biostudies-literatureFaivre BAgence Nationale de la RechercheFoundation for the National Institutes of HealthNIGMS NIH HHS2278-2289https://www.ebi.ac.uk/biostudies/studies/S-EPMC8632129biostudies-literatureUnknown18(12)Dihydrouridine (D) is a tRNA-modified base conserved throughout all kingdoms of life and assuming an important structural role. The conserved dihydrouridine synthases (Dus) carries out D-synthesis. DusA, DusB and DusC are bacterial members, and their substrate specificity has been determined in <i>Escherichia coli</i>. DusA synthesizes D20/D20a while DusB and DusC are responsible for the synthesis of D17 and D16, respectively. Here, we characterize the function of the unique <i>dus</i> gene encoding a DusB detected in Mollicutes, which are bacteria that evolved from a common Firmicute ancestor via massive genome reduction. Using <i>in vitro</i> activity tests as well as <i>in vivo E. coli</i> complementation assays with the enzyme from <i>Mycoplasma capricolum</i> (DusB<i>_MCap</RNA biologyDihydrouridine synthesis in tRNAs is under reductive evolution in Mollicutes.PMC8632129R01 GM070641ANR-15-CE11-0004-01ANR-11-LABX-0011GM70641De Crecy-Lagard VBregeon DFaivre BFontecave MVo CDFakroun SPecqueur LLombard MGuerineau VHamdane DGoyenvalle CfalseDihydrouridine synthesis in tRNAs is under reductive evolution in Mollicutes.Dihydrouridine (D) is a tRNA-modified base conserved throughout all kingdoms of life and assuming an important structural role. The conserved dihydrouridine synthases (Dus) carries out D-synthesis. DusA, DusB and DusC are bacterial members, and their substrate specificity has been determined in <i>Escherichia coli</i>. DusA synthesizes D20/D20a while DusB and DusC are responsible for the synthesis of D17 and D16, respectively. Here, we characterize the function of the unique <i>dus</i> gene encoding a DusB detected in Mollicutes, which are bacteria that evolved from a common Firmicute ancestor via massive genome reduction. Using <i>in vitro</i> activity tests as well as <i>in vivo E. coli</i> complementation assays with the enzyme from <i>Mycoplasma capricolum</i> (DusB<i>_MCap</2021-01-01T00:00:00Z2021 Dec2026-04-08T17:50:26.825Z2025-04-05T15:22:41.811ZS-EPMC86321293368536610.1080/15476286.2021.1899653