Characterization of a Highly pH Stable Chi-Class Glutathione S-Transferase from Synechocystis PCC 6803.

Tripti Pandey,Timir Tripathi,Arvind Kumar Singh,Sudhir Kumar Singh,Gaurav Chhetri,Roger A Coulombe

doi:10.1371/journal.pone.0126811

Tripti Pandey, Timir Tripathi + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0126811

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Journal: PloS one	Publication Date: May 12, 2015
Citations: 64	License type: CC BY 4.0

Affiliation: North Eastern Hill University

Abstract

Glutathione S-transferases (GSTs) are multifunctional enzymes present in virtually all organisms. Besides having an essential role in cellular detoxification, they also perform various other functions, including responses in stress conditions and signaling. GSTs are highly studied in plants and animals; however, the knowledge regarding GSTs in cyanobacteria seems rudimentary. In this study, we report the characterization of a highly pH stable GST from the model cyanobacterium- Synechocystis PCC 6803. The gene sll0067 was expressed in Escherichia coli (E. coli), and the protein was purified to homogeneity. The expressed protein exists as a homo-dimer, which is composed of about 20 kDa subunit. The results of the steady-state enzyme kinetics displayed protein’s glutathione conjugation activity towards its class specific substrate- isothiocyanate, having the maximal activity with phenethyl isothiocyanate. Contrary to the poor catalytic activity and low specificity towards standard GST substrates such as 1-chloro-2,4-dinitrobenzene by bacterial GSTs, PmGST B1-1 from Proteus mirabilis, and E. coli GST, sll0067 has broad substrate degradation capability like most of the mammalian GST. Moreover, we have shown that cyanobacterial GST sll0067 is catalytically efficient compared to the best mammalian enzymes. The structural stability of GST was studied as a function of pH. The fluorescence and CD spectroscopy in combination with size exclusion chromatography showed a highly stable nature of the protein over a broad pH range from 2.0 to 11.0. To the best of our knowledge, this is the first GST with such a wide range of pH related structural stability. Furthermore, the presence of conserved Proline-53, structural motifs such as N-capping box and hydrophobic staple further aid in the stability and proper folding of cyanobacterial GST- sll0067.

Highlights

Glutathione S-transferases (GSTs; EC 2.5.1.18) are a protein superfamily involved in cellular detoxification [1]
While the N-terminal domain provides the site for GSH binding, the C-terminal domain contributes most of the amino acid residues that interact with various hydrophobic xenobiotic substrates
The recombinant plasmid was transformed into E. coli M15 cells for functional expression

Summary

Introduction

Glutathione S-transferases (GSTs; EC 2.5.1.18) are a protein superfamily involved in cellular detoxification [1]. Characterization of Chi-Class Synechocystis GST to reduced glutathione (GSH), thereby playing an important role in the metabolism of xenobiotics including drugs, herbicides and pesticides [1, 2]. The presence of high concentration of GSH in the cytosol of cyanobacteria indicates the presence as well as the importance of enzymes that can utilize GSH in these organisms [30, 31] These observations suggest important roles of GSTs in cyanobacteria. On the basis of sequence similarity and catalytic activity, our studies suggest that sll0067 is a Chi-class GST with a high preference for isothiocyanates as substrates. It is highly pH stable and can withstand a pH variation from 2 to 11. To the best of our knowledge, we report the first GST with such an unusual structural stability over a wide range of pH

Materials and Methods

Results

Discussion