title: Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803.
abstract: Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
authors.forename: Rayakorn
authors.initials: R
authors.lastname: Yutthanasirikul
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Nagano
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Jimbo
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
authors.forename: Yukako
authors.initials: Y
authors.lastname: Hihara
authors.affiliation: GeneFrontier Corporation, Todai-Kashiwa Venture Plaza, 5-4-19 Kashiwanoha, Kashiwa 277-0882, Japan,; Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kanamori
authors.affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Ueda
authors.affiliation: Bio-AFM Frontier Research Center, Institute of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan, and.
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Haruyama
authors.affiliation: Bio-AFM Frontier Research Center, Institute of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan, and.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Konno
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8503, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Yoshida
authors.affiliation: Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8503, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Hisabori
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan,. Electronic address: nishiyama@molbiol.saitama-u.ac.jp.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Nishiyama
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D013434:Sulfenic Acids / Q000737:chemistry / Q000378:metabolism; D046939:Synechocystis / Q000737:chemistry / Q000378:metabolism*; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D009711:Nucleotides; D013434:Sulfenic Acids; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D010444:Peptide Elongation Factor Tu; D003545:Cysteine
keywords: cyanobacteria; oxidative stress; redox regulation; translation; translation elongation factor