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Debra Dunaway-mariano - One of the best experts on this subject based on the ideXlab platform.
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Investigation of the Catalytic Site within the ATP-Grasp Domain of Clostridium symbiosum Pyruvate Phosphate Dikinase*
2015Co-Authors: Brian M. Martin, Debra Dunaway-marianoAbstract:Pyruvate Phosphate Dikinase (PPDK) catalyzes the interconversion of ATP, Pi, and Pyruvate with AMP, PPi, and phosphoenolPyruvate (PEP) in three partial reac-tions as follows: 1) E-His ATP 3 E-His-PPAMP; 2) E-His-PPAMP Pi3 E-His-PAMPPPi; and 3) E-His-P Pyruvate 3 EPEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecu-tive N-terminal, central His-455, and C-terminal do-mains. The N-terminal and central His-455 domains cat-alyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide bindin
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Design, synthesis, and evaluation of inhibitors of Pyruvate Phosphate Dikinase.
The Journal of organic chemistry, 2012Co-Authors: Debra Dunaway-mariano, Patrick S. MarianoAbstract:Pyruvate Phosphate Dikinase (PPDK) catalyzes the phosphorylation reaction of Pyruvate that forms phosphoenolPyruvate (PEP) via two partial reactions: PPDK + ATP + Pi → PPDK-P + AMP + PPi and PPDK-P + Pyruvate → PEP + PPDK. Based on its role in the metabolism of microbial human pathogens, PPDK is a potential drug target. A screen of substances that bind to the PPDK ATP-grasp domain active site revealed that flavone analogues are potent inhibitors of the Clostridium symbiosum PPDK. In silico modeling studies suggested that placement of a 3–6 carbon-tethered ammonium substituent at the 3′- or 4′-positions of 5,7-dihydroxyflavones would result in favorable electrostatic interactions with the PPDK Mg-ATP binding site. As a result, polymethylene-tethered amine derivatives of 5,7-dihydroxyflavones were prepared. Steady-state kinetic analysis of these substances demonstrates that the 4′-aminohexyl-5,7-dyhydroxyflavone 10 is a potent competitive PPDK inhibitor (Ki = 1.6 ± 0.1 μM). Single turnover experiments were ...
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Swiveling Domain Mechanism in Pyruvate Phosphate Dikinase
Biochemistry, 2007Co-Authors: Kap Lim, Randy J. Read, Celia C. H. Chen, Aleksandra Tempczyk, Min Wei, Debra Dunaway-mariano, Osnat HerzbergAbstract:Pyruvate Phosphate Dikinase (PPDK) catalyzes the reversible conversion of phosphoenolPyruvate (PEP), AMP, and Pi to Pyruvate and ATP. The enzyme contains two remotely located reaction centers: the nucleotide partial reaction takes place at the N-terminal domain, and the PEP/Pyruvate partial reaction takes place at the C-terminal domain. A central domain, tethered to the N- and C-terminal domains by two closely associated linkers, contains a phosphorylatable histidine residue (His455). The molecular architecture suggests a swiveling domain mechanism that shuttles a phosphoryl group between the two reaction centers. In an early structure of PPDK from Clostridium symbiosum, the His445-containing domain (His domain) was positioned close to the nucleotide binding domain and did not contact the PEP/Pyruvate-binding domain. Here, we present the crystal structure of a second conformational state of C. symbiosum PPDK with the His domain adjacent to the PEP-binding domain. The structure was obtained by producing a...
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Examination of the Structure, Stability, and Catalytic Potential in the Engineered Phosphoryl Carrier Domain of Pyruvate Phosphate Dikinase†,‡
Biochemistry, 2006Co-Authors: Ying Lin, Debra Dunaway-mariano, Jacqueline D. Lusin, James B. AmesAbstract:Pyruvate Phosphate Dikinase (PPDK) is a multidomain protein that catalyzes the interconversion of ATP, Pyruvate, and Phosphate with AMP, phosphoenolPyruvate (PEP), and pyroPhosphate using its central domain to transport phosphoryl groups between two distant active sites. In this study, the mechanism by which the central domain moves between the two catalytic sites located on the N-terminal and C-terminal domains was probed by expressing this domain as an independent protein and measuring its structure, stability, and ability to catalyze the ATP/Phosphate partial reaction in conjunction with the engineered N-terminal domain protein (residues 1-340 of the native PPDK). The encoding gene was engineered to express the central domain as residues 381-512 of the native PPDK. The central domain was purified and shown to be soluble, monomeric (13,438 Da), and stable (deltaG = 4.3 kcal/mol for unfolding in buffer at pH 7.0, 25 degrees C) and to possess native structure, as determined by multidimensional heteronuclear NMR analysis. The main chain structure of the central domain in solution aligns closely with that of the X-ray structure of native PPDK (the root-mean-square deviation is 2.2 A). Single turnover reactions of [14C]ATP and Phosphate, carried out in the presence of equal concentrations of central domain and the N-terminal domain protein, did not produce the expected products, in contrast to efficient product formation observed for the N-terminal central domain construct (residues 1-553 of the native PPDK). These results are interpreted as evidence that the central domain, although solvent-compatible, must be tethered by the flexible linkers to the N-terminal domain for the productive domain-domain docking required for efficient catalysis.
