Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability.


Abstract

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.

Submission Details

ID: 2nrpQi2L4

Submitter: Shu-Ching Ou

Submission Date: Nov. 27, 2018, 3:44 p.m.

Version: 1

Publication Details
Axarli I;Muleta AW;Chronopoulou EG;Papageorgiou AC;Labrou NE,Biochim Biophys Acta Gen Subj (2017) Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability. PMID:27612661
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5LD0 2016-09-21 1.6 Chimeric GST
5LCZ 2016-09-21 2.33 Chimeric GST

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.9 Glutathione S-transferase A1 P04903 GSTA2_RAT