Abstract
The trypanothione synthetase (TryS) catalyses the two-step biosynthesis of trypanothione from spermidine and glutathione and is an attractive new drug target for the development of trypanocidal and antileishmanial drugs, especially since the structural information of TryS from Leishmania major has become available. Unfortunately, the TryS structure was solved without any of the substrates and lacks loop regions that are mechanistically important. This contribution describes docking and molecular dynamics simulations that led to further insights into trypanothione biosynthesis and, in particular, explains the binding modes of substrates for the second catalytic step. The structural model essentially confirm previously proposed binding sites for glutathione, ATP and two Mg2+ ions, which appear identical for both catalytic steps. The analysis of an unsolved loop region near the proposed spermidine binding site revealed a new pocket that was demonstrated to bind glutathionylspermidine in an inverted orientation. For the second step of trypanothione synthesis glutathionylspermidine is bound in a way that preferentially allows N1-glutathionylation of N8-glutathionylspermidine, classifying N8-glutathionylspermidine as the favoured substrate. By inhibitor docking, the binding site for N8-glutathionylspermidine was characterised as druggable.
Highlights
The protozoan parasites of the genera Trypanosoma and Leishmania cause neglected diseases such as Chagas’ disease, African sleeping sickness or the various forms of Leishmaniasis, which in toto still account for about 100 000 fatalities per annum [1]
As first discovered in Crithidia fasciculate [8], T(SH)2 is the proximal reductant for thioredoxinrelated proteins called tryparedoxins, which in turn reduce a variety of tryparedoxin peroxidases, which may belong to the peroxiredoxin or the glutathione peroxidase family, the ribonucleotide reductase [10] and proteins implicated in proliferative control [11]
The synthetase domains of the enzymes were superposed by MOE [24] and the Gsp analogue, ADP and both Mg2+ ions were transferred from the E. coli glutathionylspermidine synthetase (EcGspS) structure into the LmTryS model and subjected to energy minimisation by the pertinent MOE function and MFF94x force field with relaxation of surrounding residues
Summary
The protozoan parasites of the genera Trypanosoma and Leishmania cause neglected diseases such as Chagas’ disease, African sleeping sickness or the various forms of Leishmaniasis, which in toto still account for about 100 000 fatalities per annum [1] (http://www.who.int/mediacentre/factsheets/fs259/en/index. html). TryS model, this loop appears to build an additional pocket close to the site where the spermidine moiety of the Gsp-analogue binds.
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