Oxidation of a cysteine residue in elongation factor EF-Tu reversibly inhibits translation in the cyanobacterium Synechocystis sp. PCC 6803

Rayakorn Yutthanasirikul, Takanori Nagano, Haruhiko Jimbo, Yukako Hihara, Takashi Kanamori, Takuya Ueda, Takamitsu Haruyama, Hiroki Konno, Keisuke Yoshida, Toru Hisabori, Yoshitaka Nishiyama

Research output: Contribution to journalArticle

9 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)5860-5870
Number of pages11
JournalJournal of Biological Chemistry
Volume291
Issue number11
DOIs
Publication statusPublished - 2016 Mar 11
Externally publishedYes

Fingerprint

Peptide Elongation Factor Tu
Synechocystis
Peptide Elongation Factors
Cyanobacteria
Cysteine
Oxidation
Reactive Oxygen Species
Nucleotides
Peptide Elongation Factor G
Guanosine Triphosphate
Oxidation-Reduction
Sulfenic Acids
Thioredoxins
Atomic Force Microscopy
Dithiothreitol
Chromatography
Disulfides
Serine
Escherichia coli
Gel Chromatography

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Oxidation of a cysteine residue in elongation factor EF-Tu reversibly inhibits translation in the cyanobacterium Synechocystis sp. PCC 6803. / Yutthanasirikul, Rayakorn; Nagano, Takanori; Jimbo, Haruhiko; Hihara, Yukako; Kanamori, Takashi; Ueda, Takuya; Haruyama, Takamitsu; Konno, Hiroki; Yoshida, Keisuke; Hisabori, Toru; Nishiyama, Yoshitaka.

In: Journal of Biological Chemistry, Vol. 291, No. 11, 11.03.2016, p. 5860-5870.

Research output: Contribution to journalArticle

Yutthanasirikul, R, Nagano, T, Jimbo, H, Hihara, Y, Kanamori, T, Ueda, T, Haruyama, T, Konno, H, Yoshida, K, Hisabori, T & Nishiyama, Y 2016, 'Oxidation of a cysteine residue in elongation factor EF-Tu reversibly inhibits translation in the cyanobacterium Synechocystis sp. PCC 6803', Journal of Biological Chemistry, vol. 291, no. 11, pp. 5860-5870. https://doi.org/10.1074/jbc.M115.706424
Yutthanasirikul, Rayakorn ; Nagano, Takanori ; Jimbo, Haruhiko ; Hihara, Yukako ; Kanamori, Takashi ; Ueda, Takuya ; Haruyama, Takamitsu ; Konno, Hiroki ; Yoshida, Keisuke ; Hisabori, Toru ; Nishiyama, Yoshitaka. / Oxidation of a cysteine residue in elongation factor EF-Tu reversibly inhibits translation in the cyanobacterium Synechocystis sp. PCC 6803. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 11. pp. 5860-5870.
@article{4bb8a7f98717415288b52c031b17a1a1,
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.",
author = "Rayakorn Yutthanasirikul and Takanori Nagano and Haruhiko Jimbo and Yukako Hihara and Takashi Kanamori and Takuya Ueda and Takamitsu Haruyama and Hiroki Konno and Keisuke Yoshida and Toru Hisabori and Yoshitaka Nishiyama",
year = "2016",
month = "3",
day = "11",
doi = "10.1074/jbc.M115.706424",
language = "English",
volume = "291",
pages = "5860--5870",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "11",

}

TY - JOUR

T1 - Oxidation of a cysteine residue in elongation factor EF-Tu reversibly inhibits translation in the cyanobacterium Synechocystis sp. PCC 6803

AU - Yutthanasirikul, Rayakorn

AU - Nagano, Takanori

AU - Jimbo, Haruhiko

AU - Hihara, Yukako

AU - Kanamori, Takashi

AU - Ueda, Takuya

AU - Haruyama, Takamitsu

AU - Konno, Hiroki

AU - Yoshida, Keisuke

AU - Hisabori, Toru

AU - Nishiyama, Yoshitaka

PY - 2016/3/11

Y1 - 2016/3/11

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84964321585&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84964321585&partnerID=8YFLogxK

U2 - 10.1074/jbc.M115.706424

DO - 10.1074/jbc.M115.706424

M3 - Article

VL - 291

SP - 5860

EP - 5870

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 11

ER -