Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA

Minghao Chen, Shin Ichi Asai, Shun Narai, Shusuke Nambu, Naoki Omura, Yuriko Sakaguchi, Tsutomu Suzuki, Masao Ikeda-Saito, Kimitsuna Watanabe, Min Yao, Naoki Shigi, Yoshikazu Tanaka

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

Two-thiouridine (s2U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s2U is produced by the combined action of two proteins, 2-thiouridine synthetase TtuA and 2-thiouridine synthesis sulfur carrier protein TtuB, which act as a sulfur (S) transfer enzyme and a ubiquitin-like S donor, respectively. Despite the accumulation of biochemical data in vivo, the enzymatic activity by TtuA/TtuB has rarely been observed in vitro, which has hindered examination of the molecular mechanism of S transfer. Here we demonstrate by spectroscopic, biochemical, and crystal structure analyses that TtuA requires oxygen-labile [4Fe-4S]-type iron (Fe)-S clusters for its enzymatic activity, which explains the previously observed inactivation of this enzyme in vitro. The [4Fe-4S] cluster was coordinated by three highly conserved cysteine residues, and one of the Fe atomswas exposed to the active site. Furthermore, the crystal structure of the TtuA-TtuB complex was determined at a resolution of 2.5 Å, which clearly shows the S transfer of TtuB to tRNA using its C-terminal thiocarboxylate group. The active site of TtuA is connected to the outside by two channels, one occupied by TtuB and the other used for tRNA binding. Based on these observations, we propose a molecular mechanism of S transfer by TtuA using the ubiquitin-like S donor and the [4Fe-4S] cluster.

Original languageEnglish
Pages (from-to)4954-4959
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number19
DOIs
Publication statusPublished - 2017 May 9

Keywords

  • 2-thiouridine
  • Crystal structure
  • Fe-s cluster
  • Sulfur transfer
  • Trna modification

ASJC Scopus subject areas

  • General

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