Two phylogenetically divergent isocitrate dehydrogenases are encoded in Leishmania parasites. Molecular and functional characterization of Leishmania mexicana isoenzymes with specificity towards NAD+ and NADP+

Abstract

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 Km 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.