Institute of Pharmacogenetics, University of Duisburg-Essen, Essen, Germany.
PLoS One. 2013;8(3):e58318. doi: 10.1371/journal.pone.0058318. Epub 2013 Mar 7.
Primaquine, an 8-aminoquinoline, is the only drug which cures the dormant hypnozoites of persistent liver stages from P. vivax. Increasing resistance needs the discovery of alternative pathways as drug targets to develop novel drug entities. Deoxyhypusine hydroxylase (DOHH) completes hypusine biosynthesis in eukaryotic initiation factor (eIF-5A) which is the only cellular protein known to contain the unusual amino acid hypusine. Modified EIF-5A is important for proliferation of the malaria parasite. Here, we present the first successful cloning and expression of DOHH from P. vivax causing tertiary malaria. The nucleic acid sequence of 1041 bp encodes an open reading frame of 346 amino acids. Histidine tagged expression of P. vivax DOHH detected a protein of 39.01 kDa in E. coli. The DOHH protein from P. vivax shares significant amino acid identity to the simian orthologues from P. knowlesi and P. yoelii strain H. In contrast to P. falciparum only four E-Z-type HEAT-like repeats are present in P. vivax DOHH with different homology to phycocyanin lyase subunits from cyanobacteria and in proteins participating in energy metabolism of Archaea and Halobacteria. However, phycocyanin lyase activity is absent in P. vivax DOHH. The dohh gene is present as a single copy gene and transcribed throughout the whole erythrocytic cycle. Specific inhibition of recombinant P. vivax DOHH is possible by complexing the ferrous iron with zileuton, an inhibitor of mammalian 5-lipoxygenase (5-LOX). Ferrous iron in the active site of 5-LOX is coordinated by three conserved histidines and the carboxylate of isoleucine(673). Zileuton inhibited the P. vivax DOHH protein with an IC50 of 12,5 nmol determined by a relative quantification by GC/MS. By contrast, the human orthologue is only less affected with an IC50 of 90 nmol suggesting a selective iron-complexing strategy for the parasitic enzyme.
伯氨喹啉是一种 8-氨基喹啉，是唯一能治愈间日疟原虫潜伏性肝期休眠合子的药物。为了提高耐药性，需要发现替代途径作为药物靶点，以开发新的药物实体。脱氧hypusine 羟化酶（DOHH）完成真核起始因子（eIF-5A）中的 hypusine 生物合成，eIF-5A 是唯一已知含有特殊氨基酸 hypusine 的细胞蛋白。修饰后的 eIF-5A 对疟原虫的增殖很重要。在这里，我们首次成功克隆并表达了引起间日疟的间日疟原虫的 DOHH。1041bp 的核酸序列编码一个 346 个氨基酸的开放阅读框。在大肠杆菌中表达的 DOHH 标记有组氨酸，检测到一个 39.01kDa 的蛋白。来自间日疟原虫的 DOHH 蛋白与来自食蟹猴疟原虫和约氏疟原虫 H 株的灵长类同源物有显著的氨基酸同一性。与恶性疟原虫不同的是，在间日疟原虫 DOHH 中只有四个 E-Z 型 HEAT 样重复存在，与蓝藻的藻蓝蛋白裂解酶亚基和参与古菌和盐杆菌能量代谢的蛋白具有不同的同源性。然而，间日疟原虫 DOHH 中不存在藻蓝蛋白裂解酶活性。dohh 基因是单拷贝基因，在整个红细胞周期中都有转录。通过与亚铁离子络合，间苯三酚可以特异性抑制重组间日疟原虫 DOHH 的活性，间苯三酚是哺乳动物 5-脂氧合酶（5-LOX）的抑制剂。5-LOX 活性中心的亚铁离子由三个保守的组氨酸和异亮氨酸（673）的羧基配位。通过相对定量 GC/MS 测定，间苯三酚对间日疟原虫 DOHH 蛋白的 IC50 为 12.5nmol。相比之下，人类同源物只有较低的影响，IC50 为 90nmol，这表明寄生虫酶具有选择性的铁络合策略。
