Insight into the 3D structure of ADP-glucose pyrophosphorylase from rice (Oryza sativa L.)

ADP-glucose pyrophosphorylase (E.C. 2.7.7.27; AGPase) is a key regulatory enzyme that catalyzes the rate-limiting step of starch biosynthesis in higher plants. AGPase consists of pair of small (SS) and large (LS) subunits thereby constituting a heterotetrameric structure. No crystal structure of the native heterotetrameric enzyme is available for any species, thus limiting the complete understanding of structure–function relationships of this enzyme. In this study, an attempt was made to deduce the heterotetrameric assembly of AGPase in rice. Homology modeling of the three-dimensional structure of the LS and SS was performed using the Swiss Model Server, and the models were evaluated and docked using GRAMM-X to obtain the stable heterodimer orientation (LS as receptor and SS as ligand) and then the heterotetrameric orientation. The initial heterotetrameric orientation was further refined using the RosettaDock Server. MD simulation of the representative heterodimer/tetramer was performed using NAMD, which indicated that the tail-to-tail interaction of LS and SS was more stable than the head-to-head orientation, and the heterotetramer energy was also minimized to ?767,011 kcal mol^?1. Subunit–subunit interaction studies were then carried out using the programs NACCESS and Dimplot. A total of 57 interface residues were listed in SS and 63 in LS. The residues plotted by Dimplot were similar to those listed by NACCESS. Multiple sequence alignment of the sequences of LS and SS from potato, maize and rice validated the interactions inferred in the study. RMSD of 1.093 Å was obtained on superimposition of the deduced heterotetramer on the template homo-tetramer (1YP2), showing the similarity between the two structures.     

Generation,characterization, and heterologous expression of wild-type and up-regulated forms of Arabidopsis thaliana leaf ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key enzyme in starch biosynthesis of higher plants, consists of a pair of regulatory large (LS) and catalytically small (SS) subunits. In plants, these subunits are coded by multiple genes resulting in the formation of tissue-specific enzyme forms, which are differentially regulated during plant growth and development. Some AGPase isoforms differ in catalytic and regulatory properties as well as intracellular location. In an effort to gain a better understanding of the role of the leaf AGPase in carbon partitioning and its effect on plant productivity, the Arabidopsis leaf AGPase containing the mature forms of the SS and LS was expressed in a heterologous expression system and characterized enzymatically. The Arabidopsis recombinant AGPase had kinetic values for 3-phosphoglyceric acid, glucose-1-phosphate and Mg(2+) similar to those of the native enzyme. As the N-terminus of the LS has been suggested to be involved in enzyme function, the length of the N-terminal region was extended or shortened. Of the five modified LSs analyzed, only the T5 form lacking six residues of the mature N-terminus was able to form detectable levels of enzyme activity, indicating that the N-terminal region is critical for enzyme function. Two up-regulatory LS mutations that allosterically activate the potato enzyme, a stem isoform, were introduced into the corresponding Arabidopsis LS sequences and co-expressed with wild-type SS. Both modified enzymes showed up-regulatory properties, indicating that these specific residue changes were also operational in the leaf isoform.     

Allosteric regulation of the higher plant ADP-glucose pyrophosphorylase is a product of synergy between the two subunits

The higher plant ADP-glucose pyrophosphorylase (AGPase) is a heterotetramer consisting of two regulatory large subunits (LSs) and two catalytic small subunits (SSs). To further characterize the roles of these subunits in determining enzyme function, different combinations of wildtype LS (LWT) and variant forms (LUpReg1, LM345) were co-expressed with wildtype SS (SWT) and variant forms (STG-15 and Sdevo330) and their enzyme properties compared to those measured for the heterotetrameric wildtype enzyme and SS homotetrameric enzymes. Analysis of the allosteric regulatory properties of the various enzymes indicates that although the LS is required for optimal activation by 3-phosphoglyceric acid and resistance to Pi, the overall allosteric regulatory and kinetic properties are specified by both subunits. Our results show that the regulatory and kinetic properties of AGPase are not simply due to the LS modulating the properties of the SS but, instead, are a product of synergistic interaction between the two 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).     

