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Site-directed mutagenesis substituting cysteine for serine in 2-Cys peroxiredoxin (2-Cys Prx A) of Arabidopsis thaliana effectively improves its peroxidase and chaperone functions.
Ann Bot. 2015 Sep; 116(4):713-25.AB

Abstract

BACKGROUND AND AIMS

The 2-Cys peroxiredoxin (Prx) A protein of Arabidopsis thaliana performs the dual functions of a peroxidase and a molecular chaperone depending on its conformation and the metabolic conditions. However, the precise mechanism responsible for the functional switching of 2-Cys Prx A is poorly known. This study examines various serine-to-cysteine substitutions on α-helix regions of 2-Cys Prx A in Arabidopsis mutants and the effects they have on the dual function of the protein.

METHODS

Various mutants of 2-Cys Prx A were generated by replacing serine (Ser) with cysteine (Cys) at different locations by site-directed mutagenesis. The mutants were then over-expressed in Escherichia coli. The purified protein was further analysed by size exclusion chromatography, polyacrylamide gel electrophoresis, circular dichroism spectroscopy and transmission electron microscopy (TEM) and image analysis. Peroxidase activity, molecular chaperone activity and hydrophobicity of the proteins were also determined. Molecular modelling analysis was performed in order to demonstrate the relationship between mutation positions and switching of 2-Cys Prx A activity.

KEY RESULTS

Replacement of Ser(150) with Cys(150) led to a marked increase in holdase chaperone and peroxidase activities of 2-Cys Prx A, which was associated with a change in the structure of an important domain of the protein. Molecular modelling demonstrated the relationship between mutation positions and the switching of 2-Cys Prx A activity. Examination of the α2 helix, dimer-dimer interface and C-term loop indicated that the peroxidase function is associated with a fully folded α2 helix and easy formation of a stable reduced decamer, while a more flexible C-term loop makes the chaperone function less likely.

CONCLUSIONS

Substitution of Cys for Ser at amino acid location 150 of the α-helix of 2-Cys Prx A regulates/enhances the dual enzymatic functions of the 2-Cys Prx A protein. If confirmed in planta, this leads to the potential for it to be used to maximize the functional utility of 2-Cys Prx A protein for improved metabolic functions and stress resistance in plants.

Authors+Show Affiliations

Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea.Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea.Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea.Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea.Division of Applied Life Science (BK21 Program), Systems and Synthetic Agrobiotech Center (SSAC), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Research Institute of Natural Science (RINS), Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Republic of Korea and.Division of Applied Life Science (BK21 Program), Systems and Synthetic Agrobiotech Center (SSAC), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Research Institute of Natural Science (RINS), Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Republic of Korea and.Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea.Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea.Division of Applied Life Science (BK21 Program), Systems and Synthetic Agrobiotech Center (SSAC), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Research Institute of Natural Science (RINS), Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Republic of Korea and.Division of Applied Life Science (BK21 Program), Systems and Synthetic Agrobiotech Center (SSAC), Plant Molecular Biology and Biotechnology Research Center (PMBBRC), Research Institute of Natural Science (RINS), Gyeongsang National University, 501 Jinju-daero, Jinju 660-701, Republic of Korea and.Department of Bioenvironmental Chemistry, Chonbuk National University, Jeonju 561-756, Republic of Korea.Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea, bychung@kaeri.re.kr.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

