Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológica (IQUIFIB-CONICET), Junín 956, C1113AAD, Buenos Aires, Argentina.
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológica (IQUIFIB-CONICET), Junín 956, C1113AAD, Buenos Aires, Argentina.
Mol Biochem Parasitol. 2020 Nov;240:111320. doi: 10.1016/j.molbiopara.2020.111320. Epub 2020 Sep 24.
Leishmania parasites are of great relevance to public health because they are the causative agents of various long-term and health-threatening diseases in humans. Dependent on the manifestation, drugs either require difficult and lengthy administration, are toxic, expensive, not very effective or have lost efficacy due to the resistance developed by these pathogens against clinical treatments. The intermediary metabolism of Leishmania parasites is characterized by several unusual features, among which whether the Krebs cycle operates in a cyclic and/or in a non-cyclic mode is included. Our survey of the genomes of Leishmania species and monoxenous parasites such as those of the genera Crithidia and Leptomonas (http://www.tritrypdb.org) revealed that two genes encoding putative isocitrate dehydrogenases (IDHs) -with distantly related sequences- are strictly conserved among these parasites. Thus, in this study, we aimed to functionally characterize the two leishmanial IDH isoenzymes, for which we selected the genes LmxM10.0290 (Lmex_IDH-90) and LmxM32.2550 (Lmex_IDH-50) from L. mexicana. Phylogenetic analysis showed that Lmex_IDH-50 clustered with members of Subfamily I, which contains mainly archaeal and bacterial IDHs, and that Lmex_IDH-90 was a close relative of eukaryotic enzymes comprised within Subfamily II IDHs. 3-D homology modeling predicted that both IDHs exhibited the typical folding motifs recognized as canonical for prokaryotic and eukaryotic counterparts, respectively. Both IDH isoforms displayed dual subcellular localization, in the cytosol and the mitochondrion. Kinetic studies showed that Lmex_IDH-50 exclusively catalyzed the reduction of NAD, while Lmex_IDH-90 solely used NADP as coenzyme. Besides, Lmex_IDH-50 differed from Lmex_IDH-90 by exhibiting a nearly 20-fold lower apparent K value towards isocitrate (2.0 μM vs 43 μM). Our findings showed, for the first time, that the genus Leishmania differentiates not only from other trypanosomatids such as Trypanosoma cruzi and Trypanosoma brucei, but also from most living organisms, by exhibiting two functional homo-dimeric IDHs, highly specific towards NAD and NADP, respectively. It is tempting to argue that any or both types of IDHs might be directly or indirectly linked to the Krebs cycle and/or to the de novo synthesis of glutamate. Our results about the biochemical and structural features of leishmanial IDHs show the relevance of deepening our knowledge of the metabolic processes in these pathogenic parasites to potentially identify new therapeutic targets.
利什曼原虫寄生虫对公共健康具有重要意义，因为它们是人类各种长期和威胁健康疾病的病原体。根据表现形式，药物要么需要困难和漫长的给药，要么具有毒性、昂贵、效果不佳，或者由于这些病原体对临床治疗产生的耐药性而失去疗效。利什曼原虫的中间代谢具有几个不寻常的特征，其中包括克雷布斯循环是否以循环和/或非循环模式运行。我们对利什曼物种和单生性寄生虫（如克鲁斯氏锥虫属和毛滴虫属）的基因组进行了调查（http://www.tritrypdb.org），结果表明，两种编码假定的异柠檬酸脱氢酶（IDHs）的基因——具有远缘相关的序列——在这些寄生虫中严格保守。因此，在这项研究中，我们旨在对两种利什曼原虫 IDH 同工酶进行功能表征，为此我们从 L. mexicana 中选择了 LmxM10.0290（Lmex_IDH-90）和 LmxM32.2550（Lmex_IDH-50）这两个基因。系统发育分析表明，Lmex_IDH-50 与亚科 I 的成员聚类，该亚科包含主要的古细菌和细菌 IDHs，而 Lmex_IDH-90 是真核酶的近亲，属于亚科 II IDHs。3-D 同源建模预测，两种 IDH 都表现出典型的折叠模体，分别被认为是原核和真核对应物的典型。两种 IDH 同工酶都具有双重亚细胞定位，存在于细胞质和线粒体中。动力学研究表明，Lmex_IDH-50 仅催化 NAD 的还原，而 Lmex_IDH-90 仅使用 NADP 作为辅酶。此外，Lmex_IDH-50 与 Lmex_IDH-90 不同，其对异柠檬酸的表观 K 值低近 20 倍（2.0 μM 对 43 μM）。我们的发现首次表明，属利什曼原虫不仅与克鲁斯氏锥虫和布氏锥虫等其他锥虫体不同，而且与大多数生物也不同，因为它具有两种功能同源二聚体 IDH，分别高度特异性地针对 NAD 和 NADP。人们不禁要争辩说，任何一种或两种 IDH 都可能直接或间接地与克雷布斯循环和/或谷氨酸的从头合成有关。我们关于利什曼原虫 IDH 的生化和结构特征的研究结果表明，深入了解这些致病寄生虫的代谢过程对于潜在地确定新的治疗靶点具有重要意义。
