A Novel GDP-dependent Pyruvate Kinase Isozyme from Toxoplasma gondii Localizes to Both the Apicoplast and the Mitochondrion

Abstract

We previously reported a cytosolic pyruvate kinase (EC 2.7.1.40) from Toxoplasma gondii (TgPyKI) that differs from most eukaryotic pyruvate kinases in being regulated by glucose 6-phosphate rather than fructose 1,6-diphosphate. Another putative pyruvate kinase (TgPyKII) was identified from parasite genome, which exhibits 32% amino acid sequence identity to TgPyKI and retains pyruvate kinase signature motifs and amino acids essential for substrate binding and catalysis. Whereas TgPyKI is most closely related to plant/algal enzymes, phylogenetic analysis suggests a proteobacterial origin for TgPyKII. Enzymatic characterization of recombinant TgPyKII shows a high pH optimum at 8.5, and a preference for GDP as a phosphate recipient. Catalytic activity is independent of K ؉ , and no allosteric or regulatory effects were observed in the presence of fructose 1,6-diphosphate, fructose 2,6-diphosphate, glucose 6-phosphate, ribose 5-phosphate, AMP, or ATP. Unlike TgPyKI, native TgPyKII activity was exclusively associated with the membranous fraction of a T. gondii tachyzoite lysate. TgPyKII possesses a long N-terminal extension containing five putative start codons before the conserved region and localizes to both apicoplast and mitochondrion by immunofluorescence assay using native antibody and fluorescent protein fusion to the N-terminal extension. Further deletional and site-directed mutagenesis suggests that a translation product from 1st Met is responsible for the localization to the apicoplast, whereas one from 3rd Met is for the mitochondrion. This is the first study of a potential mitochondrial pyruvate kinase in any system. Toxoplasma gondii is an obligate intracellular protozoan parasite of warm-blooded animals, including humans (1). Although normally asymptomatic, toxoplasmosis is a significant problem in pregnant women infected early during gestation, immunocompromised individuals, and livestock. This parasite is a member of the phylum Apicomplexa, which includes many other parasites such as Plasmodium species responsible for malaria. Glucose is thought to be the main source of energy for the rapidly multiplying forms of both Toxoplasma and Plasmodium, which use the Embden-Meyerhof pathway for glycolytic phosphorylation (2).Pyruvate kinase catalyzes the essentially irreversible transphosphorylation from phosphoenolpyruvate (PEP) 4 to ADP-producing pyruvate (3). In most mammals and bacteria, pyruvate kinase is allosterically regulated by fructose 1,6-diphosphate (4) and thus plays a regulatory role in glycolysis. The product pyruvate feeds into many metabolic pathways, placing pyruvate kinase at a crucial metabolic intersection. Many organisms express multiple pyruvate kinase isozymes with different kinetic properties. For example, Escherichia coli bears two isozymes, type I and II, both of which are homotropically activated by the substrate PEP. The type I isozyme is also activated heterotropically by fructose 1,6-diphosphate and is inhibited by ATP (5), whereas the type II isozyme is activated by AMP and mo...

Fatty acid metabolism in the Plasmodium apicoplast: Drugs, doubts and knockouts

“…The apicoplast localization of the P. falciparum and T. gondii PKII have been confirmed by antibody labeling and epitope tagging [115,120], and the activity of T. gondii enzyme has been demonstrated in vitro [62]. Interestingly, the TgPKII displayed an exclusive preference for GDP over ADP as a phosphate acceptor [62], distinguishing it from most other pyruvate kinases. However, as TgPKII was additionally targeted to the mitochondrion [62], it is not clear how closely this substrate preference will be shared with the Plasmodium enzyme.…”

“…Like the pPTs, PKII is expected to support multiple aspects of apicoplast metabolism, providing pyruvate for both the FASII and DOXP pathways and ATP for a range of enzymatic reactions [112,117]. The apicoplast localization of the P. falciparum and T. gondii PKII have been confirmed by antibody labeling and epitope tagging [115,120], and the activity of T. gondii enzyme has been demonstrated in vitro [62]. Interestingly, the TgPKII displayed an exclusive preference for GDP over ADP as a phosphate acceptor [62], distinguishing it from most other pyruvate kinases.…”

“…In efforts to develop more potent inhibitors, a myriad of studies were then undertaken in both Plasmodium and T. gondii. Numerous compounds were tested for inhibition of parasite growth , and many FASII enzymes were characterized [49,52,55,[62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79], allowing further optimization of lead compounds against their putative targets. These studies identified several promising anti-malarial drug candidates, some of which displayed activity against bloodstage parasites at nanomolar concentrations [54,59].…”

Rewiring and regulation of cross-compartmentalized metabolism in protists

“…The retention of a mitochondrial TPI-GAPDH in oomycetes could therefore be leftover from a time when mitochondrial and plastid metabolisms were coordinately regulated in a common ancestor of oomycetes and diatoms. A GDP-dependent mitochondrial PYK has also been characterized in the apicomplexan parasite Toxoplasma gondii, but the significance of this finding is unknown (Saito et al 2008). It is important to remember that the key feature that differentiates T. gondii and the stramenopiles from animals, plants, yeast and potentially C. reinhardtii, where glycolytic enzymes readily associate with the outer mitochondrial membrane (Nakashima et al 1986;Giege et al 2003;Brandina et al 2006;Graham et al 2007;Atteia et al 2009), is that in T. gondii and the stramenopiles some glycolytic enzymes are mitochondrial matrix targeted, rather than being associated with the mitochondrial outer membrane via VDAC homologues.…”

Dual targeting of aminoacyl-tRNA synthetases to the mitochondrion and complex plastid in chlorarachniophytes

“…1A), we propose that alternatively targeted proteins must be generated by translation initiation variants: one product translated from the first Met codon is co-translationally transported to the plastid via the ER, and another product translated from the second Met codon is post-translationally imported into the mitochondrion. A similar situation is found in pyruvate kinase from the apicomplexan parasite Toxoplasma gondii (Saito et al, 2008), which has a non-photosynthetic secondary plastid of red algal origin, suggesting this may be a common solution to dual targeting in complex plastid-bearing organisms. However, ambiguous targeting sequence for mitochondria and secondary plastids have also been identified for T. gondii superoxide (A,B) The topologies correspond to the best-scoring ML tree for HisRSs (A) and GlyRSs (B), as obtained with RAxML.…”