Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites

Maeda, Takeshi; Saito, Tomoya; Oguchi, Yoshiyo; Nakazawa, Miki; Takeuchi, Tsutomu

doi:10.1007/s00436-002-0739-8

Cited by 22 publications

(11 citation statements)

References 16 publications

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“…The enzyme was characterized enzymatically, with apparent K m , V max and Hill's coefficient ( n H ) values obtained for PEP (Table 1). The values here reported are in agreement with those previously reported for this enzyme [13].…”

Section: Resultssupporting

confidence: 93%

“…This was unexpected since this enzyme contains a glutamic acid at position 438, which has been considered a hallmark of unregulated enzymes like mammalian PK-M1 [13] (Fig. 4B).…”

Section: Discussionmentioning

confidence: 99%

“…Second, microorganisms that exhibit obligate or facultative anaerobic growth, including bacteria and yeast, possess allosterically regulated PK enzymes [9], [10]. Third, a variety of parasitic protozoans, including trypanosomatids and apicomplexans, express allosterically regulable forms of pyruvate kinase which allow them to adapt their glycolytic flux to the different cell environments encountered throughout their life cycle progression [3], [11], [12], [13].…”

Section: Introductionmentioning

confidence: 99%

“…Previous characterization of cytoplasmic T. gondii pyruvate kinase ( Tg PK1), using either parasite extracts [15] or a recombinant purified enzyme [13], indicated that this enzyme was allosterically activated by glucose 6-phosphate (G6P), which enhanced its activity under PEP unsaturated conditions. Since G6P is produced in the initial step of glycolysis by a hexokinase-catalyzed reaction, this would imply a different regulatory mechanism of the glycolytic flux in Toxoplasma parasites when compared to its mammalian hosts, which are typically allosterically regulated by a metabolite produced further down the pathway, namely FBP.…”

Section: Introductionmentioning

confidence: 99%

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The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

et al. 2010

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BackgroundPyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population.Methodology/Principal FindingsWe have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers.ConclusionsWe hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two α-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

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Section: Resultssupporting

confidence: 93%

“…This was unexpected since this enzyme contains a glutamic acid at position 438, which has been considered a hallmark of unregulated enzymes like mammalian PK-M1 [13] (Fig. 4B).…”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

et al. 2010

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“…Two types of pyruvate kinase were characterized in Toxoplasma gondii [1,2]. Pyruvate kinase type-II isozyme ( Tg PyKII) was localized in both the mitochondrion and the apicoplast, whereas pyruvate kinase type-I ( Tg PyKI) was located in the cytosol.…”

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confidence: 99%

Pyruvate kinase type-II isozyme in Plasmodium falciparum localizes to the apicoplast

Maeda

Saito

Harb

et al. 2009

Parasitology International

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Bioinformatics research on Plasmodium falciparum revealed two isoforms of pyruvate kinase: type-I and type-II enzymes. The type-I enzyme shows typical glycolytic properties, while type-II enzyme is involved in fatty acid type-II biosynthesis and has been predicted to localize to the apicoplast with the targeting signal in its N-terminus. The type-I and type-II isoforms have the same evolutionary origin as Toxoplasma gondii isozymes, TgPyKI and TgPyKII, respectively; however, TgPyKII localizes to both the mitochondrion and the apicoplast. Accordingly, we made a recombinant full length of P. falciparum pyruvate kinase type-II protein using a wheat germ cell-free expression system and obtained a specific antibody against the type-II protein. Fluorescent microscopic analysis revealed that P. falciparum type-II enzyme was localized only to the apicoplast, not to the mitochondrion. The data suggest differences in localization and metabolic pathways between P. falciparum and T. gondii pyruvate kinase isoforms.

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Energy Metabolism as an Antimalarial Drug Target

Jortzik

Becker

2011

Apicomplexan Parasites

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Expression and characterization of recombinant pyruvate kinase from Toxoplasma gondii tachyzoites

Cited by 22 publications

References 16 publications

The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

Pyruvate kinase type-II isozyme in Plasmodium falciparum localizes to the apicoplast

Energy Metabolism as an Antimalarial Drug Target

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