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Pyruvate Site of Pyruvate Phosphate Dikinase: Crystal Structure of the Enzyme−PhosphonoPyruvate Complex, and Mutant Analysis†,‡
Biochemistry, 2002Co-Authors: Osnat Herzberg, Celia C. H. Chen, Aleksandra Tempczyk, Min Wei, Sijiu Liu, Andrew Howard, Debra Dunaway-marianoAbstract:Crystals of Pyruvate Phosphate Dikinase in complex with a substrate analogue inhibitor, phosphonoPyruvate (Ki = 3 μM), have been obtained in the presence of Mg2+. The structure has been determined and refined at 2.2 A resolution, revealing that the Mg2+-bound phosphonoPyruvate binds in the α/β-barrel's central channel, at the C-termini of the β-strands. The mode of binding resembles closely the previously proposed PEP substrate binding mode, inferred by the homology of the structure (but not sequence homology) to Pyruvate kinase. Kinetic analysis of site-directed mutants, probing residues involved in inhibitor binding, showed that all mutations resulted in inactivation, confirming the key role that these residues play in catalysis. Comparison between the structure of the PPDK−phosphonoPyruvate complex and the structures of two complexes of Pyruvate kinase, one with Mg2+-bound phospholactate and the other with Mg2+-oxalate and ATP, revealed that the two enzymes share some key features that facilitate commo...
Brian M. Martin - One of the best experts on this subject based on the ideXlab platform.
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Investigation of the Catalytic Site within the ATP-Grasp Domain of Clostridium symbiosum Pyruvate Phosphate Dikinase*
2015Co-Authors: Brian M. Martin, Debra Dunaway-marianoAbstract:Pyruvate Phosphate Dikinase (PPDK) catalyzes the interconversion of ATP, Pi, and Pyruvate with AMP, PPi, and phosphoenolPyruvate (PEP) in three partial reac-tions as follows: 1) E-His ATP 3 E-His-PPAMP; 2) E-His-PPAMP Pi3 E-His-PAMPPPi; and 3) E-His-P Pyruvate 3 EPEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecu-tive N-terminal, central His-455, and C-terminal do-mains. The N-terminal and central His-455 domains cat-alyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide bindin
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Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum Pyruvate Phosphate Dikinase.
The Journal of biological chemistry, 2001Co-Authors: Min Wei, Osnat Herzberg, Brian M. Martin, Marielena Mcguire, Kui Huang, Geeta Kapadia, Debra Dunaway-marianoAbstract:Abstract Pyruvate Phosphate Dikinase (PPDK) catalyzes the interconversion of ATP, Pi, and Pyruvate with AMP, PPi, and phosphoenolPyruvate (PEP) in three partial reactions as follows: 1) E-His + ATP →E-His-PP·AMP; 2) E-His-PP·AMP + Pi → E-His-P·AMP·PPi; and 3)E-His-P + Pyruvate → E·PEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecutive N-terminal, central His-455, and C-terminal domains. The N-terminal and central His-455 domains catalyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide binding domain have been unsuccessful. The object of the present study is to demonstrate Mg(II) activation of catalysis at the ATP/Pi active site, to identify the residues at the ATP/Pi active site that contribute to catalysis, and to identify roles for these residues based on their positions within the active site scaffold. First, Mg(II) activation studies of catalysis of E + ATP + Pi →E-P + AMP + PPi partial reaction were carried out using a truncation mutant (Tem533) in which the C-terminal domain is absent. The kinetics show that a minimum of 2 Mg(II) per active site is required for the reaction. The active site residues used for substrate/cofactor binding/activation were identified by site-directed mutagenesis. Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive; Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and Thr-253 and Gln-240 mutants were almost fully active. The participation of the nucleotide ribose 2′-OH and α-P in enzyme binding is indicated by the loss of productive binding seen with substrate analogs modified at these positions. The ATP, Pi, and Mg(II) ions were docked into the PPDK N-terminal domain crevice, in an orientation consistent with substrate/cofactor binding modes observed for other members of the ATP-Grasp fold enzyme superfamily and consistent with the structure-function data. On the basis of this docking model, the ATP polyPhosphate moiety is oriented/activated for pyrophosphoryl transfer through interaction with Lys-22 (γ-P), Arg-92 (α-P), and the Gly-101 to Met-103 loop (γ-P) as well as with the Mg(II) cofactors. The Pi is oriented/activated for partial reaction 2 through interaction with Arg-337 and a Mg(II) cofactor. The Mg(II) ions are bound through interaction with Asp-321, Glu-323, and Gln-335 and substrate. Residues Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of an active site loop, over the nucleotide ribose-binding site.
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Separate site catalysis by Pyruvate Phosphate Dikinase as revealed by deletion mutants.
Biochemistry, 1995Co-Authors: Marielena Mcguire, Debra Dunaway-mariano, Brian M. MartinAbstract:Previous studies had indicated that Pyruvate Phosphate Dikinase (PPDK), an enzyme which catalyzes the interconversion of adenosine 5'-triPhosphate (ATP), orthoPhosphate (P(i)), and Pyruvate with adenosine 5'-monoPhosphate (AMP), pyroPhosphate (PP(i)), and phosphoenolPyruvate (PEP), is made up of 25, 13, 18, and 35 kDa domains [Carroll, L. J., Xu, Y., Thrall, S. H., Martin, B. M. & Dunaway-Mariano, D. (1994) Biochemistry 33, 1134]. The catalytic histidine (which mediates the phosphoryl group transfers from ATP to P(i) and Pyruvate) is located on the 18 kDa domain while the 25 and 13 kDa domains appear to contain the ATP binding site and the 35 kDa domain appears to contain the Pyruvate binding site, respectively. The goal of this investigation was to examine functional interdependency of the putative ATP and Pyruvate binding domains. Two truncated forms of PPDK were created by using recombinant DNA techniques. The 35 kDa (C-terminal) deletion mutant was found to catalyze the E+ATP+P(i) E-P+AMP+PP(i) partial reaction but not the E-P+Pyruvate E+PEP partial reaction. The 25 kDa (N-terminal) deletion mutant was found to catalyze the E-P+Pyruvate E+PEP partial reaction but not the E+ATP+P(i) E-P+AMP+PP(i) partial reaction. Neither mutant catalyzes the full ATP+P(i)+Pyruvate AMP+PP(i)+PEP reaction. These results are interpreted to mean that the ATP and Pyruvate binding domains in PPDK are functionally independent, thus providing evidence for separate active sites for catalysis of the two partial reactions.
