Characterization of Ser73 in Arabidopsis thaliana Glutathione S-transferase zeta class.
Glutathione S-transferases (GSTs) are ubiquitous detoxifying superfamily enzymes. The zeta class GST from Arabidopsis thaliana (AtGSTZ) can efficiently degrade dichloroacetic acid (DCA), which is a common carcinogenic contaminant in drinking water. Ser73 in AtGSTZ is a conserved residue at Glutathione binding site (G-site). Compared with the equivalent residues in other GSTs, the catalytic and structural properties of Ser73 were poorly investigated. In this article, site-saturation mutagenesis was performed to characterize the detailed role of Ser73. The DCA dechlorinating (DCA-DC) activity showed that most of the mutants had less than 3% of the wild-type activity, except S73T and S73A showing 43.48% and 21.62% of the wild-type activity, respectively, indicating that position 73 in AtGSTZ showed low mutational substitutability. Kinetic experiments revealed that mutants S73T, S73A, and S73G showed low binding affinity and catalytic efficiency toward DCA, 1.8-, 3.1-, and 10.7-fold increases in K(m)(DCA) values and 4.0-, 9.6-, and 34.1-fold decreases in K(cat-)(DCA)/K(m)(DCA) values, respectively, compared to the wild type. Thermostability and refolding experiments showed that the wild type maintained more thermostability and recovered activity. These results demonstrated the important role of Ser73 in catalytic activity and structural stability of the enzyme. Such properties of Ser73 could be particularly crucial to the molecular evolution of AtGSTZ and might, therefore, help explain why Ser73 is conserved in all GSTs. This conclusion might provide insights into the directed evolution of the DCA-DC activity of AtGSTZ.
Tao
Sudan
S
Laboratory of Molecular Genetics, College of Life Sciences, Nankai University, Tianjin 300071, China.
Chen
Xiwen
X
Liu
Jia
J
Ming
Ming
M
Chong
Namsu
N
Chen
Defu
D
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D019143:Evolution, Molecular; D005982:Glutathione Transferase / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012694:Serine / Q000737:chemistry*
D012694:Serine; D005982:Glutathione Transferase