Site-Directed Mutagenesis of the Redox-Active Cysteines of Trypanosoma cruzi Trypanothione Reductase

Abstract

The gene for trypanothione reductase from the Silvio strain of Trypanosoma cruzi has been cloned, sequenced and overexpressed in Escherichia coli using the constitutive lpp promoter on the expression plasmid pBSTNAV. Up to 13% of the total soluble protein is enzymically active trypanothione reductase with kinetic properties similar to the enzyme purified from T. cruzi. In order to assess the catalytic role of the putative active-site cysteine residues (C53 and C58), three mutant proteins have been constructed by site-directed mutagenesis substituting alanine or serine residues for cysteine; [C53A]trypanothione reductase, [C53S]trypanothione reductase and [C58S]trypanothione reductase. Although the purified, recombinant mutant proteins were catalytically inactive with NADPH and trypanothione disulphide as substrates, all showed comparable levels of transhydrogenase activity between NADPH and thio-NADP+, suggesting that the mutant proteins had correctly folded in vivo. All three mutants showed substantially different catalytic parameters for thio-NADP+ than the wild-type enzyme, presumably as a consequence of modifying the environment of the enzyme-bound flavin, thereby altering its chemical reactivity. The purified [C58S]trypanothione reductase showed spectral properties similar to the oxidised wild-type enzyme but, unlike the wild-type enzyme, did not acquire the characteristic charge-transfer complex of the EH2 form on addition of NADPH. In contrast, in the absence of NADPH both [C53A]trypanothione reductase and [C53S]trypanothione reductase showed spectral properties similar to the EH2 form of the wild-type enzyme. These data indicate that both C53 and C58 are essential for overall catalysis, with the thiolate anion of C58 interacting with the enzyme-bound FAD and C53 interacting with the disulphide substrate. These mutants should be useful in crystallographic studies of reaction intermediates which cannot be obtained with the catalytically active native enzyme.

Modeled structure of trypanothione reductase of Leishmania infantum

“…Instead, the metabolites trypanothione (T(SH)2) and N 1 -glutathionyl-spermidine (GspdSH) and the auxiliary enzyme TR maintain the reducing environment in protozoan cells. TR and GR are both homodimeric, with a subunit molecular weight of approximately 52 kDa, and catalyze the transfer of electrons from NADPH to their specific substrates via an FAD prosthetic group and a redox-active cysteine disulfide (3,4). TR and mammalian GR share approximately 40% sequence identity and are mutually exclusive with respect to disulfide substrate specificity (1), indicating a difference in substrate binding pocket geometry.…”

Leishmania major Elongation Factor 1B Complex Has Trypanothione S-Transferase and Peroxidase Activity

“…Linoleic acid hydroperoxide (13(S)-hydroperoxy-9(Z),11(E)-octadecadieonic acid) was synthesized using soybean lipoxidase and purified as described (30). Recombinant T. cruzi trypanothione reductase was purified as previously described (31). L. major Friedlin A1 clone promastigotes and C. fasciculata chanomastigotes were cultured as described (32).…”

Synthesis and evaluation of substrate analogue inhibitors of trypanothione reductase

“…Synthesis and evaluation of substrate analogue inhibitors of trypanothione reductase substrate, TR utilises a pair of active site cysteine residues (Cys53 and Cys58), which undergo a series of disulfide exchanges with oxidised trypanothione, ultimately releasing reduced substrate and leaving the active site cysteine pair oxidised as the disulfide 20,21 . In turn, NAPDH provides the reducing power to return the enzyme to its reduced state.…”