LeishmaniaTDR1 structure, a unique trimeric glutathione transferase capable of deglutathionylation and antimonial prodrug activation

Abstract

Thiol-dependent reductase I (TDR1), an enzyme found in parasitic Leishmania species and Trypanosoma cruzi, is implicated in deglutathionylation and activation of antimonial prodrugs used to treat leishmaniasis. The 2.3 Å resolution structure of TDR1 reveals a unique trimer of subunits each containing two glutathione-S-transferase (GST) domains. The similarities of individual domains and comparisons with GST classes suggest that TDR1 evolved by gene duplication, diversification, and gene fusion; a combination of events previously unknown in the GST protein superfamily and potentially explaining the distinctive enzyme properties of TDR1. The deglutathionylation activity of TDR1 implies that glutathione itself has regulatory intracellular roles in addition to being a precursor for trypanothione, the major low mass thiol present in trypanosomatids. We propose that activation of antiparasite Sb (V)-drugs is a legacy of the deglutathionylation activity of TDR1 and involves processing glutathione adducts with concomitant reduction of the metalloid to active Sb(III) species.thioltransferase | crystal structure | protozoan biology | redox metabolism T he kinetoplastid parasites Leishmania species and Trypanosoma cruzi infect humans, proliferate intracellularly, and cause the leishmaniases and Chagas disease, respectively. These parasites possess a thiol-dependent reductase known as thiol-dependent reductase I (TDR1) (1) or, specifically in T. cruzi, Tc52 (2). In Leishmania, TDR1 is implicated in redox regulation and in mediating susceptibility to the antimonial prodrugs Pentostam and Glucantime that represent frontline treatments (3). The therapeutic activity of these drugs follows reduction of the relatively inert pentavalent metalloid to more toxic trivalent species. This process occurs slowly in vitro in the presence of low molecular mass thiols such as glutathione (GSH) or the trypanosomatid-specific polyamine-GSH conjugate called trypanothione [TðSHÞ 2 ], especially at low pH as found in the parasitophorous vacuole in which Leishmania resides intracellularly in macrophages (4, 5). However, TDR1, in the presence of glutathione, catalyzes the reduction of Sb(V) in vitro and hence can mediate activation of the antimonial prodrugs (1). This finding correlates with the observation that in Leishmania major the enzyme is significantly more abundant in the form of the parasite (the amastigote) that infects the mammalian host than in the promastigote or insect form and that the amastigotes are strikingly more sensitive to Sb(V) than promastigotes (1, 6).TDR1 belongs to the glutathione-S-transferase (GST) superfamily (1), members of which contribute to varied, important biological events, including xenobiotic detoxification, stress response, and signaling processes. GSTs are classified into distinct subgroups on the basis of sequence, structure, immunological properties, and type of substrate (7-10). However, TDR1 has distinctive and unique properties (1). For example, the enzyme consists of fused GST-type domains for which t...

Multi-analytical platform metabolomic approach to study miltefosine mechanism of action and resistance in Leishmania

“…Polyamine biosynthetic pathway provides spermidine for trypanothione biosynthesis, via conjugation with glutathione to produce glutathionyl-spermidine and trypanothione, the main anti-oxidant of the parasites against host cell oxidative burst [67], although recently, the importance of glutathione as a substantial player in the overall redox power of the parasite has been proposed [68]. Interestingly, variation in total level and the reduced: oxidized ratio of trypanothione is a typical response in antimonial treatment [69], also detected in a CE metabolomic study [56], which is in tune with partial crossresistance between these two drugs [61].…”

Interaction of ascaridole, carvacrol, and caryophyllene oxide from essential oil of Chenopodium ambrosioides L. with mitochondria in Leishmania and other eukaryotes

“…In addition, Leishmania can synthesize ascorbate as a powerful antioxidant (Manhas, Anand, Tripathi, & Madhubala, ). Therefore, we assessed the redox state of thiols in the presence of Asc, Car, and Caryo by a CMFDA assay (Sarkar et al, ) resulting in a glutathione S ‐transferase (Fyfe, Westrop, Silva, Coombs, & Hunter, ) catalyzed conjugation of MF to low molecular weight thiols. As shown, these findings are in line with superoxide radical measurements.…”

Oxidative Stress in Human Infectious Diseases – Present and Current Knowledge About Its Druggability

“…Recently it has been indicated that thiols act as reducing agents in this conversion. Furthermore, the participation of a unique parasite-specific trimeric glutathione transferase TDR1 in the activation of antimonial prodrugs has been suggested (Fyfe et al, 2012).…”