm and the potato tuber small subunit. The MP-TI small subunit variant exhibited greater heat stability than did MP. Characterization of heat stability as well as kinetic and allosteric properties suggests that MP-TI may lead to increased starch yield when expressed in monocot endosperms.     

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.     

The potato tuber,maize endosperm and a chimeric maize-potato ADP-glucose pyrophosphorylase exhibit fundamental differences in Pi inhibition

ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed.     

Purification and characterization of adenosine diphosphate glucose pyrophosphorylase from maize/potato mosaics

Adenosine diphosphate glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in starch biosynthesis. The reaction produces ADP-glucose and pyrophosphate from glucose-1-P and ATP. Investigations from a number of laboratories have shown that alterations in allosteric properties as well as heat stability of this enzyme have dramatic positive effects on starch synthesis in the potato (Solanum tuberosum) tuber and seeds of important cereals. Here, we report the characterization of purified recombinant mosaic AGPases derived from protein motifs normally expressed in the maize (Zea mays) endosperm and the potato tuber. These exhibit properties that should be advantageous when expressed in plants. We also present an in-depth characterization of the kinetic and allosteric properties of these purified recombinant AGPases. These data point to previously unrecognized roles for known allosteric effectors.     

Characterization of an autonomously activated plant ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch biosynthesis in plants and changes in its catalytic and/or allosteric properties can lead to increased starch production. Recently, a maize (Zea mays)/potato (Solanum tuberosum) small subunit mosaic, MP [Mos(1–198)], containing the first 198 amino acids of the small subunit of the maize endosperm enzyme and the last 277 amino acids from the potato tuber enzyme, was expressed with the maize endosperm large subunit and was reported to have favorable kinetic and allosteric properties. Here, we show that this mosaic, in the absence of activator, performs like a wild-type AGPase that is partially activated with 3-phosphoglyceric acid (3-PGA). In the presence of 3-PGA, enzyme properties of Mos(1–198)/SH2 are quite similar to those of the wild-type maize enzyme. In the absence of 3-PGA, however, the mosaic enzyme exhibits greater activity, higher affinity for the substrates, and partial inactivation by inorganic phosphate. The Mos(1–198)/SH2 enzyme is also more stable to heat inactivation. The different properties of this protein were mapped using various mosaics containing smaller portions of the potato small subunit. Enhanced heat stability of Mos(1–198) was shown to originate from five potato-derived amino acids between 322 and 377. These amino acids were shown previously to be important in small subunit/large subunit interactions. These five potato-derived amino acids plus other potato-derived amino acids distributed throughout the carboxyl-terminal portion of the protein are required for the enhanced catalytic and allosteric properties exhibited by Mos(1–198)/SH2.     

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.     

A polymorphic motif in the small subunit of ADP-glucose pyrophosphorylase modulates interactions between the small and large subunits

The heterotetrameric, allosterically regulated enzyme, adenosine-5'-diphosphoglucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step in starch synthesis. Despite vast differences in allosteric properties and a long evolutionary separation, heterotetramers of potato small subunit and maize large subunit have activity comparable to either parent in an Escherichia coli expression system. In contrast, co-expression of maize small subunit with the potato large subunit produces little activity as judged by in vivo activity stain. To pinpoint the region responsible for differential activity, we expressed chimeric maize/potato small subunits in E. coli. This identified a 55-amino acid motif of the potato small subunit that is critical for glycogen production when expressed with the potato large subunit. Potato and maize small subunit sequences differ at five amino acids in this motif. Replacement experiments revealed that at least four amino acids of maize origin were required to reduce staining. An AGPase composed of a chimeric potato small subunit containing the 55-amino acid maize motif with the potato large subunit exhibited substantially less affinity for the substrates, glucose-1-phosphate and ATP and an increased Ka for the activator, 3-phosphoglyceric acid. Placement of the potato motif into the maize small subunit restored glycogen synthesis with the potato large subunit. Hence, a small polymorphic motif within the small subunit influences both catalytic and allosteric properties by modulating subunit interactions.     

Heat stability of the potato tuber ADP-glucose pyrophosphorylase: role of Cys residue 12 in the small subunit

Most of the ADP-glucose pyrophosphorylases from different sources are stable to a heat treatment. We found that in the potato (Solanum tuberosum L.) tuber enzyme, the intermolecular disulfide bridge located between Cys12 of the small subunits is responsible for the stability at 60 degrees C. When this unique disulfide bond is cleaved the enzyme is stable up to 40 degrees C. Mutation of Cys12 in the small subunit into either Ala or Ser yielded enzymes with stability similar to the reduced form of the wild type. Concurrently, the enzyme with a truncated small subunit on the N-terminal was stable only up to 40 degrees C. Thus, the N-terminal is important for the stability of the enzyme because of the presence of a disulfide bond.     

