The crystal structure of <i>Toxoplasma gondii</i> nucleoside triphosphate diphosphohydrolase 1 represents a conformational intermediate in the reductive activation mechanism of the tetrameric enzyme

Krug, Ulrike; Totzauer, R.; Sträter, Norbert

doi:10.1002/prot.24288

Cited by 10 publications

(9 citation statements)

References 16 publications

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“…The analysis of the crystal structure of Toxoplasma gondii NTPDase revealed that this enzyme suffers a reductive activation, where disulfide bridge reduction causes a conformational change and active site loop closure followed by correct positioning of the active site residues and enzymatic activation (Krug et al, 2013). In order to investigate whether the T. evansi enzyme follows the same mode of action, we have tested NTPDase activity in the presence and absence of reduction agent dithioerytrol (DTT) and confirmed the same velocity rates for both measurements, indicating that unlike the enzyme from T. gondii, the T. evansi enzyme does not follow a reductive activation mechanism.…”

Section: Discussionmentioning

confidence: 99%

Kinetic and biochemical characterization of Trypanosoma evansi nucleoside triphosphate diphosphohydrolase

Weiss

Batista

Wagner

et al. 2015

Experimental Parasitology

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Section: Discussionmentioning

confidence: 99%

Kinetic and biochemical characterization of Trypanosoma evansi nucleoside triphosphate diphosphohydrolase

Weiss

Batista

Wagner

et al. 2015

Experimental Parasitology

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“…In fact, this compound is able to activate the TgNTPase in vitro. Similarly, DTT has been also shown to activate the TgNTPase protein in vitro and to trigger egress of tachyzoites [ 57 , 58 ]. Although the processes governing egress are not fully understood, intracellular calcium levels appear to trigger the abrupt exit of parasites from the PV, which is accompanied by a rapid decrease in host cell ATP mediated by TgNTPase activation [ 59 ].…”

Section: Discussionmentioning

confidence: 99%

The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress

et al. 2016

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BackgroundNTPases (also NTPDases) are enzymes with apyrase activity. They are widely distributed among eukaryotes, and also among members of the family Sarcocystidae. In Toxoplasma gondii, the TgNTPase accumulates in the dense granules, and has been commonly associated with the strain virulence. In the closely related Neospora caninum, the NcNTPase lacks nucleoside diphosphate hydrolase activity and appears to be more abundant in virulent isolates, indicating that it may contribute to the pathogenicity of neosporosis. However, so far no additional information on NcNTPase has been provided.MethodsHerein, the NcNTPase coding sequences were analysed by different in silico and de novo sequencing approaches. A comparative analysis of NcNTPase and NcGRA7 in terms of protein dynamics, secretion, phosphorylation, and mRNA expression profiles during the tachyzoite lytic cycle was also carried out. Moreover, NcNTPase immunolocalization was analysed by confocal microscopy techniques over a set number of time-points.ResultsWe describe the presence of three different loci containing three copies of the NcNTPase within the Nc-Liv genome, and report the existence of up to four different NcNTPase alleles in Nc-Liv. We also provide evidence for the occurrence of diverse protein species of the NcNTPase by two-dimensional gel electrophoresis. Both NcNTPase and NcGRA7 were similarly up-regulated and secreted during the egress and/or early invasion phases, and were phosphorylated. However, its secretion was not affected by the addition of calcium modulators such as A23187 and ethanol. NcNTPase and NcGRA7 localized in dense granules and parasitophorous vacuole membrane throughout the lytic cycle, although differed in their inmunolocalization during early invasion and egress.ConclusionsThe present study reveals the complexity of the NcNTPase loci in N. caninum. We hypothesize that the expression of different isoforms of the NcNTPase protein could contribute to parasite virulence. Our findings showed regulation of expression, secretion and phosphorylation of NcNTPase suggesting a potential role for progression through the tachyzoites lytic cycle.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1620-4) contains supplementary material, which is available to authorized users.

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“…These enzymes occur in several organisms, including Toxoplasma gondii [3], T. cruzi [4], T. evansi [5], Trichomonas vaginalis [6], and Leishmania infantum [7]. NTPDase from parasites can hydrolyze extracellular nucleoside tri- and diphosphate, thereby affecting the host's immune response [8–10].…”

Section: Introductionmentioning

confidence: 99%

Immobilization of NTPDase-1 fromTrypanosoma cruziand Development of an Online Label-Free Assay

Lima

Oliveira

Mariotini-Moura

et al. 2016

Journal of Analytical Methods in Chemistry

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The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the TcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave K M of 0.317 ± 0.044 mmol·L−1, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L−1, which is in accordance with the data for the enzyme in solution.

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The crystal structure of Toxoplasma gondii nucleoside triphosphate diphosphohydrolase 1 represents a conformational intermediate in the reductive activation mechanism of the tetrameric enzyme

Cited by 10 publications

References 16 publications

Kinetic and biochemical characterization of Trypanosoma evansi nucleoside triphosphate diphosphohydrolase

Kinetic and biochemical characterization of Trypanosoma evansi nucleoside triphosphate diphosphohydrolase

The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress

Immobilization of NTPDase-1 fromTrypanosoma cruziand Development of an Online Label-Free Assay

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