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Location of the catalytic site for phosphoenolPyruvate formation within the primary structure of Clostridium symbiosum Pyruvate Phosphate Dikinase. 1. Identification of an essential cysteine by chemical modification with [1-14C]bromoPyruvate and site
Biochemistry, 1995Co-Authors: Linda Yankie, Debra Dunaway-mariano, Patrick S. Mariano, Li Shen, Young-shik Jung, Brian M. MartinAbstract:Pyruvate Phosphate Dikinase (PPDK) catalyzes the interconversion of adenosine 5'-triPhosphate (ATP), orthoPhosphate (Pi), and Pyruvate with adenosine 5'-monoPhosphate (AMP), pyroPhosphate (PPi), and phosphoenolPyruvate (PEP). The reaction takes place according to the following steps: (1) E+ATP+P(i) E-PP.AMP.P(i), (2) E-PP.AMP.P(i) E-P+AMP+PP(i), and (3) E-P+Pyruvate E+PEP, where E represents free enzyme; E-PP, pyrophosphorylenzyme; and E-P, phosphorylenzyme. Steps 1 and 2 comprise the nucleotide partial reaction, and step 3 comprises the Pyruvate partial reaction. The present studies were carried out to locate amino acid residues within the primary structure of Clostridium symbiosum PPDK participating in the catalysis of the Pyruvate partial reaction. The enzyme was treated with the affinity label [1-14C]bromoPyruvate, reduced with NaBH4, proteolyzed with trypsin, and chromatographed on an HPLC column. The radiolabeled tryptic peptide isolate was sequenced to reveal Cys 831 as the site of alkylation. Using PCR techniques Cys 831 was replaced by Ala, and the C831A PPDK mutant formed was then subjected to kinetic analysis. Rapid quench studies of single turnover reactions on the enzyme showed that the mutant is as efficient as wild-type PPDK in catalyzing the nucleotide partial reaction while it is unable to catalyze the Pyruvate partial reaction. These results were interpreted as evidence for a role of Cys 831 in Pyruvate/PEP binding and/or catalysis.
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Substrate binding domains in Pyruvate Phosphate Dikinase.
Biochemistry, 1994Co-Authors: Lawrence J. Carroll, Sara H. Thrall, Brian M. Martin, Debra Dunaway-marianoAbstract:Proteolysis of Clostridium symbiosum Pyruvate Phosphate Dikinase (PPDK) in its free or phosphorylated state with subtilisin Carlsberg followed two different cleavage pathways. The major pathway involved initial cleavage of the holoenzyme (93 kDa) into a stable 25-kDa N-terminal fragment and transiently stable 67-kDa C-terminal fragment. The 67-kDa fragment was cleaved to generate a stable 35-kDa fragment and an unstable 30-kDa fragment (containing the catalytic histidine). Proteolytic cleavage via the minor pathway divided the holoenzyme into an unstable 37-kDa N-terminal piece (which was further cleaved to the stable 25-kDa fragment produced in the major pathway) and a transiently stable 55-kDa C-terminal fragment. The 55-kDa fragment was then cleaved to produce the stable 35-kDa fragment produced by the major pathway. The cleavage pattern of PPDK complexed with the ATP analog adenyl imidodiPhosphate was identical to that of the free enzyme, only the rate of cleavage as slower. In contrast, proteolysis of the phosphorylenzyme-oxalate complex generated the 55-kDa fragment indicating that oxalate binding induces a change in protein conformation. Treatment of PPDK with [1-14C]bromoPyruvate followed by proteolysis revealed selective radiolabeling of the stable 35-kDa fragment while similar experiments with [14C]2',3'-dialdehyde adenosine 5'-monoPhosphate resulted in selective radiolabeling of the stable 25-kDa fragment. These results were interpreted to suggest that PPDK contains several structural domains and that the catalytic histidine, the Pyruvate binding site, and the ATP binding site may be located on different domains.
Frédéric Bringaud - One of the best experts on this subject based on the ideXlab platform.
