Nucleic Acids Research, Vol 27, Issue 18 3702-3711, Copyright © 1999 by Oxford University Press
DM Landis, JL Gerlach, ET Adman and LA Loeb
Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in
cancer therapy. In the cell, 5-FU is metabolized to 5- fluorodeoxyuridylate
(5-FdUMP), a tight binding covalent inhibitor of thymidylate synthase (TS).
In order to create 5-FdUMP resistant enzymes to protect chemosensitive
normal cells and further understand mechanisms of 5-FdUMP resistance, we
have randomized four residues within the active site of TS. Our previous
studies identified alterations in residues which produce active TS with
enhanced resistance to 5-fluorouridine (5-FdUR). By remutagenizing a subset
of the 13 previously targeted residues (A197, L198, C199 and V204), an
unbiased random library can be created allowing for extensive testing of
all possible amino acid substitutions at each of the sites. Using genetic
complementation and selection in Escherichia coli, we identified the
spectrum of substitutions that yield active TS as well as those that
resulted in 5-FdUR resistant mutants of TS. The 5-FdUR resistant TS were
found to share several structural features including hydrophobic
substitutions at residue 197, retention of the wild-type leucine 198, the
alteration C199L (present in 64% of the drug-resistant library), and polar
alterations of valine 204. The catalytic activity of mutants with these
features was approximately equal to that of the wild-type TS.
ARTICLES
Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues
The Joseph Gottstein Memorial Cancer Research Laboratory, Departments of Pathology and Biochemistry, University of Washington School of Medicine, Box 357705, Seattle, WA 98195-7705, USA.
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