Seo Suyoung, Kim Minsoo, Rhee Sangkee
Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.
Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea; Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Korea.
J Biol Chem. 2025 Jun;301(6):110257. doi: 10.1016/j.jbc.2025.110257. Epub 2025 May 21.
Pseudouridine (Ψ) is one of the most abundant RNA modifications. Following RNA degradation, Ψ nucleotides are dephosphorylated and catabolized into uracil and ribose 5'-phosphate via a two-step enzymatic reaction catalyzed by enzymes present in many bacteria and eukaryotes, but not in mammals. Malfunction of Ψ catabolism has adverse physiological effects in plants. In humans, the enzyme HDHD1A dephosphorylates pseudouridine 5'-monophosphate (ΨMP), and the resulting Ψ is excreted in the urine. In this study, we determined the crystal structures of human HDHD1A (hHDHD1A) complexed with Ψ. The structure of hHDHD1A consists of a catalytic domain with a Rossmann α/β-fold and a cap domain, with a magnesium ion at the junction of the two domains. Ψ is bound to the active site in an orientation where its nucleobase, uracil-Ψ, is surrounded by the cap domain residues, and the ribose moiety is located next to the Mg-binding site. The active site is composed mainly of hydrophobic residues, but two essential charged residues, Glu23 and Lys46, are present in the vicinity of uracil-Ψ. Glu23 interacts with the Ψ-specific N1 atom, while Lys46 interacts with the O2 atom of uracil-Ψ. Mutagenesis and kinetic analysis indicated that active site residues are involved in substrate binding and/or catalysis. In addition to Ψ-specific hydrophilic interactions, shape complementarity between ΨMP and the active site pocket is a key element underlying substrate specificity in hHDHD1A. This study provided structural and functional insights into the substrate specificity of hHDHD1A for ΨMP, highlighting both similarities and differences compared to other Ψ-recognizing enzymes.
假尿苷（Ψ）是最丰富的RNA修饰之一。RNA降解后，Ψ核苷酸会发生去磷酸化，并通过许多细菌和真核生物（但哺乳动物中不存在）中的酶催化的两步酶促反应分解为尿嘧啶和5'-磷酸核糖。Ψ分解代谢功能异常在植物中具有不良生理影响。在人类中，HDHD1A酶使假尿苷5'-单磷酸（ΨMP）去磷酸化，产生的Ψ随尿液排出。在本研究中，我们确定了与Ψ复合的人类HDHD1A（hHDHD1A）的晶体结构。hHDHD1A的结构由一个具有Rossmann α/β折叠的催化结构域和一个帽结构域组成，在两个结构域的交界处有一个镁离子。Ψ以其核碱基尿嘧啶-Ψ被帽结构域残基包围、核糖部分位于镁结合位点旁边的方向结合到活性位点。活性位点主要由疏水残基组成，但在尿嘧啶-Ψ附近存在两个必需的带电荷残基Glu23和Lys46。Glu23与Ψ特异性的N1原子相互作用，而Lys46与尿嘧啶-Ψ的O2原子相互作用。诱变和动力学分析表明，活性位点残基参与底物结合和/或催化。除了Ψ特异性的亲水相互作用外，ΨMP与活性位点口袋之间的形状互补性是hHDHD1A底物特异性的关键因素。本研究提供了关于hHDHD1A对ΨMP底物特异性的结构和功能见解，突出了与其他识别Ψ的酶相比的异同。