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contribution of Pyruvate Phosphate Dikinase in the maintenance of the glycosomal atp adp balance in the trypanosoma brucei procyclic form
Journal of Biological Chemistry, 2014Co-Authors: Kamel Deramchia, Pauline Morand, Marc Biran, Yoann Millerioux, Muriel Mazet, Marion Wargnies, Jean-michel Franconi, Frédéric BringaudAbstract:Trypanosoma brucei belongs to a group of protists that sequester the first six or seven glycolytic steps inside specialized peroxisomes, named glycosomes. Because of the glycosomal membrane impermeability to nucleotides, ATP molecules consumed by the first glycolytic steps need to be regenerated in the glycosomes by kinases, such as phosphoenolPyruvate carboxykinase (PEPCK). The glycosomal Pyruvate Phosphate Dikinase (PPDK), which reversibly converts phosphoenolPyruvate into Pyruvate, could also be involved in this process. To address this question, we analyzed the metabolism of the main carbon sources used by the procyclic trypanosomes (glucose, proline, and threonine) after deletion of the PPDK gene in the wild-type (Δppdk) and PEPCK null (Δppdk/Δpepck) backgrounds. The rate of acetate production from glucose is 30% reduced in the Δppdk mutant, whereas threonine-derived acetate production is not affected, showing that PPDK function in the glycolytic direction with production of ATP in the glycosomes. The Δppdk/Δpepck mutant incubated in glucose as the only carbon source showed a 3.8-fold reduction of the glycolytic rate compared with the Δpepck mutant, as a consequence of the imbalanced glycosomal ATP/ADP ratio. The role of PPDK in maintenance of the ATP/ADP balance was confirmed by expressing the glycosomal phosphoglycerate kinase (PGKC) in the Δppdk/Δpepck cell line, which restored the glycolytic flux. We also observed that expression of PGKC is lethal for procyclic trypanosomes, as a consequence of ATP depletion, due to glycosomal relocation of cytosolic ATP production. This illustrates the key roles played by glycosomal and cytosolic kinases, including PPDK, to maintain the cellular ATP/ADP homeostasis.
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Contribution of Pyruvate Phosphate Dikinase in the maintenance of the glycosomal ATP/ADP balance in the Trypanosoma brucei procyclic form*
The Journal of biological chemistry, 2014Co-Authors: Kamel Deramchia, Pauline Morand, Marc Biran, Yoann Millerioux, Muriel Mazet, Marion Wargnies, Jean-michel Franconi, Frédéric BringaudAbstract:Trypanosoma brucei belongs to a group of protists that sequester the first six or seven glycolytic steps inside specialized peroxisomes, named glycosomes. Because of the glycosomal membrane impermeability to nucleotides, ATP molecules consumed by the first glycolytic steps need to be regenerated in the glycosomes by kinases, such as phosphoenolPyruvate carboxykinase (PEPCK). The glycosomal Pyruvate Phosphate Dikinase (PPDK), which reversibly converts phosphoenolPyruvate into Pyruvate, could also be involved in this process. To address this question, we analyzed the metabolism of the main carbon sources used by the procyclic trypanosomes (glucose, proline, and threonine) after deletion of the PPDK gene in the wild-type (Δppdk) and PEPCK null (Δppdk/Δpepck) backgrounds. The rate of acetate production from glucose is 30% reduced in the Δppdk mutant, whereas threonine-derived acetate production is not affected, showing that PPDK function in the glycolytic direction with production of ATP in the glycosomes. The Δppdk/Δpepck mutant incubated in glucose as the only carbon source showed a 3.8-fold reduction of the glycolytic rate compared with the Δpepck mutant, as a consequence of the imbalanced glycosomal ATP/ADP ratio. The role of PPDK in maintenance of the ATP/ADP balance was confirmed by expressing the glycosomal phosphoglycerate kinase (PGKC) in the Δppdk/Δpepck cell line, which restored the glycolytic flux. We also observed that expression of PGKC is lethal for procyclic trypanosomes, as a consequence of ATP depletion, due to glycosomal relocation of cytosolic ATP production. This illustrates the key roles played by glycosomal and cytosolic kinases, including PPDK, to maintain the cellular ATP/ADP homeostasis.
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Pyruvate Phosphate Dikinase and pyroPhosphate metabolism in the glycosome of trypanosoma cruzi epimastigotes
Comparative Biochemistry and Physiology B, 2004Co-Authors: Héctor Acosta, Michel Dubourdieu, Frédéric Bringaud, Wilfredo Quiñones, Ana J. Cáceres, Juan Luis ConcepciónAbstract:Abstract Pyruvate Phosphate Dikinase (PPDK) was recently reported in trypanosomatids, but its metabolic function is not yet known. The present work deals with the cellular localization and the function of the Trypanosoma cruzi enzyme. First, we show by digitonin titration and cell fractionation that the enzyme was essentially present in the glycosome matrix of the epimastigote form. Second, we address the issue of the direction of the reaction inside the glycosome for one part, our bibliographic survey evidenced a quite exergonic ΔG°′ (at least −5.2 kcal/mol at neutral pH and physiologic ionic strength); for another part, no pyrophosphatase (PPase) could be detected in fractions corresponding to the glycosomes; therefore, glycosomal PPDK likely works in the direction of Pyruvate production. Third, we address the issue of the origin of the glycosomal pyroPhosphate (PPi): several synthetic pathways known to produce PPi are already considered to be glycosomal. This work also indicates the presence of an NADP+-dependent β-oxidation of palmitoyl-CoA in the glycosome. Several Pyruvate-consuming activities, in particular alanine dehydrogenase (ADH) and Pyruvate carboxylase (PC), were detected in the glycosomal fraction. PPDK appears therefore as a central enzyme in the metabolism of the glycosome of T. cruzi by providing a link between glycolysis, fatty acid oxidation and biosynthetic PPi-producing pathways. Indeed, PPDK seems to replace pyrophosphatase in its classical thermodynamic role of displacing the equilibrium of PPi-producing reactions, as well as in its role of eliminating the toxic PPi.
