Crystallographic and mutational studies on the tRNA thiouridine synthetase TtuA
Crystallographic and mutational studies on the tRNA thiouridine synthetase TtuA
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DOI：
10.1002/prot.24273
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发表时间：
2013-07-01
影响因子：
2.9
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
--
作者：
Nakagawa, Hirofumi;Kuratani, Mitsuo;Yokoyama, Shigeyuki
在嗜热细菌中，特异性2-巯基化发生在tRNA中保守的54位胸苷（T54）上，这是高温下生存所必需的。T54 2-巯基化是通过tRNA硫尿苷合成酶TtuA和硫载体蛋白实现的。TtuA具有5个保守的CXXC/H基序和标志性PP基序，属于tRNA 2-巯基化酶的TtcA家族，目前没有结构信息。在这项研究中，我们确定了TtuA同源物的超嗜热archeon Pyrococcus horikoshii在2.1埃分辨率的晶体结构。该P. horikoshii TtuA形成同源二聚体，并且每个亚基含有催化结构域和独特的N-和C-末端锌指。催化结构域与另一种tRNA修饰酶TilS（tRNAIle 2赖氨嘧啶合成酶）的结构相似性比与另一种类型的tRNA 2-巯基化酶MnmA的结构相似性高得多。三个保守的半胱氨酸残基聚集在推定的催化位点，这是不存在于TilS。在细菌嗜热栖热菌的体内突变分析表明，三个保守的半胱氨酸残基和推定的ATP结合残基的催化结构域是重要的TtuA活性。包含催化位点和两个锌指的带正电荷的表面可能提供tRNA结合位点。Proteins 2013; 81：1232-1244. (c)2013 Wiley Periodicals，Inc.
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 angstrom 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. (c) 2013 Wiley Periodicals, Inc.