title: 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.
authors.affiliation: Department of Tropical Medicine and Parasitology, Keio University School of Medicine, 35 Shinanomachi, Tokyo 160-8582, Japan. asait@sc.itc.keio.ac.jp
authors.forename: Tomoya
authors.initials: T
authors.lastname: Saito
authors.forename: Manami
authors.initials: M
authors.lastname: Nishi
authors.forename: Muoy I
authors.initials: MI
authors.lastname: Lim
authors.forename: Bo
authors.initials: B
authors.lastname: Wu
authors.forename: Takuya
authors.initials: T
authors.lastname: Maeda
authors.forename: Hisayuki
authors.initials: H
authors.lastname: Hashimoto
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Takeuchi
authors.forename: David S
authors.initials: DS
authors.lastname: Roos
authors.forename: Takashi
authors.initials: T
authors.lastname: Asai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006153:Guanosine Diphosphate / Q000737:chemistry*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011485:Protein Binding; D020033:Protein Isoforms; D011770:Pyruvate Kinase / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014122:Toxoplasma / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D011770:Pyruvate Kinase