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Crystallization and preliminary crystallographic investigation of glycosomal Pyruvate Phosphate Dikinase from Trypanosoma brucei
Acta Crystallographica Section D Biological Crystallography, 2000Co-Authors: Lawrence W. Cosenza, Frédéric Bringaud, Théo Baltz, Frédéric M. D. VellieuxAbstract:The PPi-dependent glycosomal enzyme Pyruvate Phosphate Dikinase (PPDK) from Trypanosoma brucei is expressed in the insect stage of the parasite. Its precise function there is still unclear, but the enzyme may catalyze the `reverse reaction' of transfer of Phosphate from phosphoenolPyruvate (PEP) to generate Pyruvate as a means of scavenging large amounts of pyroPhosphate. This protein may represent a target for drug design against diseases caused by trypanosomes and related kinetoplastids. The recombinant protein is 918 amino acids long (predicted molecular mass ≃ 100 kDa and pI = 8.9). Crystallization conditions for the recombinant PPDK are reported that result in crystals that diffract X-rays to better than 3.0 A resolution. Their space group is P21212, with unit-cell parameters a = 121.17, b = 153.5, c = 65.46 A, α = β = γ = 90°. The crystals, like the protein in solution, are sensitive to temperature and fail to diffract or diffract only to low resolution after ageing for two weeks or longer.
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Crystallization and preliminary crystallographic investigation of glycosomal Pyruvate Phosphate Dikinase from Trypanosoma brucei.
Acta crystallographica. Section D Biological crystallography, 2000Co-Authors: Lawrence W. Cosenza, Frédéric Bringaud, Théo Baltz, Frédéric M. D. VellieuxAbstract:The PP(i)-dependent glycosomal enzyme Pyruvate Phosphate Dikinase (PPDK) from Trypanosoma brucei is expressed in the insect stage of the parasite. Its precise function there is still unclear, but the enzyme may catalyze the 'reverse reaction' of transfer of Phosphate from phosphoenolPyruvate (PEP) to generate Pyruvate as a means of scavenging large amounts of pyroPhosphate. This protein may represent a target for drug design against diseases caused by trypanosomes and related kinetoplastids. The recombinant protein is 918 amino acids long (predicted molecular mass approximately 100 kDa and pI = 8.9). Crystallization conditions for the recombinant PPDK are reported that result in crystals that diffract X-rays to better than 3.0 A resolution. Their space group is P2(1)2(1)2, with unit-cell parameters a = 121.17, b = 153.5, c = 65.46 A, alpha = beta = gamma = 90 degrees. The crystals, like the protein in solution, are sensitive to temperature and fail to diffract or diffract only to low resolution after ageing for two weeks or longer.
Osnat Herzberg - One of the best experts on this subject based on the ideXlab platform.
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Swiveling Domain Mechanism in Pyruvate Phosphate Dikinase
Biochemistry, 2007Co-Authors: Kap Lim, Randy J. Read, Celia C. H. Chen, Aleksandra Tempczyk, Min Wei, Debra Dunaway-mariano, Osnat HerzbergAbstract:Pyruvate Phosphate Dikinase (PPDK) catalyzes the reversible conversion of phosphoenolPyruvate (PEP), AMP, and Pi to Pyruvate and ATP. The enzyme contains two remotely located reaction centers: the nucleotide partial reaction takes place at the N-terminal domain, and the PEP/Pyruvate partial reaction takes place at the C-terminal domain. A central domain, tethered to the N- and C-terminal domains by two closely associated linkers, contains a phosphorylatable histidine residue (His455). The molecular architecture suggests a swiveling domain mechanism that shuttles a phosphoryl group between the two reaction centers. In an early structure of PPDK from Clostridium symbiosum, the His445-containing domain (His domain) was positioned close to the nucleotide binding domain and did not contact the PEP/Pyruvate-binding domain. Here, we present the crystal structure of a second conformational state of C. symbiosum PPDK with the His domain adjacent to the PEP-binding domain. The structure was obtained by producing a...
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Pyruvate Site of Pyruvate Phosphate Dikinase: Crystal Structure of the Enzyme−PhosphonoPyruvate Complex, and Mutant Analysis†,‡
Biochemistry, 2002Co-Authors: Osnat Herzberg, Celia C. H. Chen, Aleksandra Tempczyk, Min Wei, Sijiu Liu, Andrew Howard, Debra Dunaway-marianoAbstract:Crystals of Pyruvate Phosphate Dikinase in complex with a substrate analogue inhibitor, phosphonoPyruvate (Ki = 3 μM), have been obtained in the presence of Mg2+. The structure has been determined and refined at 2.2 A resolution, revealing that the Mg2+-bound phosphonoPyruvate binds in the α/β-barrel's central channel, at the C-termini of the β-strands. The mode of binding resembles closely the previously proposed PEP substrate binding mode, inferred by the homology of the structure (but not sequence homology) to Pyruvate kinase. Kinetic analysis of site-directed mutants, probing residues involved in inhibitor binding, showed that all mutations resulted in inactivation, confirming the key role that these residues play in catalysis. Comparison between the structure of the PPDK−phosphonoPyruvate complex and the structures of two complexes of Pyruvate kinase, one with Mg2+-bound phospholactate and the other with Mg2+-oxalate and ATP, revealed that the two enzymes share some key features that facilitate commo...
