Mechanism of reductive activation of potato tuber ADP-glucose pyrophosphorylase.

Journal ArticleThe potato tuber (Solanum tuberosum L.) ADP-glucose pyrophosphorylase activity is activated by a incubation with ADP-glucose and dithiothreitol or by ATP, glucose- 1-phosphate, Ca2+, and dithiothreitol. The activation was accompanied by the appearance of new sulfhydryl groups as determined with 5, 5'-dithiobis(2-nitrobenzoic acid). By analyzing the activated and nonactivated enzymes on SDS-polyacrylamide gel electrophoresis under nonreducing conditions, it was found that an intermolecular disulfide bridge between the small subunits of the potato tuber enzyme was reduced during the activation. Further experiments showed that the activation was mediated via a slow reduction and subsequent rapid conformational change induced by ADP-glucose. The activation process could be reversed by oxidation with 5, 5'-dithiobis(2-nitrobenzoic acid). Incubation with ADP-glucose and dithiothreitol could reactivate the oxidized enzyme. Chemical modification experiments with [14C]iodoacetic acid and 4-vinylpyridine determined that the intermolecular disulfide bridge was located between Cys12 of the small subunits of the potato tuber enzyme. Mutation of Cys12 in the small subunit into either Ala or Ser eliminated the requirement of DTT on the activation and prevented the formation of the intermolecular disulfide of the potato tuber enzyme. The mutants had instantaneous activation rates as the wild-type in the reduced state. A two-step activation model is proposed

Determination of dissociation constants of protein ligands by thermal shift assay

The thermal shift assay (TSA) is a powerful tool used to detect molecular interactions between proteins and ligands. Using temperature as a physical denaturant and an extrinsic fluorescent dye, the TSA tracks protein unfolding. This method precisely determines the midpoint of the unfolding transition (Tm role= presentation \u3e), which can shift upon the addition of a ligand. Though experimental protocols have been well developed, the thermal shift assay data traditionally yielded qualitative results. Quantitative methods for Kd role= presentation \u3e determination relied either on empirical and inaccurate usage of Tm role= presentation \u3e or on isothermal approaches, which do not take full advantage of the melting point precision provided by the TSA. We present a new analysis method based on a model that relies on the equilibrium system between the native and molten globule state of the protein using the van\u27t Hoff equation. We propose the Kd role= presentation \u3e can be determined by plotting Tm role= presentation \u3e values versus the logarithm of ligand concentrations and fitting the data to an equation we derived. After testing this procedure with the monomeric maltose-binding protein and an allosterically regulated homotetrameric enzyme (ADP-glucose pyrophosphorylase), we observed that binding results correlated very well with previously established parameters. We demonstrate how this method could potentially offer a broad applicability to a wide range of protein classes and the ability to detect both active and allosteric site binding compounds

Investigating the Electrostatic Role of a Critical Arginine for the Catalysis of E. Coli ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the regulatory enzyme of the pathway for starch synthesis in plants and glycogen in mammals and enteric bacteria. It exists as a 200 kDa homotetramer (α4) in enteric bacteria, and as a heterotetramer (α2β2) in plants. In both in vivo and in vitro the substrates (Glucose 1-Phosphate; Glc-1P and Adenosine 5\u27-Triphosphate; ATP) are converted into a glucose donor ADP-Glucose and a pyrophosphate (PPi) via the ADP-Glc PPase enzyme. It has been noted that some residues are conserved in homotetrameric bacterial ADP-Glc PPases, but are not in some plant forms. One of them is Arginine-32 (R32) in the Escherichia coli ADP-Glc PPase. To explore the overall role of this residue and evaluate the structural and electrostatic importance of the Arginine\u27s guanidinium group, we replaced it with Lysine (K, -amino group), Alanine (A, - methyl group), Cysteine (C, -sulfide group), Glutamic (E, - carboxylate group), Glutamine (Q, -amido group) and Leucine (L, -hydrophobic side chain) via site directed mutagenesis. We over-expressed the enzymes, purified them to homogeneity, and measured their kinetic properties. The Specific Activity (U/mg) for the mutants were as follows: WT (90.56), R32A (1.65), R32C (0.57), R32E (0.04), R32K (5.81), R32L (0.65) and R32Q (1.37). Currently, the properties of the R32H (Histidine, -imidizoleum ring) mutant are being investigated. Our results clearly indicate that this guanidinium group of the Arginine-32 residue is critical for catalysis. Modeling of the E. coli enzyme suggests that the two (2) nitrogen atoms of the guanidinium group may interact with the β and γ phosphates of the ATP, helping in the positioning of the substrates, via electrostatic interactions, and making the PPi product a more stable leaving group

