Dataset Information

Molecular basis of dihydrouridine formation on tRNA.

ABSTRACT: Dihydrouridine (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.

SUBMITTER: Yu F

PROVIDER: S-EPMC3241823 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Molecular basis of dihydrouridine formation on tRNA.

Yu Futao F Tanaka Yoshikazu Y Yamashita Keitaro K Suzuki Takeo T Nakamura Akiyoshi A Hirano Nagisa N Suzuki Tsutomu T Yao Min M Tanaka Isao I

Proceedings of the National Academy of Sciences of the United States of America 20111128 49

Dihydrouridine (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 ...[more]

PMID: 22123979

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Project description:BACKGROUND: Dihydrouridine (D) is a modified base found in conserved positions in the D-loop of tRNA in Bacteria, Eukaryota, and some Archaea. Despite the abundant occurrence of D, little is known about its biochemical roles in mediating tRNA function. It is assumed that D may destabilize the structure of tRNA and thus enhance its conformational flexibility. D is generated post-transcriptionally by the reduction of the 5,6-double bond of a uridine residue in RNA transcripts. The reaction is carried out by dihydrouridine synthases (DUS). DUS constitute a conserved family of enzymes encoded by the orthologous gene family COG0042. In protein sequence databases, members of COG0042 are typically annotated as "predicted TIM-barrel enzymes, possibly dehydrogenases, nifR3 family". RESULTS: To elucidate sequence-structure-function relationships in the DUS family, a comprehensive bioinformatic analysis was carried out. We performed extensive database searches to identify all members of the currently known DUS family, followed by clustering analysis to subdivide it into subfamilies of closely related sequences. We analyzed phylogenetic distributions of all members of the DUS family and inferred the evolutionary tree, which suggested a scenario for the evolutionary origin of dihydrouridine-forming enzymes. For a human representative of the DUS family, the hDus2 protein suggested as a potential drug target in cancer, we generated a homology model. While this article was under review, a crystal structure of a DUS representative has been published, giving us an opportunity to validate the model. CONCLUSIONS: We compared sequences and phylogenetic distributions of all members of the DUS family and inferred the phylogenetic tree, which provides a framework to study the functional differences among these proteins and suggests a scenario for the evolutionary origin of dihydrouridine formation. Our evolutionary and structural classification of the DUS family provides a background to study functional differences among these proteins that will guide experimental analyses.

Dataset Information

Molecular basis of dihydrouridine formation on tRNA.

Publications

Molecular basis of dihydrouridine formation on tRNA.

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