Molecular and biochemical characterization of D-phosphoglycerate dehydrogenase from Entamoeba histolytica -: A unique enteric protozoan parasite that possesses both phosphorylated and nonphosphorylated serine metabolic pathways
Molecular and biochemical characterization of D-phosphoglycerate dehydrogenase from Entamoeba histolytica -: A unique enteric protozoan parasite that possesses both phosphorylated and nonphosphorylated serine metabolic pathways
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DOI：
10.1111/j.1432-1033.2004.04195.x
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发表时间：
2004-07-01
期刊：
影响因子：
--
通讯作者：
Nozaki, T
中科院分区：
文献类型：
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作者：
Ali, V;Hashimoto, T;Nozaki, T
一个假定的磷酸甘油酸脱氢酶（PGDH），催化D-磷酸甘油酸氧化为3-磷酸羟基丙酮酸在所谓的磷酸化丝氨酸代谢途径，从肠道原生动物寄生虫溶组织内阿米巴的特点。急诊溶组织菌PGDH基因（EhPGDH）编码299个氨基酸的蛋白质，计算分子量为33.5kDa，等电点为8.11。EhPGDH与拟杆菌和另一种肠道原生动物尾内甲藻的PGDH具有很高的同源性。EhPGDH缺乏羧基末端丝氨酸结合结构域和在核苷酸结合结构域中含有保守的Trp 139（大肠杆菌PGDH）的13-14个氨基酸区域，这些区域显示出对四聚体至关重要，其存在于包括高等真核生物在内的其他生物中。EhPGDH利用NADH和效率较低的NADPH催化磷酸羟基丙酮酸还原为磷酸甘油酸; EhPGDH不利用2-酮戊二酸。EhPGDH的动力学参数与哺乳动物PGDH相似，例如NADH辅因子的偏好性、底物特异性和盐可逆底物抑制。与来自细菌、植物和哺乳动物的PGDH相反，EhPGDH蛋白质作为同源二聚体存在，如通过凝胶过滤色谱所证明的。急诊溶组织菌溶胞产物含有PGDH活性，每分钟每mg溶胞产物蛋白质在相反方向上利用26 nmol NADH，其占总可溶性蛋白质的0.2-0.4%。总之，这种寄生虫代表了一种独特的单细胞原生生物，具有磷酸化和非磷酸化丝氨酸代谢途径，加强了丝氨酸代谢在这种生物体中的生物学重要性。氨基酸序列比较和系统发育分析表明，E. histolytica与另一种肠道原生动物纤毛E.尾状体和拟杆菌。
A putative phosphoglycerate dehydrogenase (PGDH), which catalyzes the oxidation of D-phosphoglycerate to 3-phosphohydroxypyruvate in the so-called phosphorylated serine metabolic pathway, from the enteric protozoan parasite Entamoeba histolytica was characterized. The E. histolytica PGDH gene (EhPGDH) encodes a protein of 299 amino acids with a calculated molecular mass of 33.5 kDa and an isoelectric point of 8.11. EhPGDH showed high homology to PGDH from bacteroides and another enteric protozoan ciliate, Entodinium caudatum. EhPGDH lacks both the carboxyl-terminal serine binding domain and the 13-14 amino acid regions containing the conserved Trp139 (of Escherichia coli PGDH) in the nucleotide binding domain shown to be crucial for tetramerization, which are present in other organisms including higher eukaryotes. EhPGDH catalyzed reduction of phosphohydroxypyruvate to phosphoglycerate utilizing NADH and, less efficiently, NADPH; EhPGDH did not utilize 2-oxoglutarate. Kinetic parameters of EhPGDH were similar to those of mammalian PGDH, for example the preference of NADH cofactor, substrate specificities and salt-reversible substrate inhibition. In contrast to PGDH from bacteria, plants and mammals, the EhPGDH protein is present as a homodimer as demonstrated by gel filtration chromatography. The E. histolytica lysate contained PGDH activity of 26 nmol NADH utilized per min per mg of lysate protein in the reverse direction, which consisted 0.2-0.4% of a total soluble protein. Altogether, this parasite represents a unique unicellular protist that possesses both phosphorylated and nonphosphorylated serine metabolic pathways, reinforcing the biological importance of serine metabolism in this organism. Amino acid sequence comparison and phylogenetic analysis of various PGDH sequences showed that E. histolytica forms a highly supported monophyletic group with another enteric protozoa, cilliate E. caudatum, and bacteroides.