先前已证明恶性疟原虫(Pf)4-硝基苯磷酸酶参与维生素B1的代谢。这里,进行BLASTp搜索,我们发现来自Pf的4-硝基苯磷酸酶与来自小鼠的磷酸乙酸磷酸酶(PGP)具有显著的同源性,人类,和酵母,促使我们重新研究疟原虫酶的生化特性。因为重组PfPGP酶是不溶的,我们对来自伯氏疟原虫(Pb)的重组表达和纯化同源物进行了扩展的底物筛选和广泛的生化表征,导致鉴定2-磷酸乙二醇酯和2-磷酸-L-乳酸为PbPGP的相关生理底物。2-磷酸乙醇酸在受损DNA末端的修复过程中产生,2-磷酸-L-乳酸是丙酮酸激酶副反应的产物,两者都能有效抑制两种关键的糖酵解酶,磷酸三糖异构酶和磷酸果糖激酶。因此,PGP介导的这些毒性代谢物的清除对于细胞存活和功能至关重要。我们的结果与之前的研究有很大不同,其中PfPGP酶已被推断作用于2-磷酸-D-乳酸盐而不作用于L异构体。除了通过直接的体外酶测定来解决底物特异性冲突外,我们在伯氏疟原虫中进行了PGP基因敲除研究,证实这种保守的代谢校对酶在疟原虫中至关重要,我们的研究结果证实PbPGP是伯氏疟原虫正常生理功能的必需酶,并提示特异性抑制疟原虫PGP的药物有望用于抗疟疾治疗.
Plasmodium falciparum (Pf) 4-nitrophenylphosphatase has been shown previously to be involved in vitamin B1 metabolism. Here, conducting a BLASTp search, we found that 4-nitrophenylphosphatase from Pf has significant homology with phosphoglycolate phosphatase (PGP) from mouse, human, and yeast, prompting us to reinvestigate the biochemical properties of the Plasmodium enzyme. Because the recombinant PfPGP enzyme is insoluble, we performed an extended substrate screen and extensive biochemical characterization of the recombinantly expressed and purified homolog from Plasmodium berghei (Pb), leading to the identification of 2-phosphoglycolate and 2-phospho-L-lactate as the relevant physiological substrates of PbPGP. 2-Phosphoglycolate is generated during repair of damaged DNA ends, 2-phospho-L-lactate is a product of pyruvate kinase side reaction, and both potently inhibit two key glycolytic enzymes, triosephosphate isomerase and phosphofructokinase. Hence, PGP-mediated clearance of these toxic metabolites is vital for cell survival and functioning. Our results differ significantly from those in a previous study, wherein the PfPGP enzyme has been inferred to act on 2-phospho-D-lactate and not on the L isomer. Apart from resolving the substrate specificity conflict through direct in vitro enzyme assays, we conducted PGP gene knockout studies in P. berghei, confirming that this conserved metabolic proofreading enzyme is essential in Plasmodium In summary, our findings establish PbPGP as an essential enzyme for normal physiological function in P. berghei and suggest that drugs that specifically inhibit Plasmodium PGP may hold promise for use in anti-malarial therapies.