title: Creation and characterization of 5-fluorodeoxyuridine-resistant Arg50 loop mutants of human thymidylate synthase.
abstract: Thymidylate synthase catalyzes the reductive methylation of dUMP to dTMP and is essential for the synthesis of DNA. Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluoro-2'-deoxyuridine 5'-monophosphate, a tight binding covalent inhibitor of thymidylate synthase. Recent studies have identified 5-fluoro-2'-deoxyuridine (5-FdUR) and antifolate-resistant mutants of human thymidylate synthase (TS) that contain single residue substitutions within the highly conserved Arg50-loop, which binds the pyrimidine substrate (Y. Tong et al., J. Biol. Chem. 273: 11611-11618, 1998). We have used random sequence mutagenesis to gain structure-function information about the TS and to create novel drug-resistant mutants for gene therapy. A library of 1.5 million mutants of the Arg50-loop and the nearby residue Tyr 33 was selected to identify mutants of the human enzyme with the ability to complement a thymidylate synthase-deficient Escherichia coli strain and form colonies in the presence of 5-FdUR. E. coli-harboring plasmids that were encoding TS with single, double, and triple amino acid substitutions were identified that survive at dosages of 5-FdUR clearly lethal to E. coli harboring either wild-type thymidylate synthase or constructs encoding previously characterized drug resistant mutants. Four 5-FdUR-resistant mutants were purified to apparent homogeneity. Kinetic studies indicate that these enzymes are highly efficient. Inhibition constants (Ki) for the double mutant K47Q;D48E and the triple mutant D48E;T51S;G52C in the presence of 5-fluoro-2'-deoxyuridine 5'-monophosphate were determined to be 75 to 100 times higher, respectively, than that of the wild-type enzyme. These mutant TSs, or others similarly created and selected, could be used to protect bone marrow cells from the cytotoxic side effects of 5-FU chemotherapy.
authors.affiliation: Department of Pathology, University of Washington School of Medicine, Seattle 98195-7705, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Landis
authors.forename: C C
authors.initials: CC
authors.lastname: Heindel
authors.forename: L A
authors.initials: LA
authors.lastname: Loeb
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D001120:Arginine / Q000235:genetics*; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D005467:Floxuridine / Q000494:pharmacology*; D015723:Gene Library; D005816:Genetic Complementation Test; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis; D009154:Mutation; D013940:Thymidylate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D005467:Floxuridine; D001120:Arginine; D013940:Thymidylate Synthase