From the Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bengaluru, Karnataka 560064, India.
From the Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bengaluru, Karnataka 560064, India
J Biol Chem. 2018 Apr 20;293(16):5878-5894. doi: 10.1074/jbc.M117.816298. Epub 2018 Feb 15.
(Pf), the causative agent of malaria, has an iron-sulfur cluster-containing class I fumarate hydratase (FH) that catalyzes the interconversion of fumarate to malate, a well-known reaction in the tricarboxylic acid cycle. In humans, the same reaction is catalyzed by class II FH that has no sequence or structural homology with the class I enzyme from Fumarate is generated in large quantities in the parasite as a by-product of AMP synthesis and is converted to malate by FH and then used in the generation of the key metabolites oxaloacetate, aspartate, and pyruvate. Previous studies have identified the FH reaction as being essential to , but biochemical characterization of PfFH that may provide leads for the development of specific inhibitors is lacking. Here, we report on the kinetic characterization of purified recombinant PfFH, functional complementation of deficiency in , and mitochondrial localization in the parasite. We found that the substrate analog mercaptosuccinic acid is a potent PfFH inhibitor, with a value in the nanomolar range. The gene could not be knocked out in when transfectants were introduced into BALB/c mice; however, knockout was successful when C57BL/6 mice were used as host, suggesting that the essentiality of the gene to the parasite was mouse strain-dependent.
疟原虫（Pf）含有一个含铁硫簇的 class I 富马酸水合酶（FH），该酶可催化富马酸向苹果酸的转化，这是三羧酸循环中的一个熟知反应。在人类中，相同的反应由没有与来自 Pf 的 class I 酶的序列或结构同源性的 class II FH 催化。富马酸在寄生虫中大量产生，作为 AMP 合成的副产物，然后由 FH 转化为苹果酸，然后用于关键代谢物草酰乙酸、天冬氨酸和丙酮酸的生成。先前的研究已经确定 FH 反应对 Pf 至关重要，但是 PfFH 的生化特征分析缺乏可能为开发特异性抑制剂提供线索。在这里，我们报告了纯化的重组 PfFH 的动力学特征、在 Pf 中的功能互补以及在寄生虫中的线粒体定位。我们发现，底物类似物巯基琥珀酸是一种有效的 PfFH 抑制剂，其 值在纳摩尔范围内。当转染子被引入 BALB/c 小鼠时，无法敲除 基因；然而，当使用 C57BL/6 小鼠作为宿主时， 基因的敲除是成功的，这表明该基因对寄生虫的必需性依赖于小鼠品系。
