Expanding the Plant GSTome Through Directed Evolution: DNA Shuffling for the Generation of New Synthetic Enzymes With Engineered Catalytic and Binding Properties

Abstract

Glutathione transferases (GSTs, EC. 2.5.1.18) are inducible multifunctional enzymes that are essential in the detoxification and degradation of toxic compounds. GSTs have considerable biotechnological potential. In the present work, a new method for the generation of synthetic GSTs was developed. Abiotic stress treatment of Phaseolus vulgaris and Glycine max plants led to the induction of total GST activity and allowed the creation of a GST-enriched cDNA library using degenerated GST-specific primers and reverse transcription-PCR. This library was further diversified by employing directed evolution through DNA shuffling. Activity screening of the evolved library led to the identification of a novel tau class GST enzyme (PvGmGSTUG). The enzyme was purified by affinity chromatography, characterized by kinetic analysis, and its structure was determined by X-ray crystallography. Interestingly, PvGmGSTUG displayed enhanced glutathione hydroperoxidase activity, which was significantly greater than that reported so far for natural tau class GSTs. In addition, the enzyme displayed unusual cooperative kinetics toward 1-chloro-2,4-dinitrochlorobenzene (CDNB) but not toward glutathione. The present work provides an easy approach for the simultaneous shuffling of GST genes from different plants, thus allowing the directed evolution of plants GSTome. This may permit the generation of new synthetic enzymes with interesting properties that are valuable in biotechnology.

Directed Evolution of a Glutathione Transferase for the Development of a Biosensor for Alachlor Determination

“…The ability of GSTs to conjugate GSH with xenobiotics has been exploited for the development of enzyme-based biosensors for the determination and monitoring of such compounds in biological and environmental samples [16][17][18][19][20][21][22]. A range of different GST isoenzymes has been used so far for the fabrication of optical, electrochemical or potentiometric biosensors for measuring pesticides, such as the herbicide atrazine [16], the insecticides DDT [17], malathion [18], dieldrin and spiromesifen [19], α-endosulfan [20], and molinate [21].…”

Drugs, host proteins and viral proteins: how their promiscuities shape antiviral design

“…The above two reviews provide comprehensive references regarding the promiscuous behaviour of this enzyme, with another example being a GST from Agrobacterium tumefacians which also showed very high peroxidase activity (Skopelitou et al ., 2012). More recently, DNA shuffling has been used to alter the promiscuity of the plant GSTome (Chronopoulou et al ., 2018).…”

Directed Evolution of Phi Class Glutathione Transferases Involved in Multiple-Herbicide Resistance of Grass Weeds and Crops

“…Protein engineering is a powerful tool that aims to generate novel proteins/enzymes with industrial, therapeutic and research potential. It enables the development of molecular tools for the manipulation of detoxifying enzymatic properties and it can also provide insights into the evolution of resistance mechanisms in the xenobiotic metabolism [37][38][39][40][41][42]. GSTs are designated as a versatile tool for protein engineering due to their catalytic multifunction, substrate specificity, structural characteristics, ease in heterologous expression and stability [38,42].…”

“…Furthermore, DNA shuffling of homologous tau class GST genes from Glycine max resulted in mutants with unusual allosteric kinetics and enhanced detoxifying potential towards this herbicide [40]. In another work, a library of tau class GSTs from abiotic stress-treated Phaseolus vulgaris and Glycine max plants was constructed, thus producing a novel enzyme with increased glutathione hydroperoxidase activity and unusual kinetics towards 1-chloro-2,4-dinitrochlorobenzene (CDNB) [41]. More recently, DNA shuffling of three homologous tau class glutathione transferases resulted in a GST variant with enhanced catalytic activity towards the herbicide alachlor [42].…”