26141131

Citation

Lee, Eun Mi, et al. "Site-directed Mutagenesis Substituting Cysteine for Serine in 2-Cys Peroxiredoxin (2-Cys Prx A) of Arabidopsis Thaliana Effectively Improves Its Peroxidase and Chaperone Functions." Annals of Botany, vol. 116, no. 4, 2015, pp. 713-25.
Lee EM, Lee SS, Tripathi BN, et al. Site-directed mutagenesis substituting cysteine for serine in 2-Cys peroxiredoxin (2-Cys Prx A) of Arabidopsis thaliana effectively improves its peroxidase and chaperone functions. Ann Bot. 2015;116(4):713-25.
Lee, E. M., Lee, S. S., Tripathi, B. N., Jung, H. S., Cao, G. P., Lee, Y., Singh, S., Hong, S. H., Lee, K. W., Lee, S. Y., Cho, J. Y., & Chung, B. Y. (2015). Site-directed mutagenesis substituting cysteine for serine in 2-Cys peroxiredoxin (2-Cys Prx A) of Arabidopsis thaliana effectively improves its peroxidase and chaperone functions. Annals of Botany, 116(4), 713-25. https://doi.org/10.1093/aob/mcv094
Lee EM, et al. Site-directed Mutagenesis Substituting Cysteine for Serine in 2-Cys Peroxiredoxin (2-Cys Prx A) of Arabidopsis Thaliana Effectively Improves Its Peroxidase and Chaperone Functions. Ann Bot. 2015;116(4):713-25. PubMed PMID: 26141131.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Site-directed mutagenesis substituting cysteine for serine in 2-Cys peroxiredoxin (2-Cys Prx A) of Arabidopsis thaliana effectively improves its peroxidase and chaperone functions. AU - Lee,Eun Mi, AU - Lee,Seung Sik, AU - Tripathi,Bhumi Nath, AU - Jung,Hyun Suk, AU - Cao,Guang Ping, AU - Lee,Yuno, AU - Singh,Sudhir, AU - Hong,Sung Hyun, AU - Lee,Keun Woo, AU - Lee,Sang Yeol, AU - Cho,Jae-Young, AU - Chung,Byung Yeoup, Y1 - 2015/07/02/ PY - 2015/02/27/received PY - 2015/05/08/accepted PY - 2015/7/5/entrez PY - 2015/7/5/pubmed PY - 2016/6/18/medline KW - 2-Cys Prx A protein KW - 2-Cys peroxiredoxin KW - Arabidopsis thaliana KW - ROS KW - chaperone KW - homology modelling KW - molecular dynamics simulation KW - peroxidase KW - reactive oxygen species KW - site-directed mutagenesis SP - 713 EP - 25 JF - Annals of botany JO - Ann Bot VL - 116 IS - 4 N2 - BACKGROUND AND AIMS: The 2-Cys peroxiredoxin (Prx) A protein of Arabidopsis thaliana performs the dual functions of a peroxidase and a molecular chaperone depending on its conformation and the metabolic conditions. However, the precise mechanism responsible for the functional switching of 2-Cys Prx A is poorly known. This study examines various serine-to-cysteine substitutions on α-helix regions of 2-Cys Prx A in Arabidopsis mutants and the effects they have on the dual function of the protein. METHODS: Various mutants of 2-Cys Prx A were generated by replacing serine (Ser) with cysteine (Cys) at different locations by site-directed mutagenesis. The mutants were then over-expressed in Escherichia coli. The purified protein was further analysed by size exclusion chromatography, polyacrylamide gel electrophoresis, circular dichroism spectroscopy and transmission electron microscopy (TEM) and image analysis. Peroxidase activity, molecular chaperone activity and hydrophobicity of the proteins were also determined. Molecular modelling analysis was performed in order to demonstrate the relationship between mutation positions and switching of 2-Cys Prx A activity. KEY RESULTS: Replacement of Ser(150) with Cys(150) led to a marked increase in holdase chaperone and peroxidase activities of 2-Cys Prx A, which was associated with a change in the structure of an important domain of the protein. Molecular modelling demonstrated the relationship between mutation positions and the switching of 2-Cys Prx A activity. Examination of the α2 helix, dimer-dimer interface and C-term loop indicated that the peroxidase function is associated with a fully folded α2 helix and easy formation of a stable reduced decamer, while a more flexible C-term loop makes the chaperone function less likely. CONCLUSIONS: Substitution of Cys for Ser at amino acid location 150 of the α-helix of 2-Cys Prx A regulates/enhances the dual enzymatic functions of the 2-Cys Prx A protein. If confirmed in planta, this leads to the potential for it to be used to maximize the functional utility of 2-Cys Prx A protein for improved metabolic functions and stress resistance in plants. SN - 1095-8290 UR - https://www.unboundmedicine.com/medline/citation/26141131/Site_directed_mutagenesis_substituting_cysteine_for_serine_in_2_Cys_peroxiredoxin__2_Cys_Prx_A__of_Arabidopsis_thaliana_effectively_improves_its_peroxidase_and_chaperone_functions_ L2 - https://academic.oup.com/aob/article-lookup/doi/10.1093/aob/mcv094 DB - PRIME DP - Unbound Medicine ER -