Work

Functional characterization and directed evolution of Cicer arietinum glutathione transferases for enhanced hydroperoxidase activity and ligandin function

Year: 2025

Type: article

Abstract: Tau class glutathione transferases (GSTUs) play essential roles in plant defense by facilitating the nucleophilic attack of glutathione (GSH) to a wide range of electrophilic xenobiotics. In addition to their conjugating activity, these enzymes possess hydroperoxidase function, enabling the detoxification of harmful organic hydroperoxides into less reactive alcohols. In this study, we identified three closely related GST isoenzymes (96–98% sequence identity) from Cicer arietinum ( Ca GSTUs) through computational homology screening. Full-length cDNAs encoding these GSTs were cloned, recombinantly produced in E. coli , and purified for functional characterization. Enzyme kinetics were evaluated using model substrates, cumene hydroperoxide (CuOOH) and 1-chloro-2,4-dinitrobenzene (CDNB), revealing that Ca GSTU1-1 displayed superior hydroperoxidase activity and thermal stability. Based on these properties, Ca GSTU1-1 was selected as the parental scaffold for directed evolution via DNA shuffling, using the homologous Glycine max isoenzyme Gm GSTU4-4. Screening of the generated chimeric library resulted in the identification of a new variant, CaGm GSTU, which demonstrated a fourfold enhancement in catalytic turnover and efficiency toward both substrates. Additionally, CaGm GSTU exhibited altered ligand-binding characteristics, including increased affinity for selected pesticides. Structural modeling and viscosity-dependence kinetics indicated that these enhancements were primarily driven by changes in enzyme flexibility. Given the widespread toxicity of hydroperoxides and related pollutants, CaGm GSTU represents a promising tool for detoxification applications in environmental and agricultural biotechnology. • Tau GSTUs catalyze GSH conjugation and detoxify harmful hydroperoxides. • Three Ca GSTUs from Cicer arietinum were cloned and biochemically characterized. • Ca GSTU1-1 displayed high hydroperoxidase activity and thermal resilience. • Directed evolution produced CaGm GSTU with 4× enhanced catalytic efficiency. • CaGm GSTU showed improved pesticide affinity and flexibility-driven function.

Source: Phytochemistry

DOI: 10.1016/j.phytochem.2025.114692

Authors: Anni Kontouri, Nikolaos Georgakis, Anastassios C. Papageorgiou, Nikolaos Ε. Labrou

Institutions: Agricultural University of Athens, Åbo Akademi University, University of Turku

Language: English

Topic: Glutathione Transferases and Polymorphisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $4,350

Funders: European Commission, European Social Fund