Molecular evolution of Theta-class glutathione transferase for enhanced activity with the anticancer drug 1,3-bis-(2-chloroethyl)-1-nitrosourea and other alkylating agents.

Anna-Karin Larsson, Abeer Shokeer, Bengt Mannervik

Index: Arch. Biochem. Biophys. 497(1-2) , 28-34, (2010)

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Abstract

Glutathione transferase (GST) displaying enhanced activity with the cytostatic drug 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and structurally related alkylating agents was obtained by molecular evolution. Mutant libraries created by recursive recombination of cDNA coding for human and rodent Theta-class GSTs were heterologously expressed in Escherichia coli and screened with the surrogate substrate 4-nitrophenethyl bromide (NPB) for enhanced alkyltransferase activity. A mutant with a 70-fold increased catalytic efficiency with NPB, compared to human GST T1-1, was isolated. The efficiency in degrading BCNU had improved 170-fold, significantly more than with the model substrate NPB. The enhanced catalytic activity of the mutant GST was also 2-fold higher with BCNU than wild-type mouse GST T1-1, which is 80-fold more efficient than wild-type human GST T1-1. We propose that GSTs catalyzing inactivation of anticancer drugs may find clinical use in protecting sensitive normal tissues to toxic side-effects in treated patients, and as selectable markers in gene therapy.Copyright 2010 Elsevier Inc. All rights reserved.