Mutational analysis of parasite trypanothione reductase: acquisition of glutathione reductase activity in a triple mutant

Abstract

African trypanosomes contain a cyclic derivative of oxidized glutathione, N1,N8-bis(glutathionyl)spermidine, termed trypanothione. This is the substrate for the parasite enzyme trypanothione reductase, a key enzyme in disulfide/dithiol redox balance and a target enzyme for trypanocidal therapy. Trypanothione reductase from these and related trypanosomatid parasites is structurally homologous to host glutathione reductase but the two enzymes show mutually exclusive substrate specificities. To assess the basis of host vs parasite enzyme recognition for their disulfide substrates, the interaction of bound glutathione with active-site residues in human red cell glutathione reductase as defined by prior X-ray analysis was used as the starting point for mutagenesis of three residues in trypanothione reductase from Trypanosoma congolense, a cattle parasite. Mutation of three residues radically alters enzyme specificity and permits acquisition of glutathione reductase activity at levels 10(4) higher than in wild-type trypanothione reductase.

Structure of glutathione reductase from escherichia coli at 1.86 Å resolution: Comparison with the enzyme from human erythrocytes

“…insertion of an intersubunit disulfide bridge (Scrutton et al, 1988), the identification of catalytically important residues (Deonarain et al, 1989; Scrutton et al, 1990aScrutton et al, , 1992, the switch of the coenzyme specificity from NADP to NAD (Scrutton et al, 1990b), and the specificity change from glutathione to trypanothione (Henderson et al, 1991) and the reverse (Sullivan et al, 1991). Because the 2 enzyme species have only 52% amino acid residues in common (Greer & Perham, 1986), understanding the engineering results on the GR,,, necessitates detailed structural knowledge of this enzyme.…”

“…Among them were the Reprint requests to: Georg E. Schulz, Institut fur Organische Chemie und Biochemie, Albertstr. 21, 79104 Freiburg im Breisgau, Germany; e-mail: schulz@bio2.chemie.uni-freiburg.de.Abbreviations: B-factor, crystallographic temperature factor; GK,,, glutathione reductase from Escherichia coli; GR,,,, human glutathione reductase; GSSG, oxidized glutathione; NCS, noncrystallographic symmetry; R-factor, crystallographic reliability factor; u, standard deviation; MIR, multiple isomorphous replacement.insertion of an intersubunit disulfide bridge (Scrutton et al, 1988), the identification of catalytically important residues (Deonarain et al, 1989; Scrutton et al, 1990aScrutton et al, , 1992, the switch of the coenzyme specificity from NADP to NAD (Scrutton et al, 1990b), and the specificity change from glutathione to trypanothione (Henderson et al, 1991) and the reverse (Sullivan et al, 1991). Because the 2 enzyme species have only 52% amino acid residues in common (Greer & Perham, 1986), understanding the engineering results on the GR,,, necessitates detailed structural knowledge of this enzyme.…”

Substrate Specificity of Trypanothione Reductase

“…The K,,, value observed for trypanothione was 22 pM in potassium phosphate, pH 7.5. Values for the K,,, of trypanothione for 7: cruzi trypano- [23]). This finding suggests that both compounds bind, with the remaining glutamic acid residue in the Glu-I1 position, and that there is little preference for binding based on the asymmetry of the polyamine, in agreement with the results with nortrypanothione and homotrypanothione analogues described above.…”

“…Trypanothione reductase was assayed at 25°C in 50 mM potassium phosphate, pH 7.5, 1 mM EDTA and 100 pM NADPH in 1 ml. (Hepes buffer used in many other studies was avoided due to its inhibitory behaviour with trypanothione reductase [23]). The concentrations of trypanothione and all analogues were determined by end-point titrations with NADPH, assuming an c , , , for NADPH of 6.22mM -' cm-' [23].…”

“…(Hepes buffer used in many other studies was avoided due to its inhibitory behaviour with trypanothione reductase [23]). The concentrations of trypanothione and all analogues were determined by end-point titrations with NADPH, assuming an c , , , for NADPH of 6.22mM -' cm-' [23]. Initial-rate measurements were made at least in duplicate at six or more substrate concentrations, typically between 0.25 K,, and 5 K,,,.…”