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Pyruvate site of Pyruvate Phosphate Dikinase crystal structure of the enzyme phosphonoPyruvate complex and mutant analysis
Biochemistry, 2002Co-Authors: Osnat Herzberg, Celia C. H. Chen, Aleksandra Tempczyk, Min Wei, Sijiu Liu, Andrew Howard, Debra DunawaymarianoAbstract:Crystals of Pyruvate Phosphate Dikinase in complex with a substrate analogue inhibitor, phosphonoPyruvate (Ki = 3 μM), have been obtained in the presence of Mg2+. The structure has been determined and refined at 2.2 A resolution, revealing that the Mg2+-bound phosphonoPyruvate binds in the α/β-barrel's central channel, at the C-termini of the β-strands. The mode of binding resembles closely the previously proposed PEP substrate binding mode, inferred by the homology of the structure (but not sequence homology) to Pyruvate kinase. Kinetic analysis of site-directed mutants, probing residues involved in inhibitor binding, showed that all mutations resulted in inactivation, confirming the key role that these residues play in catalysis. Comparison between the structure of the PPDK−phosphonoPyruvate complex and the structures of two complexes of Pyruvate kinase, one with Mg2+-bound phospholactate and the other with Mg2+-oxalate and ATP, revealed that the two enzymes share some key features that facilitate commo...
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Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum Pyruvate Phosphate Dikinase.
The Journal of biological chemistry, 2001Co-Authors: Min Wei, Osnat Herzberg, Brian M. Martin, Marielena Mcguire, Kui Huang, Geeta Kapadia, Debra Dunaway-marianoAbstract:Abstract Pyruvate Phosphate Dikinase (PPDK) catalyzes the interconversion of ATP, Pi, and Pyruvate with AMP, PPi, and phosphoenolPyruvate (PEP) in three partial reactions as follows: 1) E-His + ATP →E-His-PP·AMP; 2) E-His-PP·AMP + Pi → E-His-P·AMP·PPi; and 3)E-His-P + Pyruvate → E·PEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecutive N-terminal, central His-455, and C-terminal domains. The N-terminal and central His-455 domains catalyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide binding domain have been unsuccessful. The object of the present study is to demonstrate Mg(II) activation of catalysis at the ATP/Pi active site, to identify the residues at the ATP/Pi active site that contribute to catalysis, and to identify roles for these residues based on their positions within the active site scaffold. First, Mg(II) activation studies of catalysis of E + ATP + Pi →E-P + AMP + PPi partial reaction were carried out using a truncation mutant (Tem533) in which the C-terminal domain is absent. The kinetics show that a minimum of 2 Mg(II) per active site is required for the reaction. The active site residues used for substrate/cofactor binding/activation were identified by site-directed mutagenesis. Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive; Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and Thr-253 and Gln-240 mutants were almost fully active. The participation of the nucleotide ribose 2′-OH and α-P in enzyme binding is indicated by the loss of productive binding seen with substrate analogs modified at these positions. The ATP, Pi, and Mg(II) ions were docked into the PPDK N-terminal domain crevice, in an orientation consistent with substrate/cofactor binding modes observed for other members of the ATP-Grasp fold enzyme superfamily and consistent with the structure-function data. On the basis of this docking model, the ATP polyPhosphate moiety is oriented/activated for pyrophosphoryl transfer through interaction with Lys-22 (γ-P), Arg-92 (α-P), and the Gly-101 to Met-103 loop (γ-P) as well as with the Mg(II) cofactors. The Pi is oriented/activated for partial reaction 2 through interaction with Arg-337 and a Mg(II) cofactor. The Mg(II) ions are bound through interaction with Asp-321, Glu-323, and Gln-335 and substrate. Residues Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of an active site loop, over the nucleotide ribose-binding site.
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Location of the Phosphate binding site within Clostridium symbiosum Pyruvate Phosphate Dikinase.
Biochemistry, 1998Co-Authors: Marielena Mcguire, Osnat Herzberg, Kui Huang, Geeta Kapadia, Debra Dunaway-marianoAbstract:Pyruvate Phosphate Dikinase (PPDK) catalyzes the interconversion of ATP, Pi, and Pyruvate with AMP, PPi, and PEP in three partial reactions: (1) E + ATP f E‚ATP f E-PP‚AMP, (2) E-PP‚ AMP + Pi f E-PP‚AMP‚Pi f E-P‚AMP‚PPi, and (3) E-P + Pyruvate f E-P‚Pyruvate f E‚PEP. The Clostridium symbiosum PPDK structure consists of N-terminal, central, and C-terminal domains. The N-terminal and central domains catalyze partial reactions 1 and 2 whereas the C-terminal and central domains catalyze partial reaction 3. The goal of the present work is to determine where on the N-terminal domain catalysis of partial reactions 1 and 2 occurs and, in particular, where the P i binding site is located. Computer modeling studies implicated Arg337 as a key residue for Pi binding. This role was tested by site-directed mutagenesis. The R337A PPDK was shown to be impaired in catalysis of the forward (kcat 300-fold lower) and reverse (kcat 30-fold lower) full reactions. Time courses for the single turnover reactions were measured to show that catalysis of partial reaction 1 is 5-fold slower in the mutant, catalysis of the second partial reaction is 140-fold slower in the mutant, and catalysis of the third partial reaction is unaffected. With the exception of the mutation site, the crystal structure of the R337A PPDK closely resembles the structure of the wild-type protein. Thus, the altered kinetic properties observed for this mutant are attributed solely to the elimination of the interaction between substrate and the guanidinium group of the Arg337 side chain. On the basis of these findings we propose that the Pi binding site is located within the crevice of the PPDK N-terminal domain, at a site that is flanked by the ATP ‚-P and the Mg 2+ cofactor.
