Decoding Substrate Selectivity of an Archaeal RlmCD-like Methyltransferase Through Its Salient Traits

Sayan Saha, Shankar Prasad Kanaujia
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Abstract

5-Methyluridine (m5U) rRNA modifications frequently occur at U747 and U1939 (Escherichia coli numbering) in domains II and IV of the 23S rRNA in Gram-negative bacteria, with the help of S-adenosyl-l-methionine (SAM)-dependent rRNA methyltransferases (MTases), RlmC and RlmD, respectively. In contrast, Gram-positive bacteria utilize a single SAM-dependent rRNA MTase, RlmCD, to modify both corresponding sites. Notably, certain archaea, specifically within the Thermococcales group, have been found to possess two genes encoding SAM-dependent archaeal (tRNA and rRNA) m5U (Arm5U) MTases. Among these, a tRNA-specific Arm5U MTase (PabTrmU54) has already been characterized. This study focused on the structural and functional characterization of the rRNA-specific Arm5U MTase from the hyperthermophilic archaeon Pyrococcus horikoshii (PhRlmCD). An in-depth structural examination revealed a dynamic hinge movement induced by the replacement of the iron–sulfur cluster with disulfide bonds, obstructing the substrate-binding site. It revealed distinctive characteristics of PhRlmCD, including elongated positively charged loops in the central domain and rotational variations in the TRAM domain, which influence substrate selectivity. Additionally, the results suggested that two potential mini-rRNA fragments interact in a similar manner with PhRlmCD at a positively charged cleft at the interface of domains and facilitate dual MTase activities akin to the protein RlmCD. Altogether, these observations showed that Arm5U MTases originated from horizontal gene transfer events, most likely from Gram-positive bacteria.

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通过古菌 RlmCD 类甲基转移酶的显著特征解码其底物选择性
在革兰氏阴性细菌中,5-甲基尿嘧啶(m5U)rRNA 的修饰经常发生在 23S rRNA 结构域 II 和 IV 中的 U747 和 U1939(大肠杆菌编号),分别由依赖 S-腺苷-l-蛋氨酸(SAM)的 rRNA 甲基转移酶(MTase)RlmC 和 RlmD 进行修饰。相比之下,革兰氏阳性细菌利用单一的依赖 SAM 的 rRNA MT 酶 RlmCD 来修饰两个相应的位点。值得注意的是,某些古细菌,特别是热球菌属(Thermococcales)中的古细菌,被发现拥有两个编码依赖 SAM 的古细菌(tRNA 和 rRNA)m5U(Arm5U)MT 酶的基因。其中,一种特异性 tRNA 的 Arm5U MT 酶(PabTrmU54)已经定性。本研究重点研究了嗜热古菌 Pyrococcus horikoshii(PhRlmCD)的 rRNA 特异性 Arm5U MT 酶的结构和功能特征。对其结构的深入研究发现,铁硫簇被二硫键取代后产生了动态铰链运动,阻碍了底物结合位点。该研究揭示了 PhRlmCD 的独特特征,包括中央结构域带正电的拉长环和 TRAM 结构域的旋转变化,这些都会影响底物的选择性。此外,研究结果表明,两个潜在的迷你 RNA 片段以类似的方式与 PhRlmCD 在结构域界面的带正电的裂隙中相互作用,并促进类似于蛋白质 RlmCD 的双重 MT 酶活性。这些观察结果表明,Arm5U MT酶起源于水平基因转移事件,很可能来自革兰氏阳性细菌。
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