Studies of the Kinetic Mechanism of Maize Endosperm ADP-Glucose Pyrophosphorylase Uncovered Complex Regulatory Properties

ADP-glucose pyrophosphorylase catalyzes the synthesis of ADP-glucose (ADP-Glc) from Glc-1-phosphate (G-1-P) and ATP. Kinetic studies were performed to define the nature of the reaction, both in the presence and absence of allosteric effector molecules. When 3-phosphoglycerate (3-PGA), the putative physiological activator, was present at a saturating level, initial velocity studies were consistent with a Theorell-Chance BiBi mechanism and product inhibition data supported sequential binding of ATP and G-1-P, followed by ordered release of pyrophosphate and ADP-Glc. A sequential mechanism was also followed when 3-PGA was absent, but product inhibition patterns changed dramatically. In the presence of 3-PGA, ADP-Glc is a competitive inhibitor with respect to ATP. In the absence of 3-PGA—with or without 5.0 mm inorganic phosphate—ADP-Glc actually stimulated catalytic activity, acting as a feedback product activator. By contrast, the other product, pyrophosphate, is a potent inhibitor in the absence of 3-PGA. In the presence of subsaturating levels of allosteric effectors, G-1-P serves not only as a substrate but also as an activator. Finally, in the absence of 3-PGA, inorganic phosphate, a classic inhibitor or antiactivator of the enzyme, stimulates enzyme activity at low substrate by lowering the K_M values for both substrates.Plant ADP-Glc pyrophosphorylase (AGPase) catalyzes an important, rate-limiting step in starch biosynthesis: the reversible formation of ADP-Glc from ATP and Glc-1-P (G-1-P). Most AGPases are regulated by effector molecules derived from the prevalent carbon metabolism pathway, with inorganic phosphate (P_i) and 3-phosphoglycerate (3-PGA) being the most studied effectors of higher plants. Interestingly, the barley (Hordeum vulgare) endosperm form of AGPase is unique among higher plant homologs in its insensitivity to both 3-PGA and P_i (Kleczkowski et al., 1993a). Heat lability (as often found for endosperm AGPases) and reductive activation (for those AGPases harboring an N-terminal Cys residue in the small subunit) are also important mechanisms by which AGPases are regulated (Fu et al., 1998; Tiessen et al., 2002).Transgenic plant studies emphasize the importance of allosteric effectors in controlling enzyme activity and, in turn, starch yield. For example, expressing an allosterically enhanced Escherichia coli AGPase resulted in a 35% increase in potato (Solanum tuberosum) tuber starch (Stark et al., 1992) and a 22% to 25% increase in maize (Zea mays) seed starch (Wang et al., 2007). Rice (Oryza sativa) seed weight was increased up to 11% by expression of a second E. coli-derived AGPase mutant with altered allosteric properties (Sakulsingharoj et al., 2004). In another example, expressing a maize AGPase variant with less sensitivity to P_i and enhanced heat stability led to a 38% increase in wheat (Triticum aestivum) yield (Smidansky et al., 2002), a 23% increase in rice yield (Smidansky et al., 2003), and up to a 68% increase in maize yield (L.C. Hannah, unpublished data). Increases in these cases were due to enhanced seed number. Finally, transgenic expression of an allosterically altered potato tuber AGPase enhanced Arabidopsis (Arabidopsis thaliana) leaf transitory starch turnover and improved growth characteristics (Obana et al., 2006) and enhanced the fresh weight of aerial parts of lettuce (Lactuca sativa) plants (Lee et al., 2009).In higher plants, AGPase is a heterotetramer, consisting of two large and two small subunits; by contrast, most bacterial AGPases are homotetramers. Crystal structures of a bacterial AGPase and a nonnative, small subunit homotetramer derived from the potato tuber enzyme have been described recently (Jin et al., 2005; Cupp-Vickery et al., 2008). Unfortunately, since both structures were determined in the presence of high sulfate concentrations, both enzymes are in inactive forms.While AGPase allosteric regulation has received a great deal of attention, the kinetic mechanism has been defined completely only for two cases: the homotetrameric form from the bacterium Rhodospirillum rubrum and the plant heterotetrameric enzyme from barley leaf (Paule and Preiss, 1971; Kleczkowski et al., 1993b). The kinetic mechanism is sequential in both cases, with ATP the first substrate bound and ADP-Glc the final product released. Despite this similarity, there are important differences, most notably the existence of an isomerization step following ADP-Glc release, so that this product and ATP bind to different forms of the barley enzyme. This isomerization step is absent from the bacterial enzyme. Interestingly, isoforms of the closely related nucleoside diphospho-Glc family exhibit fundamentally different kinetic mechanisms. Some UDP-Glc pyrophosphorylases catalyze a sequential BiBi mechanism (Elling, 1996), while others, such as dTDP-Glc and CDP-Glc pyrophosphorylases from Salmonella, employ a ping-pong mechanism (Lindqvist et al., 1993, 1994).Because of the mechanistic diversity exhibited by pyrophosphorylases in general and by the two well-characterized AGPases in particular, we investigated the kinetic mechanism of the recombinant maize endosperm AGPase. We were particularly interested in the roles played by allosteric effectors that appear to be critically important in catalytic efficiency and, thus, starch content. Surprisingly, patterns of initial velocity at varying substrate concentrations as well as product inhibition behavior were identical to those observed for the homotetrameric bacterial enzyme (Paule and Preiss, 1971) and differed significantly from the heterotetrameric barley leaf enzyme (Kleczkowski et al., 1993b). Moreover, we found that both G-1-P and ADP-Glc could stimulate AGPase catalytic activity beyond that expected for simple substrate effects. We also found that the classic inhibitor, P_i, actually enhanced AGPase activity at low substrate concentrations but inhibited activity at high substrate levels. A model is presented to account for this observation. Finally, we determined that 3-PGA only stimulates AGPase activity by 2.5-fold if care is taken to saturate with substrates during assays.     