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Disruption of Pyruvate Phosphate Dikinase in Brucella ovis PA CO2-dependent and independent strains generates attenuation in the mouse model
Veterinary Research, 2020Co-Authors: Nieves Vizcaíno, Lara Pérez-etayo, Raquel Conde-Álvarez, Maite Iriarte, Ignacio Moriyón, Amaia Zúñiga-ripaAbstract:AbstractBrucella ovis is a non-zoonotic rough Brucella that causes genital lesions, abortions and increased perinatal mortality in sheep and is responsible for important economic losses worldwide. Research on virulence factors of B. ovis is necessary for deciphering the mechanisms that enable this facultative intracellular pathogen to establish persistent infections and for developing a species-specific vaccine, a need in areas where the cross-protecting ovine smooth B. melitensis Rev1 vaccine is banned. Although several B. ovis virulence factors have been identified, there is little information on its metabolic abilities and their role in virulence. Here, we report that deletion of Pyruvate Phosphate Dikinase (PpdK, catalyzing the bidirectional conversion Pyruvate ⇌ phosphoenolPyruvate) in B. ovis PA (virulent and CO2-dependent) impaired growth in vitro. In cell infection experiments, although showing an initial survival higher than that of the parental strain, this ppdK mutant was unable to multiply. Moreover, when inoculated at high doses in mice, it displayed an initial spleen colonization higher than that of the parental strain followed by a marked comparative decrease, an unusual pattern of attenuation in mice. A homologous mutant was also obtained in a B. ovis PA CO2-independent construct previously proposed for developing B. ovis vaccines to solve the problem that CO2-dependence represents for large scale production. This CO2-independent ppdK mutant reproduced the growth defect in vitro and the multiplication/clearance pattern in mouse spleens, and is thus an interesting vaccine candidate for the immunoprophylaxis of B. ovis ovine brucellosis.
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disruption of Pyruvate Phosphate Dikinase in brucella ovis pa co 2 dependent and independent strains generates attenuation in the mouse model
Veterinary Research, 2020Co-Authors: Nieves Vizcaíno, Maite Iriarte, Ignacio Moriyón, Lara Perezetayo, Raquel Condealvarez, Amaia ZunigaripaAbstract:Brucella ovis is a non-zoonotic rough Brucella that causes genital lesions, abortions and increased perinatal mortality in sheep and is responsible for important economic losses worldwide. Research on virulence factors of B. ovis is necessary for deciphering the mechanisms that enable this facultative intracellular pathogen to establish persistent infections and for developing a species-specific vaccine, a need in areas where the cross-protecting ovine smooth B. melitensis Rev1 vaccine is banned. Although several B. ovis virulence factors have been identified, there is little information on its metabolic abilities and their role in virulence. Here, we report that deletion of Pyruvate Phosphate Dikinase (PpdK, catalyzing the bidirectional conversion Pyruvate ⇌ phosphoenolPyruvate) in B. ovis PA (virulent and CO2-dependent) impaired growth in vitro. In cell infection experiments, although showing an initial survival higher than that of the parental strain, this ppdK mutant was unable to multiply. Moreover, when inoculated at high doses in mice, it displayed an initial spleen colonization higher than that of the parental strain followed by a marked comparative decrease, an unusual pattern of attenuation in mice. A homologous mutant was also obtained in a B. ovis PA CO2-independent construct previously proposed for developing B. ovis vaccines to solve the problem that CO2-dependence represents for large scale production. This CO2-independent ppdK mutant reproduced the growth defect in vitro and the multiplication/clearance pattern in mouse spleens, and is thus an interesting vaccine candidate for the immunoprophylaxis of B. ovis ovine brucellosis.
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The Fast-Growing Brucella suis Biovar 5 Depends on PhosphoenolPyruvate Carboxykinase and Pyruvate Phosphate Dikinase but Not on Fbp and GlpX Fructose-1,6-Bisphosphatases or Isocitrate Lyase for Full Virulence in Laboratory Models.
Frontiers in microbiology, 2018Co-Authors: Amaia Zúñiga-ripa, Raquel Conde-Álvarez, Maite Iriarte, Thibault Barbier, Jean-jacques Letesson, Leticia Lázaro-antón, María J. De Miguel, Pilar M. Muñoz, Ignacio MoriyónAbstract:Bacteria of the genus Brucella infect a range of vertebrates causing a worldwide extended zoonosis. The best-characterized brucellae infect domestic livestock, behaving as stealthy facultative intracellular parasites. This stealthiness depends on envelope molecules with reduced pathogen-associated molecular patterns, as revealed by the low lethality and ability to persist in mice of these bacteria. Infected cells are often engorged with brucellae without signs of distress, suggesting that stealthiness could also reflect an adaptation of the parasite metabolism to use local nutrients without harming the cell. To investigate this, we compared key metabolic abilities of B. abortus 2308 Wisconsin (2308W), a cattle biovar 1 virulent strain, and B. suis 513, the reference strain of the ancestral biovar 5 found in wild rodents. B. suis 513 used a larger number of C substrates and showed faster growth rates in vitro, two features similar to those of B. microti, a species phylogenomically close to B. suis biovar 5 that infects voles. However, whereas B. microti shows enhanced lethality and reduced persistence in mice, B. suis 513 was similar to B. abortus 2308W in this regard. Mutant analyses showed that B. suis 513 and B. abortus 2308W were similar in that both depend on phosphoenolPyruvate synthesis for virulence but not on the classical gluconeogenic fructose-1,6-bisphosphatases Fbp-GlpX or on isocitrate lyase (AceA). However, B. suis 513 used Pyruvate Phosphate Dikinase (PpdK) and phosphoenolPyruvate carboxykinase (PckA) for phosphoenolPyruvate synthesis in vitro while B. abortus 2308W used only PpdK. Moreover, whereas PpdK dysfunction causes attenuation of B. abortus 2308W in mice, in B. suis 513 attenuation occurred only in the double PckA-PpdK mutant. Also contrary to what occurs in B. abortus 2308, a B. suis 513 malic enzyme (Mae) mutant was not attenuated, and this independence of Mae and the role of PpdK was confirmed by the lack of attenuation of a double Mae-PckA mutant. Altogether, these results decouple fast growth rates from enhanced mouse lethality in the brucellae, suggest that an Fbp-GlpX-independent gluconeogenic mechanism is ancestral in this group and show differences in central C metabolic steps that may reflect a progressive adaptation to intracellular growth.
