Mapping the functional synthetase domain of trypanothione synthetase from Leishmania major

Abstract

Trypanothione synthetase (TryS) is a protein unique to the Kinetoplastida and has been shown to be essential in Trypanosoma brucei by RNAi techniques [1,2]. TryS is the sole enzyme responsible for the biosynthesis of trypanothione in the human pathogenic parasites T. brucei, Trypanosoma cruzi and Leishmania major [3–5]. The protein is thought to have two domains: an ATP-dependent synthetase domain that generates the intermediate, glutathionylspermidine, and the final product, trypanothione, from glutathione and spermidine; and an amidase domain, which can hydrolyse glutathionylspermidine and trypanothione to the original substrates. There has only been one previous study that succeeded in resolving these domains from the homologous enzyme glutathionylspermidine synthetase (GspS) from Escherichia coli [6]. GspS typically shares about 48% amino acid similarity and 32% identity with the TryS proteins from the various species. To date, the threedimensional crystal structure for GspS and TryS has not been elucidated and although a short communication outlining the optimized NMR conditions for the N-terminal amidase domain of GspS from E. coli was published a number of years ago [7], no subsequent data have been released. Little is known about the active sites of TryS, although site-directed mutagenesis has highlighted an essential Cys residue for the amidase activity of GspS from Crithidia fasciculata which is conserved across all species of GspS and TryS [8]. In addition, the important roles of Arg-553 and Arg-613 for synthetase activity were recently identified in TryS from C. fasciculata [9]. Our attempts to crystallise the full length TryS from the various species of trypanosomatids have so far been met with limited suc