title: 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.
abstract: OBJECTIVE
  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.
  
  
  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.affiliation: Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea.
authors.forename: Eun Mi
authors.initials: EM
authors.lastname: Lee
authors.affiliation: Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea.
authors.forename: Seung Sik
authors.initials: SS
authors.lastname: Lee
authors.affiliation: Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea.
authors.forename: Bhumi Nath
authors.initials: BN
authors.lastname: Tripathi
authors.affiliation: Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea.
authors.forename: Hyun Suk
authors.initials: HS
authors.lastname: Jung
authors.affiliation: 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.
authors.forename: Guang Ping
authors.initials: GP
authors.lastname: Cao
authors.affiliation: 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.
authors.forename: Yuno
authors.initials: Y
authors.lastname: Lee
authors.affiliation: Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea.
authors.forename: Sudhir
authors.initials: S
authors.lastname: Singh
authors.affiliation: Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea.
authors.forename: Sung Hyun
authors.initials: SH
authors.lastname: Hong
authors.affiliation: 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.
authors.forename: Keun Woo
authors.initials: KW
authors.lastname: Lee
authors.affiliation: 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.
authors.forename: Sang Yeol
authors.initials: SY
authors.lastname: Lee
authors.affiliation: Department of Bioenvironmental Chemistry, Chonbuk National University, Jeonju 561-756, Republic of Korea.
authors.forename: Jae-Young
authors.initials: JY
authors.lastname: Cho
authors.affiliation: Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup 580-185, Republic of Korea, bychung@kaeri.re.kr.
authors.forename: Byung Yeoup
authors.initials: BY
authors.lastname: Chung
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009195:Peroxidase / Q000235:genetics / Q000378:metabolism; D054464:Peroxiredoxins / Q000235:genetics* / Q000378:metabolism; D012694:Serine / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D018832:Molecular Chaperones; D012694:Serine; C493631:2-Cys peroxiredoxin A, Arabidopsis; D054464:Peroxiredoxins; D009195:Peroxidase; D003545:Cysteine
keywords: 2-Cys Prx A protein; 2-Cys peroxiredoxin; Arabidopsis thaliana; ROS; chaperone; homology modelling; molecular dynamics simulation; peroxidase; reactive oxygen species; site-directed mutagenesis