Abstract
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'-O-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.