Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA
Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA
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DOI：
10.1073/pnas.1615585114
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发表时间：
2017-05-09
影响因子：
11.1
通讯作者：
Tanaka, Yoshikazu
中科院分区：
文献类型：
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作者：
Chen, Minghao;Asai, Shin-ichi;Tanaka, Yoshikazu
转移 RNA (tRNA) 54 位的二硫尿苷 (s(2)U) 是一种转录后修饰，可使嗜热细菌在高温环境中生存。 s(2)U 由两种蛋白质 2-硫尿苷合成酶 TtuA 和 2-硫尿苷合成硫载体蛋白 TtuB 联合作用产生，它们分别充当硫 (S) 转移酶和泛素样 S 供体。尽管体内生化数据不断积累，但体外观察到TtuA/TtuB的酶活性却很少，这阻碍了S转移分子机制的研究。在这里，我们通过光谱、生化和晶体结构分析证明，TtuA 需要氧不稳定的 [4Fe-4S] 型铁 (Fe)-S 簇来发挥其酶活性，这解释了之前观察到的该酶在体外失活的情况。 [4Fe-4S]簇由三个高度保守的半胱氨酸残基配位，其中一个Fe原子暴露于活性位点。此外，TtuA-TtuB 复合物的晶体结构以 2.5 埃的分辨率测定，清楚地显示了 TtuB 使用其 C 末端硫代羧酸酯基团向 tRNA 进行 S 转移。 TtuA 的活性位点通过两个通道与外部相连，一个通道被 TtuB 占据，另一个用于 tRNA 结合。基于这些观察，我们提出了 TtuA 使用类泛素 S 供体和 [4Fe-4S] 簇进行 S 转移的分子机制。
Two-thiouridine (s(2)U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s(2)U 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 angstrom, 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.