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

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

Functional demonstrations of starch binding domains present in Ostreococcus tauri starch synthases isoforms

Abstract Background: Starch‑binding domains are key modules present in several enzymes involved in polysaccharide metabolism. These non‑catalytic modules have already been described as essential for starch‑binding and the cata‑ lytic activity of starch synthase III from the higher plant Arabidopsis thaliana. In Ostreococcus tauri, a unicellular green alga of the Prasinophyceae family, there are three SSIII isoforms, known as Ostta SSIII‑A, SSIII‑B and SSIII‑C. Results: In this work, using in silico and in vitro characterization techniques, we have demonstrated that Ostta SSIII‑ A, SSIII‑B and SSIII‑C contain two, three and no starch‑binding domains, respectively. Additionally, our phylogenetic analysis has indicated that OsttaSSIII‑B, presenting three N‑terminal SBDs, is the isoform more closely related to higher plant SSIII. Furthermore, the sequence alignment and homology modeling data gathered showed that both the main 3‑D structures of all the modeled domains obtained and the main amino acid residues implicated in starch binding are well conserved in O. tauri SSIII starch‑binding domains. In addition, adsorption assays showed that OsttaSSIII‑A D2 and SSIII‑B D2 domains are the two that make the greatest contribution to amylose and amylopectin binding, while OsttaSSIII‑B D1 is also important for starch binding. Conclusions: The results presented here suggest that differences between OsttaSSIII‑A and SSIII‑B SBDs in the number of and binding of amino acid residues may produce differential affinities for each isoform to polysaccharides. Increasing the knowledge about SBDs may lead to their employment in biomedical and industrial applications. Keywords: Ostreococcus tauri, Starch‑binding domains, Starch synthase, Homology modeling, Adsorption assayFil: Barchiesi, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Hedin, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Gomez Casati, Diego Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Ballicora, Miguel. Loyola University Chicago; Estados UnidosFil: Busi, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentin

Practical Spectrophotometric Assay for the \u3cem\u3edapE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-Succinyl-L,L-Diaminopimelic Acid Desuccinylase, a Potential Antibiotic Target

A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [± 0.2] μM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] μM, phenylboronic acid (IC50 = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] μM. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics

A Critical Inter-Subunit Interaction for the Transmission of the Allosteric Signal in the Agrobacterium Tumefaciens ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase is a key regulatory enzyme involved in starch and glycogen synthesis in plants and bacteria, respectively. It has been hypothesized that inter-subunit communications are important for the allosteric effect in this enzyme. However, no specific interactions have been identified as part of the regulatory signal. The enzyme from Agrobacterium tumefaciens is a homotetramer allosterically regulated by fructose 6-phosphate and pyruvate. Three pairs of distinct subunit-subunit interfaces are present. Here we focus on an interface that features two symmetrical interactions between Arg11 and Asp141 from one subunit with residues Asp141 and Arg11 of the neighbor subunit, respectively. Previously, scanning mutagenesis showed that a mutation at the Arg11 position disrupted the activation of the enzyme. Considering the distance of these residues from the allosteric and catalytic sites, we hypothesized that the interaction between Arg11 and Asp141 is critical for allosteric signaling rather than effector binding. To prove our hypothesis, we mutated those two sites (D141A, D141E, D141N, D141R, R11D, and R11K) and performed kinetic and binding analysis. Mutations that altered the charge affected the regulation the most. To prove that the interaction per se (rather than the presence of specific residues) is critical, we partially rescued the R11D protein by introducing a second mutation (R11D/D141R). This could not restore the activator effect on kcat, but it did rescue the effect on substrate affinity. Our results indicate the critical functional role of Arg11 and Asp141 to relay the allosteric signal in this subunit interface

