Background: Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. Methods: A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). Results: Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher kcat/Km and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free formand in complex with GSH were determined to 1.6 Å and 2.3 Å resolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. Conclusions: DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. General significance: Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors.
Submitter: Shu-Ching Ou
Submission Date: Nov. 27, 2018, 3:44 p.m.
|Number of data points||96|
|Proteins||Glutathione S-transferase A1|
|Assays/Quantities/Protocols||Experimental Assay: Thermal Stability: Tm ; Experimental Assay: Adsorption with BES-GSH: Rate of Adsorption ; Experimental Assay: Adsorption with BES-GSH: Dissociation Constant ; Experimental Assay: Kinetic Constant: kcat ; Experimental Assay: Kinetic Constant: Km: CDNB ; Experimental Assay: Kinetic Constant: Km: GSH ; Derived Quantity: SD of Kinetic Constant: kcat ; Derived Quantity: SD of Kinetic Constant: Km: GSH ; Derived Quantity: SD of Kinetic Constant: Km: CDNB ; Derived Quantity: Kinetic Constant: kcat/Km: GSH ; Derived Quantity: Kinetic Constant: kcat/Km: CDNB ; Derived Quantity: SD of Thermal Stability: Tm ; Derived Quantity: SD of Adsorption with BES-GSH: Rate of Adsorption ; Derived Quantity: SD of Adsorption with BES-GSH: Dissociation Constant|
|Libraries||Variants for Complex ; Variants for Protein|