A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion
A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion
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DOI：
10.1074/jbc.m709015200
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发表时间：
2008-05-16
影响因子：
4.8
通讯作者：
Asai, Takashi
中科院分区：
文献类型：
--
作者：
Saito, Tomoya;Nishi, Manami;Asai, Takashi
我们以前报道了弓形虫胞浆型丙酮酸激酶(EC2.7.1.40)，它不同于大多数真核丙酮酸激酶，它受葡萄糖6-磷酸而不是1，6-二磷酸果糖的调节。从寄生虫基因组中鉴定出另一种可能的丙酮酸激酶(TgPyKII)，其氨基酸序列与TgPyKI的同源性为32%，并保留了丙酮酸激酶的特征基序和底物结合和催化所必需的氨基酸。虽然TgPyKI与植物/藻类酶关系最密切，但系统发育分析表明TgPyKII起源于变形细菌。重组TgPyKII的酶学性质表明，其最适pH为8.5，并且偏爱GDP作为磷酸盐受体。催化活性与K+无关，在1，6-二磷酸果糖、2，6-二磷酸果糖、6-磷酸葡萄糖、5-磷酸核糖、AMP或ATP存在时，没有观察到变构或调节作用。与TgPyKI不同，天然的TgPyKII活性仅与弓形虫速殖子裂解物的膜部分有关。TgPyKII具有一个长的N端延伸，在保守区之前含有5个假定的起始密码子，并通过天然抗体的免疫荧光分析和N端延伸的荧光蛋白融合定位于质外体和线粒体。进一步的缺失和定点突变表明，来自第一个Met的翻译产物负责定位到顶体，而来自第三个Met的一个翻译产物负责线粒体的定位。这是第一次在任何系统中对潜在的线粒体丙酮酸激酶进行研究。
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.