Active site in RrmJ, a heat shock-induced methyltransferase
Active site in RrmJ, a heat shock-induced methyltransferase
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DOI：
10.1074/jbc.m205423200
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发表时间：
2002-11-01
影响因子：
4.8
通讯作者：
Jakob, U
中科院分区：
文献类型：
--
作者：
Hager, J;Staker, BL;Jakob, U
热休克蛋白 RrmJ (FtsJ) 从真细菌到真核生物都高度保守，负责 23 S rRNA A 环中普遍保守的碱基 U2552 的 2'-O-核糖甲基化。这种甲基化的缺失发生在核糖体成熟过程的后期，似乎会导致 50 S 核糖体亚基不稳定和过早解离。为了了解 2'-O-核糖甲基转移反应的机制，我们表征了 RrmJ 的酶参数，并对 RrmJ 进行了位点特异性诱变。与 VP39（一种来自痘苗病毒的结构相关的 2'-O-甲基转移酶）进行的基于结构的序列比对指导了我们的诱变研究。我们分析了 RrmJ 突变体的体内功能，并表征了纯化蛋白质的体外甲基转移反应。 RrmJ 的活性位点似乎是由两个赖氨酸残基 Lys-38 和 Lys-164 以及带负电的残基 Asp-124 组成的催化三联体形成。另一个高度保守的残基 Glu-199 存在于 RrmJ 和 VP39 的活性位点中，似乎在体内甲基转移反应中仅发挥次要作用。基于这些结果，提出了 RrmJ 甲基转移活性的反应机制。
The heat shock protein RrmJ (FtsJ), highly conserved from eubacteria to eukarya, is responsible for the 2'-O-ribose methylation of the universally conserved base U2552 in the A-loop of the 23 S rRNA. Absence of this methylation, which occurs late in the maturation process of the ribosome, appears to cause the destabilization and premature dissociation of the 50 S ribosomal subunit. To understand the mechanism of 2'-O-ribose methyltransfer reactions, we characterized the enzymatic parameters of RrmJ and conducted site-specific mutagenesis of RrmJ. A structure based sequence alignment with VP39, a structurally related 2'-O-methyltransferase from vaccinia virus, guided our mutagenesis studies. We analyzed the function of our RrmJ mutants in vivo and characterized the methyltransfer reaction of the purified proteins in vitro. The active site of RrmJ appears to be formed by a catalytic triad consisting of two lysine residues, Lys-38 and Lys-164, and the negatively charged residue Asp-124. Another highly conserved residue, Glu-199, that is present in the active site of RrmJ and VP39 appears to play only a minor role in the methyltransfer reaction in vivo. Based on these results, a reaction mechanism for the methyltransfer activity of RrmJ is proposed.