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Molecular Cloning, Expression and Enzymatic Characterization of Tetrahymena thermophila Glutathione-S-Transferase Mu 34

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Abstract

Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079 mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190 µmol/min/mg) as substrate. Enzyme kinetic analysis revealed Km values of 0.68 mM with GSH and 0.40 mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25 °C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage.

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Acknowledgements

This work was funded by the Anadolu University Scientific Research Projects Commission (AUBAP) with grant number 1001F45, which was given to Muhittin Arslanyolu. This project was awarded a European Cooperation in Science and Technology (COST) Grant under the Action BM1102 by the Scientific and Technological Research Council of Türkiye (TÜBİTAK) and AUBAP.

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  1. Department of Biology, Faculty of Sciences, Eskisehir Technical University, Yunusemre Campus, Eskisehir, 26470, Turkey

    Handan Açelya Kapkaç & Muhittin Arslanyolu

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HAK carried out each experiment, examined the results, and prepared the manuscript. MA conceptualized and supervised the project and revised the manuscript. All authors reviewed the manuscript.

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Correspondence to Muhittin Arslanyolu.

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Kapkaç, H.A., Arslanyolu, M. Molecular Cloning, Expression and Enzymatic Characterization of Tetrahymena thermophila Glutathione-S-Transferase Mu 34. Protein J 43, 613–626 (2024). https://doi.org/10.1007/s10930-024-10204-1

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