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.     

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.     

Subcellular localization of ADPglucose pyrophosphorylase in developing wheat endosperm and analysis of the properties of a plastidial isoform

The intracellular location of ADPglucose pyrophosphorylase (AGPase) in wheat during endosperm development was investigated by analysis of the recovery of marker enzymes from amyloplast preparations. Amyloplast preparations contained 20-28% of the total endosperm activity of two plastidial marker enzymes and less than 0.8% of the total endosperm activity of two cytosolic marker enzymes. Amylo plasts prepared at various stages of development, from 8-30 d post anthesis, contained between 2% and 10% of the total AGPase activity; this implies that between 7% and 40% of the AGPase in wheat endosperm is plastidial during this period of development. Two proteins were recognized by antibodies to both the large and small subunits of wheat AGPase. The larger of the two AGPases was the major form of the enzyme in whole cell extracts, and the smaller, less abundant, form of AGPase was enriched in plastid preparations. The results are consistent with data from other graminaceous endosperms, suggesting that there are distinct plastidial and cytosolic isoforms of AGPase composed of different subunits. The plastidial isoform of AGPase from wheat endosperm is relatively insensitive to the allosteric regulators 3-phosphoglycerate and inorganic orthophos phate compared with plastidial AGPase from other species. Amyloplast AGPase showed no sensitivity to physiological concentrations of inorganic orthophosphate. 15 mM 3-phosphoglycerate caused no stimulation of the pyrophosphorolytic reaction, and only 2-fold stimulation of the ADPglucose synthesizing reaction.     

Heat stability and allosteric properties of the maize endosperm ADP-glucose pyrophosphorylase are intimately intertwined

ADP-glucose (Glc) pyrophosphorylase (AGPase), a key regulatory enzyme in starch biosynthesis, is highly regulated. Transgenic approaches in four plant species showed that alterations in either thermal stability or allosteric modulation increase starch synthesis. Here, we show that the classic regulators 3-phosphoglyceric acid (3-PGA) and inorganic phosphate (Pi) stabilize maize (Zea mays) endosperm AGPase to thermal inactivation. In addition, we show that glycerol phosphate and ribose-5-P increase the catalytic activity of maize AGPase to the same extent as the activator 3-PGA, albeit with higher K(a) (activation constant) values. Activation by fructose-6-P and Glc-6-P is comparable to that of 3-PGA. The reactants ATP and ADP-Glc, but not Glc-1-P and pyrophosphate, protect AGPase from thermal inactivation, a result consistent with the ordered kinetic mechanism reported for other AGPases. 3-PGA acts synergistically with both ATP and ADP-Glc in heat protection, decreasing the substrate concentration needed for protection and increasing the extent of protection. Characterization of a series of activators and inhibitors suggests that they all bind at the same site or at mutually exclusive sites. Pi, the classic "inhibitor" of AGPase, binds to the enzyme in the absence of other metabolites, as determined by thermal protections experiments, but does not inhibit activity. Rather, Pi acts by displacing bound activators and returning the enzyme to its activity in their absence. Finally, we show from thermal inactivation studies that the enzyme exists in two forms that have significantly different stabilities and do not interconvert rapidly.     

