Hu You, Jaroch Marshall, Sun Guangxin, Dedon Peter C, de Crécy-Lagard Valérie, Bruner Steven D
Department of Chemistry, University of Florida, Gainesville, Florida 32611, United States.
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611, United States.
Biochemistry. 2025 Jan 21;64(2):458-467. doi: 10.1021/acs.biochem.4c00524. Epub 2024 Dec 7.
Post-transcriptional modifications at the anticodon stem-loop of tRNAs are key to the translation function. Metabolic pathways to these modifications often incorporate complex enzymology. A notable example is the hypermodified nucleoside, queuosine, found at the wobble position of Asn, Asp, His, and Tyr encoding tRNAs. The epoxyqueuosine reductase, QueH, catalyzes the final step in the biosynthetic pathway to queuosine. The metalloenzyme catalyzes a two-electron reduction of epoxyqueuosine to provide the modified tRNA. The structure of QueH from has previously been determined and unexpectedly contains two metal binding motifs in the active site. This includes a predicted 4Fe-4S cluster, along with a single-metal binding site coordinated by two cysteines along an aspartate carboxylate. In this report, we describe the structural and biochemical analysis of the QueH metal binding sites along with the chemistry of epoxide deoxygenation. To probe the active-site architecture, enzyme mutants of metal binding residues were structurally and biochemically characterized. In addition, structural and binding experiments were used to probe interactions of QueH with tRNA and the in vivo role of QueH and variants in Q-tRNA synthesis was evaluated. Overall, this work provides insight into the chemical mechanism of the final step of the queuosine biosynthetic pathway.
转运RNA（tRNA）反密码子茎环处的转录后修饰是翻译功能的关键。这些修饰的代谢途径通常涉及复杂的酶学过程。一个显著的例子是超修饰核苷 queuosine，它存在于编码天冬酰胺、天冬氨酸、组氨酸和酪氨酸的tRNA的摆动位置。环氧queuosine还原酶QueH催化了queuosine生物合成途径的最后一步。这种金属酶催化环氧queuosine的双电子还原反应，从而产生修饰后的tRNA。此前已确定了来自[具体来源未给出]的QueH的结构，其活性位点意外地包含两个金属结合基序。这包括一个预测的4Fe-4S簇，以及一个由两个半胱氨酸沿着一个天冬氨酸羧酸盐配位的单金属结合位点。在本报告中，我们描述了QueH金属结合位点的结构和生化分析以及环氧化物脱氧反应的化学过程。为了探究活性位点结构，对金属结合残基的酶突变体进行了结构和生化表征。此外，利用结构和结合实验来探究QueH与tRNA的相互作用，并评估了QueH及其变体在Q-tRNA合成中的体内作用。总体而言，这项工作为queuosine生物合成途径最后一步的化学机制提供了深入见解。