Iodine Staining of Escherichia coli Expressing Genes Involved in the Synthesis of Bacterial Glycogen

The presence of intracellular glycogen can be detected by the following iodine staining technique. Cells with glycogen stain dark brown, whereas in its absence they remain with a pale yellowish color. It is hypothesized that iodine atoms fit into helical coils formed by the α-polyglucan to form a coloured glycogen-iodine complex. Here, we have studied the expression of Streptococcus mutans (S. mutans) genes that control the biosynthesis of this polysaccharide. Thus, we expressed glgC and glgD genes coding for both ADP-Glc pyrophosphorylase subunits in Escherichia coli (E. coli) AC70R1-504 cells to complement the deficient accumulation of glycogen by this strain. In control cells or in those where an inactive protein was expressed, the synthesis of the polysaccharide was undetectable by this iodine staining technique.Fil: Demonte, Ana M.. Universidad Nacional del Litoral; ArgentinaFil: Asención Diez, Matías Damián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; ArgentinaFil: Ballicora, Miguel A.. Loyola University Chicago. Department of Chemistry & Biochemistry; Estados UnidosFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Agrobiotecnologia del Litoral; Argentina. Universidad Nacional del Litoral; Argentin

Allosteric Control of Substrate Specificity of the Escherichia coli ADP-Glucose Pyrophosphorylase

The substrate specificity of enzymes is crucial to control the fate of metabolites to different pathways. However, there is growing evidence that many enzymes can catalyze alternative reactions. This promiscuous behavior has important implications in protein evolution and the acquisition of new functions. The question is how the undesirable outcomes of in vivo promiscuity can be prevented. ADP-glucose pyrophosphorylase from Escherichia coli is an example of an enzyme that needs to select the correct substrate from a broad spectrum of alternatives. This selection will guide the flow of carbohydrate metabolism toward the synthesis of reserve polysaccharides. Here, we show that the allosteric activator fructose-1,6-bisphosphate plays a role in such selection by increasing the catalytic efficiency of the enzyme toward the use of ATP rather than other nucleotides. In the presence of fructose-1,6-bisphosphate, the kcat/S0.5 for ATP was near ~600-fold higher that other nucleotides, whereas in the absence of activator was only ~3-fold higher. We propose that the allosteric regulation of certain enzymes is an evolutionary mechanism of adaptation for the selection of specific substrates.Fil: Ebrecht, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. University of Chicago; Estados UnidosFil: Solamen, Ligin. University of Chicago; Estados UnidosFil: Hill, Benjamin L.. University of Chicago; Estados UnidosFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Olsen, Kenneth W.. University of Chicago; Estados UnidosFil: Ballicora, Miguel A.. University of Chicago; Estados Unido

The Crystal Structure of Nitrosomonas Europaea Sucrose Synthase Reveals Critical Conformational Changes and Insights into the Sucrose Metabolism in Prokaryotes

In this paper we report the first crystal structure of a prokaryotic sucrose synthase from the non-photosynthetic bacterium Nitrosomonas europaea. The obtained structure was in an open form, whereas the only other available structure from the plant Arabidopsis thaliana was in a closed conformation. Comparative structural analysis revealed a “hinge-latch” combination, which is critical to transition between the open and closed forms of the enzyme. The N. europaea sucrose synthase shares the same fold as the GT-B family of the retaining glycosyltransferases. In addition, a triad of conserved homologous catalytic residues in the family showed to be functionally critical in the N. europaea sucrose synthase (Arg567, Lys572, Glu663). This implies that sucrose synthase shares not only a common origin with the GT-B family, but also a similar catalytic mechanism. The enzyme preferred transferring glucose from ADP-glucose rather than UDP-glucose like the eukaryotic counterparts. This predicts that these prokaryotic organisms have a different sucrose metabolic scenario from plants. Nucleotide preference determines where the glucose moiety is targeted after sucrose is degraded