The Mechanism of Pseudouridine Synthase I as Deduced from Its Interaction with 5-fluorouracil-tRNA

tRNA pseudouridine synthase I (Ψ SI) catalyzes the conversion of uridine to Ψ at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. Ψ SI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNAPhecontaining FUra in place of Ura) to form a putative analog of a steady-state intermediate in the normal reaction pathway. Previously, we proposed that a conserved aspartate of the enzyme serves as a nucleophilic catalyst in both the normal enzyme reaction and in the formation of a covalent complex with FUra-tRNA. The covalent adduct between FUra-tRNA and Ψ SI was isolated and disrupted by hydrolysis and the FUra-tRNA was recovered. The target FU39 of the recovered FUra-tRNA was modified by the addition of water across the 5,6-double bond of the pyrimidine base to form 5,6-dihydro-6-hydroxy-5-fluorouridine. We deduced that the conserved aspartate of the enzyme adds to the 6-position of the target FUra to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage to form the observed product. Assuming that an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for Ψ S.