Analysis of allosteric effector binding sites of potato ADP-glucose pyrophosphorylase through reverse genetics

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme of bacterial glycogen and plant starch synthesis as it controls carbon flux via its allosteric regulatory behavior. Unlike the bacterial enzyme that is composed of a single subunit type, the plant AGPase is a heterotetrameric enzyme (alpha2beta2) with distinct roles for each subunit type. The large subunit (LS) is involved mainly in allosteric regulation through its interaction with the catalytic small subunit (SS). The LS modulates the catalytic activity of the SS by increasing the allosteric regulatory response of the hetero-oligomeric enzyme. To identify regions of the LS involved in binding of effector molecules, a reverse genetics approach was employed. A potato (Solanum tuberosum L.) AGPase LS down-regulatory mutant (E38A) was subjected to random mutagenesis using error-prone polymerase chain reaction and screened for the capacity to form an enzyme capable of restoring glycogen production in glgC(-) Escherichia coli. Dominant mutations were identified by their capacity to restore glycogen production when the LS containing only the second site mutations was co-expressed with the wild-type SS. Sequence analysis showed that most of the mutations were decidedly nonrandom and were clustered at conserved N- and C-terminal regions. Kinetic analysis of the dominant mutant enzymes indicated that the K(m) values for cofactor and substrates were comparable with the wild-type AGPase, whereas the affinities for activator and inhibitor were altered appreciably. These AGPase variants displayed increased resistance to P(i) inhibition and/or greater sensitivity toward 3-phosphoglyceric acid activation. Further studies of Lys-197, Pro-261, and Lys-420, residues conserved in AGPase sequences, by site-directed mutagenesis suggested that the effectors 3-phosphoglyceric acid and P(i) interact at two closely located binding sites.     

Catalytic implications of the higher plant ADP-glucose pyrophosphorylase large subunit

ADP-glucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, is composed of a pair of catalytic small subunits (SSs) and a pair of catalytically disabled large subunits (LSs). The N-terminal region of the LS has been known to be essential for the allosteric regulatory properties of the heterotetrameric enzyme. To gain further insight on the role of this region and the LS itself in enzyme function, the six proline residues found in the N-terminal region of the potato tuber AGPase were subjected to scanning mutagenesis. The wildtype and various mutant heterotetramers were expressed using our newly developed host-vector system, purified, and their kinetic parameters assessed. While P(17)L, P(26)L, and P(55)L mutations only moderately affected the kinetic properties, P(52)L and P(66)L gave rise to significant and contrasting changes in allosteric properties: P(66)L enzyme displayed up-regulatory properties toward 3-PGA while the P(52)L enzyme had down-regulatory properties. Unlike the other mutants, however, various mutations at P(44) led to only moderate changes in regulatory properties, but had severely impaired catalytic rates, apparent substrate affinities, and responsiveness to metabolic effectors, indicating Pro-44 or the LS is essential for optimal catalysis and activation of the AGPase heterotetramer. The catalytic importance of the LS is further supported by photoaffinity labeling studies, which revealed that the LS binds ATP at the same efficiency as the SS. These results indicate that the LS, although considered having no catalytic activity, may mimic many of the catalytic events undertaken by the SS and, thereby, influences net catalysis of the heterotetrameric enzyme.     

Insights into subunit interactions in the heterotetrameric structure of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase, 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. The LS is involved in mainly allosteric regulation through its interaction with the catalytic SS. Recently the crystal structure of the SS homotetramer has been solved, but no crystal structure of the native heterotetrameric enzyme is currently available. In this study, we first modeled the three-dimensional structure of the LS to construct the heterotetrameric enzyme. Because the enzyme has a 2-fold symmetry, six different dimeric (either up-down or side-by-side) interactions were possible. Molecular dynamics simulations were carried out for each of these possible dimers. Trajectories obtained from molecular dynamics simulations of each dimer were then analyzed by the molecular mechanics/Poisson-Boltzmann surface area method to identify the most favorable dimers, one for up-down and the other for side-by-side. Computational results combined with site directed mutagenesis and yeast two hybrid experiments suggested that the most favorable heterotetramer is formed by LS-SS (side-by-side), and LS-SS (up-down). We further determined the order of assembly during the heterotetrameric structure formation. First, side-by-side LS-SS dimers form followed by the up-down tetramerization based on the relative binding free energies.     

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.     

Direct appraisal of the potato tuber ADP-glucose pyrophosphorylase large subunit in enzyme function by study of a novel mutant form

The higher plant ADP-glucose pyrophosphorylase is a heterotetramer consisting of two subunit types, which have evolved at different rates from a common ancestral gene. The potato tuber small subunit (SS) displays both catalytic and regulatory properties, whereas the exact role of the large subunit (LS), which contains substrate and effector binding sites, remains unresolved. We identified a mutation, S302N, which increased the solubility of the recombinant potato tuber LS and, in turn, enabling it to form a homotetrameric structure. The LS302N homotetramer possesses very little enzyme activity at a level 100-fold less than that seen for the unactivated SS homotetramer. Unlike the SS enzyme, however, the LS302N homotetramer enzyme is neither activated by the effector 3-phosphoglycerate nor inhibited by P(i). When combined with the catalytically silenced SS, S D143N, however, the LS302N-containing enzyme shows significantly enhanced catalytic activity and restored 3-PGA activation. This unmasking of catalytic and regulatory potential of the LS is conspicuously evident when the activities of the resurrected L(K41R.T51K.S302N) homotetramer are compared with its heterotetrameric form assembled with S D143N. Overall, these results indicate that the LS possesses catalytic and regulatory properties only when assembled with SS and that the net properties of the heterotetrameric enzyme is a product of subunit synergy.     

