Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues.

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Landis DM,Gerlach JL,Adman ET,Loeb LA

doi

10.1093/nar/27.18.3702

keywords:

subject

Has Abstract

pub_date

1999-09-15 00:00:00

pages

3702-11

issue

18

eissn

0305-1048

issn

1362-4962

pii

gkc559

journal_volume

27

pub_type

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