Xr. Gu et al., The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA, P NAS US, 96(25), 1999, pp. 14270-14275
Citations number
25
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
tRNA pseudouridine synthase I (Psi SI) catalyzes the conversion of uridine
to Psi at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. Psi S
I forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNA(Phe) contai
ning FUra in place of Ura) to form a putative analog of a steady-state inte
rmediate in the normal reaction pathway. Previously, we proposed that a con
served aspartate of the enzyme serves as a nucleophilic catalyst in both th
e normal enzyme reaction and in the formation of a covalent complex with FU
ra-tRNA. The covalent adduct between FUra-tRNA and Psi 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-fluor
ouridine. 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 t
hat an analogous covalent complex is formed in the normal reaction, we have
deduced a complete mechanism for Psi S.