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The Fast-Growing Brucella suis Biovar 5 Depends on PhosphoenolPyruvate Carboxykinase and Pyruvate Phosphate Dikinase but Not on Fbp and GlpX Fructose-1,6-Bisphosphatases or Isocitrate Lyase for Full Virulence in Laboratory Models
Frontiers Media S.A., 2018Co-Authors: Amaia Zúñiga-ripa, Raquel Conde-Álvarez, Maite Iriarte, Thibault Barbier, Jean-jacques Letesson, Leticia Lázaro-antón, María J. De Miguel, Pilar M. Muñoz, Ignacio MoriyónAbstract:Bacteria of the genus Brucella infect a range of vertebrates causing a worldwide extended zoonosis. The best-characterized brucellae infect domestic livestock, behaving as stealthy facultative intracellular parasites. This stealthiness depends on envelope molecules with reduced pathogen-associated molecular patterns, as revealed by the low lethality and ability to persist in mice of these bacteria. Infected cells are often engorged with brucellae without signs of distress, suggesting that stealthiness could also reflect an adaptation of the parasite metabolism to use local nutrients without harming the cell. To investigate this, we compared key metabolic abilities of Brucella abortus 2308 Wisconsin (2308W), a cattle biovar 1 virulent strain, and B. suis 513, the reference strain of the ancestral biovar 5 found in wild rodents. B. suis 513 used a larger number of C substrates and showed faster growth rates in vitro, two features similar to those of B. microti, a species phylogenomically close to B. suis biovar 5 that infects voles. However, whereas B. microti shows enhanced lethality and reduced persistence in mice, B. suis 513 was similar to B. abortus 2308W in this regard. Mutant analyses showed that B. suis 513 and B. abortus 2308W were similar in that both depend on phosphoenolPyruvate synthesis for virulence but not on the classical gluconeogenic fructose-1,6-bisphosphatases Fbp-GlpX or on isocitrate lyase (AceA). However, B. suis 513 used Pyruvate Phosphate Dikinase (PpdK) and phosphoenolPyruvate carboxykinase (PckA) for phosphoenolPyruvate synthesis in vitro while B. abortus 2308W used only PpdK. Moreover, whereas PpdK dysfunction causes attenuation of B. abortus 2308W in mice, in B. suis, 513 attenuation occurred only in the double PckA-PpdK mutant. Also contrary to what occurs in B. abortus 2308, a B. suis 513 malic enzyme (Mae) mutant was not attenuated, and this independence of Mae and the role of PpdK was confirmed by the lack of attenuation of a double Mae-PckA mutant. Altogether, these results decouple fast growth rates from enhanced mouse lethality in the brucellae and suggest that an Fbp-GlpX-independent gluconeogenic mechanism is ancestral in this group and show differences in central C metabolic steps that may reflect a progressive adaptation to intracellular growth
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Brucella abortus Depends on Pyruvate Phosphate Dikinase and Malic Enzyme but Not on Fbp and GlpX Fructose-1,6-Bisphosphatases for Full Virulence in Laboratory Models
Journal of bacteriology, 2014Co-Authors: Amaia Zúñiga-ripa, Raquel Conde-Álvarez, Maite Iriarte, Thibault Barbier, Estrella Martínez-gómez, Leyre Palacios-chaves, Yolanda Gil-ramírez, María-jesús Grilló, Jean-jacques Letesson, Ignacio MoriyónAbstract:The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular parasites and thus are able to adjust their metabolism to the extra- and intracellular environments encountered during an infectious cycle. However, this aspect of Brucella biology is imperfectly understood, and the nutrients available in the intracellular niche are unknown. Here, we investigated the central pathways of C metabolism used by Brucella abortus by deleting the putative fructose-1,6-bisphosphatase (fbp and glpX), phosphoenolPyruvate carboxykinase (pckA), Pyruvate Phosphate Dikinase (ppdK), and malic enzyme (mae) genes. In gluconeogenic but not in rich media, growth of ΔppdK and Δmae mutants was severely impaired and growth of the double Δfbp-ΔglpX mutant was reduced. In macrophages, only the ΔppdK and Δmae mutants showed reduced multiplication, and studies with the ΔppdK mutant confirmed that it reached the replicative niche. Similarly, only the ΔppdK and Δmae mutants were attenuated in mice, the former being cleared by week 10 and the latter persisting longer than 12 weeks. We also investigated the glyoxylate cycle. Although aceA (isocitrate lyase) promoter activity was enhanced in rich medium, aceA disruption had no effect in vitro or on multiplication in macrophages or mouse spleens. The results suggest that B. abortus grows intracellularly using a limited supply of 6-C (and 5-C) sugars that is compensated by glutamate and possibly other amino acids entering the Krebs cycle without a critical role of the glyoxylate shunt.
