A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion

Tomoya Saito, Manami Nishi, Muoy I. Lim, Bo Wu, Takuya Maeda, Hisayuki Hashimoto, Tsutomu Takeuchi, David S. Roos, Takashi Asai

Research output: Contribution to journalArticle

35 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)14041-14052
Number of pages12
JournalJournal of Biological Chemistry
Volume283
Issue number20
DOIs
Publication statusPublished - 2008 May 16

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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