ADP-glucose pyrophosphorylase from wheat endosperm. Purification and characterization of an enzyme with novel regulatory properties

ADP-glucose pyrophosphorylase (AGPase; EC 2.7.7.27) was purified and characterized from two wheat (Triticum aestivum L.) tissues: leaf and endosperm. The leaf enzyme, purified over 1,300-fold, was found to be a heterotetramer composed of subunits of 51 and 54 kDa and possessing regulatory properties typical of AGPases from photosynthetic tissues, being mainly regulated by 3-phosphoglycerate (activator; A0.5=0.01 mM) and orthophosphate (inhibitor; I0.5=0.2 mM). Conversely, the enzyme from wheat endosperm was insensitive to activation by 3-phosphoglycerate and other metabolites. It was, however, inhibited by orthophosphate (I0.5=0.7 mM), ADP (I0.5=3.2 mM) and fructose-1,6-bisphosphate (0.5 = 1.5 mM). All of these inhibitory actions were reversed by 3-phosphoglycerate and fructose-6-phosphate. The endosperm enzyme was found to be a heterotetramer composed of subunits of 52 and 53 kDa, which were recognized by antiserum raised to spinach leaf AGPase. The results suggest that wheat endosperm AGPase possesses distinctive regulatory properties that are relevant in vivo.     

Quantification of the Contribution of CO2, HCO3-, and External Carbonic Anhydrase to Photosynthesis at Low Dissolved Inorganic Carbon in Chlorella saccharophila

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-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'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.     

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.     

Rapid purification of the potato ADP-glucose pyrophosphorylase by polyhistidine-mediated chromatography

In an attempt to obtain facile methods to purify the heterotetrameric ADP-glucose pyrophosphorylase (AGPase), polyhistidine tags were attached to either the large (LS) or small (SS) subunits of this oligomeric enzyme. The addition of polyhistidine tag to the N-terminus of the LS or SS and co-expression with its unmodified counterpart subunit resulted in substantial induction of enzyme activity. In contrast, attachment of a polyhistidine-containing peptide through the use of a commercially available pET vector or addition of polyhistidine tags to the C-terminal ends of either subunit resulted in poor expression and/or production of enzyme activity. Preliminary experiment showed that these polyhistidine N-terminal-tagged enzymes interacted with Ni-NTA-agarose, indicating that immobilized metal affinity chromatography (IMAC) would be useful for efficient purification of the heterotetrameric AGPases. When ion-exchange chromatography step was employed prior to the IMAC, the polyhistidine-tagged AGPases were purified to near homogeneity. Comparison of kinetic parameters between AGPases with and without the polyhistidine tags revealed that attachment of the polyhistidine did not alter the allosteric and catalytic properties of the enzymes. These results indicate that polyhistidine tags will be useful for the rapid purification of preparative amounts of AGPases for biochemical and physical studies.     

The contribution of adenosine 5′-diphosphoglucose pyrophosphorylase to the control of starch synthesis in potato tubers

The aim of this work was to investigate the extent to which starch synthesis in potato (Solanum tuberosum L.) tubers is controlled by the activity of ADPglucose pyrophosphorylase (EC 2.7.7.27; AGPase). In order to do this, fluxes of carbohydrate metabolism were measured in tubers that had reduced AGPase activity as a result of the expression of a cDNA encoding the B subunit in the antisense orientation. Reduction in AGPase activity led to a reduction in starch accumulation, and an increase in sucrose accumulation. The control coefficient of AGPase on starch accumulation in intact plants was estimated to be around 0.3. The fluxes of carbohydrate metabolism were measured in tuber discs from wild-type and transgenic plants by investigating the metabolism of [U-¹⁴C]glucose. In tuber discs, the control coefficient of AGPase over starch synthesis was estimated as 0.55, while the control coefficient of the enzyme over sucrose synthesis was −0.47. The values obtained suggest that AGPase activity exerts appreciable control over tuber metabolism in potato. Received: 24 February 1999 / Accepted: 8 April 1999