Recent advances in protein engineering and biotechnological applications of glutathione transferases.

Glutathione transferases (GSTs, EC 2.5.1.18) are a widespread family of enzymes that play a central role in the detoxification, metabolism, and transport or sequestration of endogenous or xenobiotic compounds. During the last two decades, delineation of the important structural and catalytic features of GSTs has laid the groundwork for engineering GSTs, involving both rational and random approaches, aiming to create new variants with new or altered properties. These approaches have expanded the usefulness of native GSTs, not only for understanding the fundamentals of molecular detoxification mechanisms, but also for the development medical, analytical, environmental, and agricultural applications. This review article attempts to summarize successful examples and current developments on GST engineering, highlighting in parallel the recent knowledge gained on their phylogenetic relationships, structural/catalytic features, and biotechnological applications.

Inhibition Analysis and High-Resolution Crystal Structure of Mus musculus Glutathione Transferase P1-1.

Multidrug resistance is a significant barrier that makes anticancer therapies less effective. Glutathione transferases (GSTs) are involved in multidrug resistance mechanisms and play a significant part in the metabolism of alkylating anticancer drugs. The purpose of this study was to screen and select a lead compound with high inhibitory potency against the isoenzyme GSTP1-1 from Mus musculus (MmGSTP1-1). The lead compound was selected following the screening of a library of currently approved and registered pesticides that belong to different chemical classes. The results showed that the fungicide iprodione [3-(3,5-dichlorophenyl)-2,4-dioxo-N-propan-2-ylimidazolidine-1-carboxamide] exhibited the highest inhibition potency (ΙC50 = 11.3 ± 0.5 µΜ) towards MmGSTP1-1. Kinetics analysis revealed that iprodione functions as a mixed-type inhibitor towards glutathione (GSH) and non-competitive inhibitor towards 1-chloro-2,4-dinitrobenzene (CDNB). X-ray crystallography was used to determine the crystal structure of MmGSTP1-1 at 1.28 Å resolution as a complex with S-(p-nitrobenzyl)glutathione (Nb-GSH). The crystal structure was used to map the ligand-binding site of MmGSTP1-1 and to provide structural data of the interaction of the enzyme with iprodione using molecular docking. The results of this study shed light on the inhibition mechanism of MmGSTP1-1 and provide a new compound as a potential lead structure for future drug/inhibitor development.

Comparative Analysis of Two Stress-Inducible tau Class Glutathione Transferases from Glycine max Revealed Significant Catalytic and Structural Diversification.

BACKGROUND: Glutathione transferases (GSTs, EC. 2.5.1.18) form a large group of multifunctional enzymes that are involved in the metabolism and inactivation of a wide range of endogenous and xenobiotic compound as well as in cell regulation and response to several biotic and abiotic stresses. OBJECTIVES: In the present work, we report the comparative analysis of the structural and functional features of two isoenzymes (GmGSTU5-5 and GmGSTU8-8) of the glutathione transferase (GST) family from Glycine max. METHODS: Full-length cDNA clones of GmGSTU5-5 and GmGSTU8-8 were derived from RT-PCR of RNA isolated from soybean seedlings and were cloned into a T7 expression vector. Τhe recombinant enzymes were expressed in E. coli and purified by affinity chromatography. Substrate specificity, kinetic and inhibition analysis were carried out towards a range of different xenobiotic compounds and GSH analogues. The thermal stability of the enzymes was also evaluated using activity assays and differential scanning fluorimetry. RESULTS: Analysis of substrate specificity using a range of thiol substrates and electrophilic compounds suggested that both isoenzymes display broad and overlapping specificities. They are capable of detoxifying major stress-induced toxic products. Study of their ligandin-binding properties by kinetic analysis and molecular modelling indicated that both GmGSTU5-5 and GmGSTU8-8 bind a range of secondary metabolites and plant hormones, suggesting a role in transport or storage of bioactive compounds. Thermostability analysis showed that GmGSTU5-5 and GmGSTU8-8 display extraordinary thermal stability, compared to other plant GSTs. CONCLUSION: Our results suggest that GmGSTU5-5 and GmGSTU8-8 display different or overlapping substrate specificities and kinetic properties. The biological role of GmGSTU5-5 and GmGSTU8-8 may be relevant to the detoxification of toxic compounds or the binding of bioactive metabolites that function in cell regulation and stress defence mechanisms.