Crystallographic and mutational studies on the tRNA thiouridine synthetase TtuA

Authors

  • Hirofumi Nakagawa,

    1. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
    2. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
    3. Laboratory of Structural Biology, Graduate School of Science, The University of Tokyo, Tokyo, Japan
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  • Mitsuo Kuratani,

    1. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
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  • Sakurako Goto-Ito,

    1. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
    2. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
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  • Takuhiro Ito,

    1. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
    2. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
    3. Laboratory of Structural Biology, Graduate School of Science, The University of Tokyo, Tokyo, Japan
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  • Kazushige Katsura,

    1. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
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  • Takaho Terada,

    1. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
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  • Mikako Shirouzu,

    1. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
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  • Shun-ichi Sekine,

    1. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
    2. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
    3. Laboratory of Structural Biology, Graduate School of Science, The University of Tokyo, Tokyo, Japan
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  • Naoki Shigi,

    Corresponding author
    1. Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan
    • Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
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  • Shigeyuki Yokoyama

    Corresponding author
    1. RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan
    2. Laboratory of Structural Biology, Graduate School of Science, The University of Tokyo, Tokyo, Japan
    • Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan
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Errata

This article is corrected by:

  1. Errata: Crystallographic and mutational studies on the tRNA thiouridine synthetase TtuA Volume 82, Issue 7, 1551, Article first published online: 11 June 2014

  • Present address: Structural Biology Laboratory, Life Science Division, Synchrotron Radiation Research Organization and Institute of Molecular and Cellular Bioscience, The University of Tokyo, Tokyo, 113-0033, Japan.

Correspondence to: N. Shigi, Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan. E-mail: naoki-shigi@aist.go.jp or S. Yokoyama, Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. yokoyama@riken.jp

Abstract

In thermophilic bacteria, specific 2-thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2-thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur-carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2-thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 Å resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N- and C-terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNAIle2 lysidine synthetase), than to the other type of tRNA 2-thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP-binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA-binding site. Proteins 2013; 81:1232–1244. © 2013 Wiley Periodicals, Inc.

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