Giordana Lucila, Sosa Máximo Hernán, Leroux Alejandro E, Mendoza Elkin F Rodas, Petray Patricia, Nowicki Cristina
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 Investigaciones Farmacológicas en alianza estratégica con UBA-CONICET (ININFA) Junín 956, C1113AAD, Buenos Aires, Argentina.
Mol Biochem Parasitol. 2018 Jan;219:67-76. doi: 10.1016/j.molbiopara.2017.11.001. Epub 2017 Nov 8.
Leishmania parasites cause a broad spectrum of clinical manifestations in humans and the available clinical treatments are far from satisfactory. Since these pathogens require large amounts of NADPH to maintain intracellular redox homeostasis, oxidoreductases that catalyze the production of NADPH are considered as potential drug targets against these diseases. In the sequenced genomes of most Leishmania spp. two putative malic enzymes (MEs) with an identity of about 55% have been identified. In this work, the ME from L. major (LmjF24.0770, Lmj_ME-70) and its less similar homolog from L. mexicana (LmxM.24.0761, Lmex_ME-61) were cloned and functionally characterized. Both MEs specifically catalyzed NADPH production, but only Lmex_ME-61 was activated by l-aspartate. Unlike the allosterically activated human ME, Lmex_ME-61 exhibited typical hyperbolic curves without any sign of cooperativity in the absence of l-aspartate. Moreover, Lmex_ME-61 and Lmj_ME-70 differ from higher eukaryotic homologs in that they display dimeric instead of tetrameric molecular organization. Homology modeling analysis showed that Lmex_ME-61 and Lmj_ME-70 notably differ in their surface charge distribution; this feature encompasses the coenzyme binding pockets as well. However, in both isozymes, the residues directly involved in the coenzyme binding exhibited a good degree of conservation. Besides, only Lmex_ME-61 and its closest homologs were immunodetected in cell-free extracts from L. mexicana, L. amazonensis and L. braziliensis promastigotes. Our findings provide a first glimpse into the biochemical properties of leishmanial MEs and suggest that MEs could be potentially related to the metabolic differences among the species of Leishmania parasites.
利什曼原虫寄生虫在人类中会引发广泛的临床表现，而现有的临床治疗方法远不能令人满意。由于这些病原体需要大量的烟酰胺腺嘌呤二核苷酸磷酸（NADPH）来维持细胞内的氧化还原稳态，催化NADPH生成的氧化还原酶被视为针对这些疾病的潜在药物靶点。在大多数利什曼原虫物种的测序基因组中，已鉴定出两种推定的苹果酸酶（MEs），它们的同一性约为55%。在这项研究中，克隆了来自硕大利什曼原虫的ME（LmjF24.0770，Lmj_ME - 70）及其来自墨西哥利什曼原虫的相似度较低的同源物（LmxM.24.0761，Lmex_ME - 61），并对其进行了功能表征。两种MEs都特异性地催化NADPH的生成，但只有Lmex_ME - 61被L - 天冬氨酸激活。与别构激活的人类ME不同，Lmex_ME - 61在没有L - 天冬氨酸的情况下呈现典型的双曲线，没有任何协同性的迹象。此外，Lmex_ME - 61和Lmj_ME - 70与高等真核生物同源物的不同之处在于它们呈现二聚体而非四聚体的分子结构。同源建模分析表明，Lmex_ME - 61和Lmj_ME - 70在其表面电荷分布上有显著差异；这一特征也包括辅酶结合口袋。然而，在这两种同工酶中，直接参与辅酶结合的残基表现出高度保守性。此外，在来自墨西哥利什曼原虫、亚马逊利什曼原虫和巴西利什曼原虫前鞭毛体的无细胞提取物中，仅免疫检测到Lmex_ME - 61及其最接近的同源物。我们的研究结果初步揭示了利什曼原虫MEs的生化特性，并表明MEs可能与利什曼原虫寄生虫物种间的代谢差异有关。
