Forced evolution of glutathione S-transferase to create a more efficient drug detoxication enzyme.

Abstract

Glutathione S-transferases (EC 2.5.1.18) in mammalian cells catalyze the conjugation, and thus, the detoxication of a structurally diverse group of electrophilic environmental carcinogens and alkylating drugs, including the antineoplastic nitrogen mustards. We proposed that structural alteration of the nonspecific electrophile-binding site would produce mutant enzymes with increased efficiency for detoxication of a single drug and that these mutants could serve as useful somatic transgenes to protect healthy human cells against single alkylating agents used in cancer chemotherapy protocols. Random mutagenesis of three regions (residues 9-14, 102-112, and 210-220), which together compose the glutathione S-transferase electrophile-binding site, followed by selection ofEscherichia coli expressing the enzyme library with the nitrogen mustard mechlorethamine (20-500 ,uM), yielded mutant enzymes that showed significant improvement in catalytic efficiency for mechlorethamine conjugation (up to 15-fold increase in ka,t and up to 6-fold increase in kct/Km) and that confer up to 31-fold resistance, which is 9-fold greater drug resistance than that conferred by the wild-type enzyme. The results suggest a general strategy for modification of drug-and carcinogen-metabolizing enzymes to achieve desired resistance in both prokaryotic and eukaryotic plant and animal cells.The glutathione S-transferases (EC 2.5.1.18) are a family of enzymes that are responsible for the detoxication of a broad class of electrophiles. In both prokaryotic and eukaryotic cells, these enzymes have been shown to catalyze the conjugation of the tripeptide glutathione to a variety of compounds, resulting in products that are generally less reactive (1-3). High levels of expression of several related glutathione S-transferase isozymes enable these enzymes to protect the cell from numerous structurally diverse electrophiles present in our environment (4-6) and used in cancer chemotherapy (1, 2).Several reports have shown that chronic treatment of cultured cells (7,8) or clinical tumors (9) with alkylating agents results in resistant subpopulations of cells that express alpha class glutathione S-transferases at levels higher than those in the sensitive parental populations. In studies where transfected cDNAs were used to compare cell populations that differed in the single variable of glutathione S-transferase expression, the recombinant rat 1-1 isozyme conferred as much as 3-fold resistance to chlorambucil in mouse fibroblasts (10), and this fold resistance overlaps the fold resistance seen in clinical studies of nitrogen mustard resistance (11,12). Consistent with the observed drug resistance, glutathione S-transferases have been shown to catalyze the conjugation of the nitrogen mustards chlorambucil and melphalan to glutathione (13)(14)(15).Each subunit of a glutathione S-transferase dimer contains an independent active site composed of a G site for binding glutathione and an H site for binding hydrophobic electrophiles (2). Each subunit contain...

Forced Evolution of a Herbicide Detoxifying Glutathione Transferase

“…Surprisingly, the affinity and turnover of the enzyme toward other even closely related xenobiotic substrates was largely unaffected. A more common experience of forced evolution of GSTs would be that the enhancement of one activity is at the expense of another (20). In fact, the switching in catalytic efficiency toward different GST substrates following mutagenesis has been demonstrated in rational design studies based on protein structural information (19).…”

“…Mammalian GSTs have been the subject of several attempts to engineer their detoxifying activities using both gene shuffling and directed evolution with various degrees of success (19). Using random mutagenesis selectively directed to the active site domains responsible for drug binding, a rat GST was engineered such that it conferred a 30-fold increase in the tolerance of E. coli to the alkylating toxin merchlorethamine when expressed in the bacteria (20). When analyzed, the mutant rat GST showed a 6-fold increase in catalytic efficiency in detoxifying merchlorethamine but a major decrease in CDNB conjugation as compared with the parent enzyme.…”

Bacterial glutathione S-transferases: what are they good for?

“…For example, random mutagenesis of a rat alpha class GST gene yielded mutants encoding enzymes with increased resistance to mechlorethamine, a DNA-alkylating drug used in chemotherapy, after selection in E. coli (26). A somewhat related strategy was used to characterize structure-activity relationships of a human alpha class GST gene.…”

Evaluation of Perylenediimide Derivatives for Potential Therapeutic Benefits on Cancer Chemotherapy

“…Eventually, the increased ROS levels cause cytotoxicity on tumor cells leading to death and tissue destruction (8).The toxicity of non-radiated photosensitizer is a critical issue for these applications, because the administered compound directly interacts with metabolic detoxification system enzymes, and upon irradiation, the resulting product may become less effective or more toxic to the organism. As a defense mechanism, the detoxification system enzymes are evolved and can be induced to protect the organism against toxic effects of chemicals that may be harmful to their survival (9). Among these enzymes, mammalian glutathione transferase (GST, EC 2.5.1.18) is one of the most important detoxification enzymes that catalyze the nucleophilic addition of glutathione to diverse electrophilic molecules (10) and solubilize them to facilitate the transport of toxic substances from cells.…”

“…The most commonly analyzed perylene derivatives were from hypericin (1,3,4,6,8,13-hexahydroxy-10,11-dimethylphenanthro [1,10,9,8-opqra]perylene-7,14-dione), which is the most potent, naturally occurring pro-oxidant that facilitate, or induce, the apoptosis and necrosis in a wide spectrum of cancer cell types (4). Moreover, these compounds have shown some antiviral, antidepressant, and antibacterial activities, and still in use as part of natural food supplements available in the markets (4)(5)(6).…”