Abstract
BackgroundDihydrouridine (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”.ResultsTo 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.ConclusionsWe 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.
Highlights
Dihydrouridine (D) is a modified base found in conserved positions in the D-loop of tRNA in Bacteria, Eukaryota, and some Archaea
Sequence database searches and retrieval of members of the dihydrouridine synthases (DUS) family To identify a complete set of DUS sequences, we used full-length sequences of experimentally characterized DUS from E. coli and S. cerevisiae to carry out exhaustive PSI-BLAST [14] searches of the database and retrieved all sequences reported with e-value better than 1e-25
The clustering confirmed sequence similarity of KOG1799 and KOG1436 to DUS. This analysis has confirmed that the TM0096 protein from Thermatoga maritima, annotated as “a putative flavin oxidoreductase”, whose crystal structure has been solved in complex with a flavin mononucleotide by the Structural GenomiX consortium (Protein Data Bank ID 1vhn) [16], is a member of the DUS family
Summary
Dihydrouridine (D) is a modified base found in conserved positions in the D-loop of tRNA in Bacteria, Eukaryota, and some Archaea. Dihydrouridine (D; 5,6-dihydro-uridine) is one of the posttranscriptionally modified nucleosides. It is a product of the reduction of uridine (U), and can be further modified to 5-methyldihydrouridine (m5D). D is commonly present in the tRNA from Bacteria, Eukaryota, and some Archaea [1]. It was identified in six positions in the “Dloop” of the tRNA (16, 17, 20a, 20b) and in position 47 in the variable loop. A conformational change caused by this modification probably increases flexibility and dynamics of RNA regions that participate in 3D interactions. The knowledge of the exact role of D in mediating RNA function is still limited
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