2024-03-29T02:28:16Zhttps://eprints.lib.hokudai.ac.jp/dspace-oai/requestoai:eprints.lib.hokudai.ac.jp:2115/493522022-11-17T02:08:08Zhdl_2115_20074hdl_2115_161Molecular basis of dihydrouridine formation on tRNAYu, FutaoTanaka, YoshikazuYamashita, KeitaroSuzuki, TakeoNakamura, AkiyoshiHirano, NagisaSuzuki, TsutomuYao, MinTanaka, Isaoprotein-tRNA complexRNA modificationsubstrate recognitionX-ray crystallography467Dihydrouridine (D) is a highly conserved modified base found in tRNAs from all domains of life. Dihydrouridine synthase (Dus) catalyzes the D formation of tRNA through reduction of uracil base with flavin mononucleotide (FMN) as a cofactor. Here, we report the crystal structures of Thermus thermophilus Dus (TthDus), which is responsible for D formation at positions 20 and 20a, in complex with tRNA and with a short fragment of tRNA (D-loop). Dus interacts extensively with the D-arm and recognizes the elbow region composed of the kissing loop interaction between T- and D-loops in tRNA, pulling U20 into the catalytic center for reduction. Although distortion of the D-loop structure was observed upon binding of Dus to tRNA, the canonical D-loop/T-loop interaction was maintained. These results were consistent with the observation that Dus preferentially recognizes modified rather than unmodified tRNAs, indicating that Dus introduces D20 by monitoring the complete L-shaped structure of tRNAs. In the active site, U20 is stacked on the isoalloxazine ring of FMN, and C5 of the U20 uracil ring is covalently cross-linked to the thiol group of Cys93, implying a catalytic mechanism of D20 formation. In addition, the involvement of a cofactor molecule in uracil ring recognition was proposed. Based on a series of mutation analyses, we propose a molecular basis of tRNA recognition and D formation catalyzed by Dus.National Academy of SciencesJournal Articleapplication/pdfapplication/pdfhttp://hdl.handle.net/2115/49352https://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/49352/2/PNAS108-49_19593-19598.pdfhttps://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/49352/1/Supporting_Information.pdf0027-8424Proceedings of the National Academy of Sciences of the United States of America1084919593195982011-12-06enginfo:doi/10.1073/pnas.1112352108author