Redox enzyme engineering: conversion of human glutathione reductase into a trypanothione reductase

Abstract

The substrate specificity of the human enzyme glutathione reductase was changed from its natural substrate glutathione to trypanothione [N1,N8-bis(glutathionyl)spermidine] by site-directed mutagenesis of two residues. The glutathione analogue, trypanothione, is the natural substrate for trypanothione reductase, an enzyme found in trypanosomatids and leishmanias, the causative agents of diseases such as African sleeping sickness, Chagas disease, and Oriental sore. The rational bases for our mutational experiments were the availability of a high-resolution X-ray structure for human glutathione reductase with bound substrates, the active site sequence comparisons of human glutathione reductase and the trypanothione reductases from Trypanosoma congolense and Trypanosoma cruzi, a complementary set of mutants in T. congolense trypanothione reductase, and the properties of substrate analogues of trypanothione. Mutation of two residues, A34----E34 and R37----W37, in the glutathione-binding site of human glutathione reductase switches human glutathione reductase into a trypanothione reductase with a preference for trypanothione over glutathione by a factor of 700 using kcat/Km as a criterion.

A computer model-built structure of the T. congolense trypanothione reductase in complex with NADP

“…The binding of the fourth carboxylate group close to Lys-67 in GR is conserved in the model, according to the common characteristics of both substrates in this region. This analysis is compatible with the changes in enzyme specificity that have been experimentally observed by site-directed mutagenesis in human GR: If two amino acids are altered, Ala-34 + Asp and Arg-37 + Trp, the substrate specificity of GR is switched from glutathione to trypanothione [24].…”

The Battle against Trypanosomiasis and Leishmaniasis: Metal-based and Natural Product Inhibitors of Trypanothione Reductase

“…This conclusion is consistent with considerations of the net charges of the endogenous substrates of these enzymes (GSSG has an overall 2-charge at physiological pH, whereas T(S) 2 has a 1+ charge). Site directed mutation studies led to the construction of mutant GR's that were able to reduce T(S) 2 [196][197][198] and a mutant TR that was capable of reducing GSSG [190].…”

Redesign of the substrate specificity ofescherichia coliaspartate aminotransferase to that ofescherichia colityrosine aminotransferase by homology modeling and site-directed mutagenesis

“…Subtilisin, a serine protease and probably the best studied example, has been engineered to have increased thermal stability (Bryan et al, 1987;Cunningham &Wells, 1987;Pantoliano et al, 1989), resistance to oxidation (Estell et al, 1985), altered substrate specificity (Estell et al, 1986;Wells et al, 1987), and has been converted to a cysteine protease displaying novel peptide ligase activity (Abrahmsen et al, 1991;Jackson et al, 1994). Glutathione reductase has been converted to a trypanothione reductase (Bradley et al, 1991;Henderson et al, 1991) and has had its cofactor specificity converted from NADPH to NADH (Scrutton et al, 1990). Similarly, lactate dehydrogenase has been altered to switch cofactor specificity (NADH to NADPH;Feeney et al, 1990), and additionally has been engineered to produce a malate dehydrogenase that has higher activity than the naturally occurring malate dehydrogenase enzyme from the same species (Wilks et al, 1988).…”