5ld0
From Proteopedia
Chimeric GST
Structural highlights
Function[GSTA1_HUMAN] Conjugation of reduced glutathione to a wide number of exogenous and endogenous hydrophobic electrophiles.[1] Publication Abstract from PubMedBACKGROUND: Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyse 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 form and in complex with GSH were determined to 1.6A and 2.3A 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 optimised 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 segments 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. Directed Evolution of Glutathione Transferases Towards a Selective Glutathione-Binding Site and Improved Oxidative Stability.,Axarli I, Muleta AW, Chronopoulou EG, Papageorgiou AC, Labrou NE Biochim Biophys Acta. 2016 Sep 6. pii: S0304-4165(16)30326-9. doi:, 10.1016/j.bbagen.2016.09.004. PMID:27612661[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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