title: The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA.
abstract: tRNA pseudouridine synthase I (PsiSI) catalyzes the conversion of uridine to Psi at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. PsiSI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNA(Phe) containing 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 PsiSI 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 PsiS.
authors.affiliation: Departments of Biochemistry, University of California, San Francisco, CA 94143-0448, USA.
authors.forename: X
authors.initials: X
authors.lastname: Gu
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
mesh_terms: D005472:Fluorouracil / Q000737:chemistry / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012343:RNA, Transfer; D019751:Intramolecular Transferases; C023199:tRNA-pseudouridine synthase I; D005472:Fluorouracil