Unraveling the Activation Mechanism of the Potato Tuber ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase regulates the synthesis of glycogen in bacteria and of starch in plants. The enzyme from plants is mainly activated by 3-phosphoglycerate and is a heterotetramer comprising two small and two large subunits. Here, we found that two highly conserved residues are critical for triggering the activation of the potato tuber ADP-glucose pyrophosphorylase, as shown by site-directed mutagenesis. Mutations in the small subunit, which bears the catalytic function in this potato tuber form, had a more dramatic effect on disrupting the allosteric activation than those introduced in the large subunit, which is mainly modulatory. Our results strongly agree with a model where the modified residues are located in loops responsible for triggering the allosteric activation signal for this enzyme, and the sensitivity to this activation correlates with the dynamics of these loops. In addition, previous biochemical data indicates that the triggering mechanism is widespread in the enzyme family, even though the activator and the quaternary structure are not conserved.     

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.     

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.     

ADP-Glucose Pyrophosphorylase: A Regulatory Enzyme for Plant Starch Synthesis

In plants, the synthesis of starch occurs by utilizing ADP-glucose as the glucosyl donor for the elongation of alpha-1,4-glucosidic chains. In photosynthetic bacteria the synthesis of glycogen follows a similar pathway. The first committed step in these pathways is the synthesis of ADP-glucose in a reaction catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). Generally, this enzyme is allosterically regulated by intermediates of the major carbon assimilatory pathway in the respective organism. In oxygenic photosynthesizers, ADPGlc PPase is mainly regulated by 3-phosphoglycerate (activator) and inorganic orthophosphate (inhibitor), interacting in four different patterns. Recent reports have shown that in higher plants, some of the enzymes could also be redox regulated. In eukaryotes, the enzyme is a heterotetramer comprised of two distinct subunits, a catalytic and a modulatory subunit. The latter has been proposed as related to variations in regulation of the enzyme in different plant tissues. Random and site-directed mutagenesis experiments of conserved amino acids revealed important residues for catalysis and regulation. Prediction of the ADPGlc PPase secondary structure suggests that it shares a common folding pattern to other sugar-nucleotide pyrophosphorylases, and they evolved from a common ancestor.     

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 Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.     

Discovery of Novel Allosteric Effectors Based on the Predicted Allosteric Sites for Escherichia coli D-3-Phosphoglycerate Dehydrogenase

D-3-phosphoglycerate dehydrogenase (PGDH) from Escherichia coli catalyzes the first critical step in serine biosynthesis, and can be allosterically inhibited by serine. In a previous study, we developed a computational method for allosteric site prediction using a coarse-grained two-state Gō Model and perturbation. Two potential allosteric sites were predicted for E. coli PGDH, one close to the active site and the nucleotide binding site (Site I) and the other near the regulatory domain (Site II). In the present study, we discovered allosteric inhibitors and activators based on site I, using a high-throughput virtual screen, and followed by using surface plasmon resonance (SPR) to eliminate false positives. Compounds 1 and 2 demonstrated a low-concentration activation and high-concentration inhibition phenomenon, with IC₅₀ values of 34.8 and 58.0 µM in enzymatic bioassays, respectively, comparable to that of the endogenous allosteric effector, L-serine. For its activation activity, compound 2 exhibited an AC₅₀ value of 34.7 nM. The novel allosteric site discovered in PGDH was L-serine- and substrate-independent. Enzyme kinetics studies showed that these compounds influenced K_m, k_cat, and k_cat/K_m. We have also performed structure-activity relationship studies to discover high potency allosteric effectors. Compound 2-2, an analog of compound 2, showed the best in vitro activity with an IC₅₀ of 22.3 µM. Compounds targeting this site can be used as new chemical probes to study metabolic regulation in E. coli. Our study not only identified a novel allosteric site and effectors for PGDH, but also provided a general strategy for designing new regulators for metabolic enzymes.     

The Role of Allosteric Coupling on Thermal Activation of Thermo-TRP Channels

Thermo-transient receptor potential channels display outstanding temperature sensitivity and can be directly gated by low or high temperature, giving rise to cold- and heat-activated currents. These constitute the molecular basis for the detection of changes in ambient temperature by sensory neurons in animals. The mechanism that underlies the temperature sensitivity in thermo-transient receptor potential channels remains unknown, but has been associated with large changes in standard-state enthalpy (ΔH^o) and entropy (ΔS^o) upon channel gating. The magnitude, sign, and temperature dependence of ΔH^o and ΔS^o, the last given by an associated change in heat capacity (ΔC_p), can determine a channel’s temperature sensitivity and whether it is activated by cooling, heating, or both, if ΔC_p makes an important contribution. We show that in the presence of allosteric gating, other parameters, besides ΔH^o and ΔS^o, including the gating equilibrium constant, the strength- and temperature dependence of the coupling between gating and the temperature-sensitive transitions, as well as the ΔH^o/ΔS^o ratio associated with them, can also determine a channel’s temperature-dependent activity, and even give rise to channels that respond to both cooling and heating in a ΔC_p-independent manner.     

