A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion |
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Authors: | Saito Tomoya Nishi Manami Lim Muoy I Wu Bo Maeda Takuya Hashimoto Hisayuki Takeuchi Tsutomu Roos David S Asai Takashi |
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Institution: | Department of Tropical Medicine and Parasitology, Keio University School of Medicine, 35 Shinanomachi, Tokyo 160-8582, Japan. asait@sc.itc.keio.ac.jp |
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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. |
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