Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.
Department of Molecular Science & Technology, Ajou University, Suwon, Korea.
J Biol Chem. 2022 May;298(5):101869. doi: 10.1016/j.jbc.2022.101869. Epub 2022 Mar 26.
Pseudouridine, one major RNA modification, is catabolized into uracil and ribose-5'-phosphate by two sequential enzymatic reactions. In the first step, pseudouridine kinase (PUKI) phosphorylates pseudouridine to pseudouridine 5'-monophosphate. High-fidelity catalysis of pseudouridine by PUKI prevents possible disturbance of in vivo pyrimidine homeostasis. However, the molecular basis of how PUKI selectively phosphorylates pseudouridine over uridine with >100-fold greater efficiency despite minor differences in their K values has not been elucidated. To investigate this selectivity, in this study we determined the structures of PUKI from Escherichia coli strain B (EcPUKI) in various ligation states. The structure of EcPUKI was determined to be similar to PUKI from Arabidopsis thaliana, including an α/β core domain and β-stranded small domain, with dimerization occurring via the β-stranded small domain. In a binary complex, we show that Ser30 in the substrate-binding loop of the small domain mediates interactions with the hallmark N1 atom of pseudouridine nucleobase, causing conformational changes in its quaternary structure. Kinetic and fluorescence spectroscopic analyses also showed that the Ser30-mediated interaction is a prerequisite for conformational changes and subsequent catalysis by EcPUKI. Furthermore, S30A mutation or EcPUKI complexed with other nucleosides homologous to pseudouridine but lacking the pseudouridine-specific N1 atom did not induce such conformational changes, demonstrating the catalytic significance of the proposed Ser30-mediated interaction. These analyses provide structural and functional evidence for a pseudouridine-dependent conformational change of EcPUKI and its functional linkage to catalysis.
假尿嘧啶核苷是一种主要的 RNA 修饰物，通过两个连续的酶促反应代谢为尿嘧啶和核糖-5'-磷酸。在第一步中，假尿嘧啶核苷激酶 (PUKI) 将假尿嘧啶磷酸化生成假尿嘧啶 5'-单磷酸。PUKI 对假尿嘧啶的高保真催化可防止体内嘧啶平衡可能受到干扰。然而，尽管其 K 值存在微小差异，但 PUKI 如何选择性地将假尿嘧啶磷酸化，使其效率比尿嘧啶高 100 倍以上，其分子基础尚未阐明。为了研究这种选择性，本研究通过确定不同连接状态下来自大肠杆菌菌株 B (EcPUKI) 的 PUKI 结构，对其进行了研究。EcPUKI 的结构与来自拟南芥的 PUKI 相似，包括一个 α/β 核心结构域和 β 折叠的小结构域，通过 β 折叠的小结构域发生二聚化。在二元复合物中，我们表明小结构域底物结合环中的 Ser30 介导与假尿嘧啶核苷碱基标志性 N1 原子的相互作用，导致其四元结构发生构象变化。动力学和荧光光谱分析也表明，Ser30 介导的相互作用是 EcPUKI 构象变化和随后催化的先决条件。此外，S30A 突变或与其他与假尿嘧啶同源但缺乏假尿嘧啶特异性 N1 原子的核苷形成复合物的 EcPUKI 不会诱导这种构象变化，证明了所提出的 Ser30 介导的相互作用的催化意义。这些分析为 EcPUKI 的假尿嘧啶依赖性构象变化及其与催化的功能联系提供了结构和功能证据。