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.     

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.     

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。     

Insights of interaction between small and large subunits of ADP-glucose pyrophosphorylase from bread wheat (Triticum aestivum L.)

Lack of knowledge of three dimensional structures of small and large subunits of ADP- glucose pyrophosphorylase (AGPase) in wheat has hindered efforts to understand the binding specifities of substrate and catalytic mechanism. Thus, to understand the structure activity relationship, 3D structures were built by homology modelling based on crystal structure of potato tuber ADP-glucose pyrophosphorylase. Selected models were refined by energy minimization and further validated by Procheck and Prosa-web analysis. Ramachandran plot showed that overall main chain and side chain parameters are favourable. Moreover, Z-score of the models from Prosa-web analysis gave the conformation that they are in the range of the template. Interaction analysis depicts the involvement of six amino acids in hydrogen bonding (AGP-SThr422-AGP-LMet138, AGP- SArg420-AGP-LGly47, AGP-SSer259-AGP-LSer306, AGP-SGlu241-AGP-LIle311, AGPSGln113- AGP-LGlu286 and AGP-SGln70-AGP-LLys291). Fifteen amino acids of small subunit were able to make hydrophobic contacts with seventeen amino acids of large subunit. Furthermore, decrease in the solvent accessible surface area in the amino acids involved in interaction were also reported. All the distances were formed in between 2.27 to 3.78Å. The present study focussed on heterodimeric structure of (AGPase). This predicted complex not only enhance our understanding of the interaction mechanism between these subunits (AGP-L and AGP-S) but also enable to further study to obtain better variants of this enzyme for the improvement of the plant yield.     

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.     

Subtle structural differences crucial for function in similarly engineered ADP-glucose pyrophosphorylase larger subunit in rice and maize

ADP ?C glucose pyrophosphorylase (AGPase) is a key enzyme for starch synthesis in plants. Heterotetrameric plant AGPase is encoded by two genes, Shrunken-2 (Sh2) and Brittle-2 (Bt2). The Sh2 gene encodes regulatory larger subunit and the Bt2 gene encodes smaller subunit having catalytic properties. A specific mutation in Sh2 gene involving insertion of six nucleotides, without changing the reading frame, resulted in the insertion of two additional amino acid residues serine and tyrosine at specific position at carboxyl end and also in an increase in seed weight up to 11?C17%. No increase in seed weight with the same insertion in larger subunit of AGPase enzyme in rice was observed even though the rice and maize subunits have 93% of sequence similarity. In this study, the predicted 3D-structures of larger subunit of normal as well as mutated AGPase in maize and rice, were analyzed and superposed. The segment of six amino acid residues long secondary structure just before the site of insertion of additional amino acids (serine and tyrosine) got reduced in case of mutated maize but not in mutated rice. Therefore, the six residue sequence and corresponding subtle secondary structural difference might be the key factors for functional disparity of engineered larger subunits in the two crops.     

Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase

The higher plant ADP-glucose (ADPG) pyrophosphorylase (AGPase), composed of two small subunits and two large subunits (LSs), produces ADPG, the sole substrate for starch biosynthesis from α-D-glucose 1-phosphate and ATP. This enzyme controls a key step in starch synthesis as its catalytic activity is activated by 3-phosphoglycerate (3-PGA) and inhibited by orthophosphate (Pi). Previously, two mutations in the LS of potato AGPase (PLS), PLS-E38K and PLS-G101N, were found to increase sensitivity to 3-PGA activation and tolerance to Pi inhibition. In the present study, the double mutated enzyme (PLS-E38K/G101N) was evaluated. In a complementation assay of ADPG synthesis in an Escherichia coli mutant defective in the synthesis of ADPG, expression of PLS-E38K/G101N mediated higher glycogen production than wild-type potato AGPase (PLS-WT) and the single mutant enzymes, PLS-E38K and PLS-G101N, individually. Purified PLS-E38K/G101N showed higher sensitivity to 3-PGA activation and tolerance to Pi inhibition than PLS-E38K or PLS-G101N. Moreover, the enzyme activities of PLS-E38K, PLS-G101N, and PLS-E38K/G101N were more readily stimulated by other major phosphate-ester metabolites, such as fructose 6-phosphate, fructose 2,6-bisphosphate, and ribose 5-phosphate, than was that of PLS-WT. Hence, although the specific enzyme activities of the LS mutants toward 3-PGA were impaired to some extent by the mutations, our results suggest that their enhanced allosteric regulatory properties and the broadened effector selectivity gained by the same mutations not only offset the lowered enzyme catalytic turnover rates but also increase the net performance of potato AGPase in vivo in view of increased glycogen production in bacterial cells.     

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.     

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.