The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter

The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of d- and l-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by l-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well.     

A Novel Mechanism of Transposon-Mediated Gene Activation

Transposable Insertion Sequences (IS elements) have been shown to provide various benefits to their hosts via gene activation or inactivation under stress conditions by appropriately inserting into specific chromosomal sites. Activation is usually due to derepression or introduction of a complete or partial promoter located within the element. Here we define a novel mechanism of gene activation by the transposon IS5 in Escherichia coli. The glycerol utilization operon, glpFK, that is silent in the absence of the cAMP-Crp complex, is activated by IS5 when inserted upstream of its promoter. High-level expression is nearly constitutive, only mildly dependent on glycerol, glucose, GlpR, and Crp, and allows growth at a rate similar to or more rapid than that of wild-type cells. Expression is from the glpFK promoter and dependent on (1) the DNA phase, (2) integration host factor (IHF), and (3) a short region at the 3′ end of IS5 harboring a permanent bend and an IHF binding site. The lacZYA operon is also subject to such activation in the absence of Crp. Thus, we have defined a novel mechanism of gene activation involving transposon insertion that may be generally applicable to many organisms.     

Mechanism of Filamentation-Induced Allosteric Activation of the SgrAI Endonuclease

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The Escherichia coli adenylate cyclase complex: Activation by phosphoenolpyruvate

A model for the regulation of the activity of Escherichia coli adenylate cyclase is presented. It is proposed that Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) interacts in a regulatory sense with the catalytic unit of adenylate cyclase. The phosphoenolpyruvate (PEP)-dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state of adenylate cyclase. The pyruvate or sugar-dependent dephosphorylation of Enzyme I is correlated with a low activity state of adenylate cyclase. Evidence in support of the proposed model involves the observation that Enzyme I mutants have low cAMP levels and that PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase, Kinetic studies indicate that various ligands have opposing effects on adenylate cyclase. While PEP activates the enzyme, either glucose or pyruvate inhibit it. The unique relationships of PEP and Enzyme I to adenylate cyclase activity are discussed.     

Characterization of Pyruvate Uptake in Escherichia coli K-12

The monocarboxylate pyruvate is an important metabolite and can serve as sole carbon source for Escherichia coli. Although specific pyruvate transporters have been identified in two bacterial species, pyruvate transport is not well understood in E. coli. In the present study, pyruvate transport was investigated under different growth conditions. The transport of pyruvate shows specific activities depending on the growth substrate used as sole carbon source, suggesting the existence of at least two systems for pyruvate uptake: i) one inducible system and probably highly specific for pyruvate and ii) one system active under non-induced conditions. Using the toxic pyruvate analog 3-fluoropyruvate, a mutant was isolated unable to grow on and transport pyruvate. Further investigation revealed that a revertant selected for growth on pyruvate regained the inducible pyruvate transport activity. Characterization of pyruvate excretion showed that the pyruvate transport negative mutant accumulated pyruvate in the growth medium suggesting an additional transport system for pyruvate excretion. The here presented data give valuable insight into the pyruvate metabolism and transport of E. coli suggesting the presence of at least two uptake systems and one excretion system to balance the intracellular level of pyruvate.     

Inhibition of Escherichia coli Biodegradative Threonine Dehydratase by Pyruvate

Pyruvate inhibits Escherichia coli K-12 biodegradative threonine dehydratase activity by a mechanism distinct from product inhibition by alpha-ketobutyrate and catabolite inactivation by intermediary metabolites.     

Role of a Novel PH-Kinase Domain Interface in PKB/Akt Regulation: Structural Mechanism for Allosteric Inhibition

Protein kinase B (PKB/Akt) belongs to the AGC superfamily of related serine/threonine protein kinases. It is a key regulator downstream of various growth factors and hormones and is involved in malignant transformation and chemo-resistance. Full-length PKB protein has not been crystallised, thus studying the molecular mechanisms that are involved in its regulation in relation to its structure have not been simple. Recently, the dynamics between the inactive and active conformer at the molecular level have been described. The maintenance of PKB''s inactive state via the interaction of the PH and kinase domains prevents its activation loop to be phosphorylated by its upstream activator, phosphoinositide-dependent protein kinase-1 (PDK1). By using a multidisciplinary approach including molecular modelling, classical biochemical assays, and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM), a detailed model depicting the interaction between the different domains of PKB in its inactive conformation was demonstrated. These findings in turn clarified the molecular mechanism of PKB inhibition by AKT inhibitor VIII (a specific allosteric inhibitor) and illustrated at the molecular level its selectivity towards different PKB isoforms. Furthermore, these findings allude to the possible function of the C-terminus in sustaining the inactive conformer of PKB. This study presents essential insights into the quaternary structure of PKB in its inactive conformation. An understanding of PKB structure in relation to its function is critical for elucidating its mode of activation and discovering how to modulate its activity. The molecular mechanism of inhibition of PKB activation by the specific drug AKT inhibitor VIII has critical implications for determining the mechanism of inhibition of other allosteric inhibitors and for opening up opportunities for the design of new generations of modulator drugs.