PMID:18326043

Citation	Saito, T, Nishi, M, Lim, MI, Wu, B, Maeda, T, Hashimoto, H, Takeuchi, T, Roos, DS and Asai, T (2008) A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion. J. Biol. Chem. 283:14041-52
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.
Links	PubMed PMC2376232 Online version:10.1074/jbc.M709015200
Keywords	Amino Acid Sequence; Animals; Guanosine Diphosphate/chemistry; Kinetics; Mitochondria/metabolism; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; Phylogeny; Protein Binding; Protein Isoforms; Pyruvate Kinase/chemistry; Recombinant Proteins/chemistry; Sequence Homology, Amino Acid; Toxoplasma/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
TOXGO:B1B1P9	part_of	GO:0005739: mitochondrion	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete
TOXGO:B1B1P9	part_of	GO:0020011: apicoplast	ECO:0000314: direct assay evidence used in manual assertion		C		Seeded From UniProt	complete
TOXGO:B1B1P9		GO:0020011: apicoplast	ECO:0000314:		C		Figure 4	complete CACAO 8381
TOXGO:B1B1P9		GO:0005739: mitochondrion	ECO:0000314:		C		Figure 4	complete CACAO 8382

References

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