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Proc Natl Acad Sci U S A. 1999 Dec 7;96(25):14270-5.

The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA.

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1
Departments of Biochemistry, University of California, San Francisco, CA 94143-0448, USA.

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.

PMID:
10588695
PMCID:
PMC24426
[Indexed for MEDLINE]
Free PMC Article
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