Eun Mi Lee1, Seung Sik Lee1, Bhumi Nath Tripathi1, Hyun Suk Jung2, Guang Ping Cao3, Yuno Lee3, Sudhir Singh1, Sung Hyun Hong1, Keun Woo Lee3, Sang Yeol Lee3, Jae-Young Cho4, Byung Yeoup Chung5. 1. Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea. 2. Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea. 3. 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. 4. Department of Bioenvironmental Chemistry, Chonbuk National University, Jeonju 561-756, Republic of Korea. 5. Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea, bychung@kaeri.re.kr.
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.
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: Jung Ro Lee; Seung Sik Lee; Ho Hee Jang; Young Mee Lee; Jin Ho Park; Seong-Cheol Park; Jeong Chan Moon; Soo Kwon Park; Sun Young Kim; Sun Yong Lee; Ho Byoung Chae; Young Jun Jung; Woe Yeon Kim; Mi Rim Shin; Gang-Won Cheong; Min Gab Kim; Kee Ryeon Kang; Kyun Oh Lee; Dae-Jin Yun; Sang Yeol Lee Journal: Proc Natl Acad Sci U S A Date: 2009-03-17 Impact factor: 11.205
Authors: T Rogalla; M Ehrnsperger; X Preville; A Kotlyarov; G Lutsch; C Ducasse; C Paul; M Wieske; A P Arrigo; J Buchner; M Gaestel Journal: J Biol Chem Date: 1999-07-02 Impact factor: 5.157
Authors: Jeong Chan Moon; Young-Sool Hah; Woe Yeon Kim; Bae Gyo Jung; Ho Hee Jang; Jung Ro Lee; Sun Young Kim; Young Mee Lee; Min Gyu Jeon; Choong Won Kim; Moo Je Cho; Sang Yeol Lee Journal: J Biol Chem Date: 2005-06-07 Impact factor: 5.157
Authors: Daria M Svistunova; Jillian N Simon; Elzbieta Rembeza; Mark Crabtree; Wyatt W Yue; Peter L Oliver; Mattéa J Finelli Journal: Free Radic Biol Med Date: 2018-10-31 Impact factor: 7.376