A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion.
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.
Saito
Tomoya
T
Department of Tropical Medicine and Parasitology, Keio University School of Medicine, 35 Shinanomachi, Tokyo 160-8582, Japan. asait@sc.itc.keio.ac.jp
Nishi
Manami
M
Lim
Muoy I
MI
Wu
Bo
B
Maeda
Takuya
T
Hashimoto
Hisayuki
H
Takeuchi
Tsutomu
T
Roos
David S
DS
Asai
Takashi
T
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*
D020033:Protein Isoforms; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D011770:Pyruvate Kinase