Dansereau Stephen J, Shekhtman Alexander, Epifano Francesco, Genovese Salvatore, Fiorito Serena, Begley Thomas J, Sheng Jia
Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States.
The RNA Institute, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States.
Biochemistry. 2025 Jun 17;64(12):2640-2648. doi: 10.1021/acs.biochem.5c00053. Epub 2025 May 25.
tRNA-2-selenouridine synthase (SelU) is a tRNA-modifying enzyme that is instrumental to bacterial translation by exploiting certain chalcogens. Specifically, this enzyme catalyzes the geranylation of 2-thiouridine at the wobble position of three bacterial tRNAs to enhance the recognition of codons ending in guanosine over adenosine using geranyl pyrophosphate as the cofactor. In addition, SelU is also the working enzyme for a selenation process at the same tRNA position in the presence of selenophosphate. How this enzyme conducts two mechanistically different reactions is a fundamentally interesting question. In order to gain more details about the substrate recognition of SelU, in this work, we identified a small natural compound simamycin (5'-geranyluridine) with strong interactions with this enzyme. Further, through biophysical affinity assays and NMR structural studies, we postulated an allosteric mechanism of SelU catalysis involving cooperativity among each domain and a conformational rearrangement around the active site of its N-terminal domain. This conclusion is supported by the bimolecular quenching constants, number of binding sites, and thermodynamic parameters calculated for this compound complexed with the N-terminal domain of SelU.
tRNA-2-硒代尿苷合酶（SelU）是一种tRNA修饰酶，通过利用某些硫族元素对细菌翻译起重要作用。具体而言，该酶催化三种细菌tRNA摆动位置上的2-硫代尿苷的香叶基化反应，以香叶基焦磷酸为辅因子，增强对以鸟苷结尾而非腺苷结尾的密码子的识别。此外，在亚硒酸磷酸存在的情况下，SelU也是同一tRNA位置硒化过程的作用酶。该酶如何进行两种机制不同的反应是一个极具根本意义的有趣问题。为了更详细地了解SelU的底物识别情况，在这项工作中，我们鉴定出一种与该酶有强烈相互作用的天然小分子化合物西马霉素（5'-香叶基尿苷）。此外，通过生物物理亲和力测定和核磁共振结构研究，我们推测了SelU催化的变构机制，该机制涉及每个结构域之间的协同作用以及其N端结构域活性位点周围的构象重排。与SelU的N端结构域复合的该化合物的双分子猝灭常数、结合位点数和热力学参数支持了这一结论。
