J Biol Chem. 2008 Mar 6 [Epub ahead of print]
A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion
Saito T, Nishi M, Lim MI, Wu B, Maeda T, Hashimoto H, Takeuchi T, Roos DS, Asai T.
Department of Tropical Medicine and Parasitology, Keio University School of Medicine, Tokyo 1608582.
We previously reported a cytosolic pyruvate kinase (EC 184.108.40.206) 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. While 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 suggest that a translate from 1st methionine (Met) is responsible for the localization to the apicoplast whereas one from 3rd Met is for the mitochondrion. This is the first report of a potential mitochondrial pyruvate kinase in any system.
PMID: 18326043 [PubMed - as supplied by publisher]