Vol. 57, Issue 2, 359-366, February 2000

Drug-Resistant Variants of Escherichia coli Thymidylate Synthase: Effects of Substitutions at Pro-254

Corinne Fantz, David Shaw, William Jennings, Antonia Forsthoefel, Maria Kitchens, Jason Phan, Wladek Minor, Lukasz Lebioda, Franklin G. Berger, and H. Trent Spencer

Departments of Biological Sciences (D.S., W.J., A.F., M.K., F.G.B., H.T.S.) and Chemistry and Biochemistry (C.F., J.P., L.L.), University of South Carolina and the South Carolina Cancer Center (H.T.S.), Columbia, South Carolina; and Department of Molecular Pharmacology and Biophysics (W.M.), University of Virginia, Charlottesville, Virginia.

Drug-resistant variants of thymidylate synthase (TS) can potentially be used in gene therapy applications to decrease the myelosuppressive side effects of TS-directed anticancer agents or to select genetically modified cells in vivo. Mutations of proline 303 of human TS confer resistance to TS-directed fluoropyrimidines and antifolates (Kitchens et al., 1999). We generated the corresponding variants in Escherichia coli TS (ecTS), position 254, to better understand the mechanism by which mutations at this residue confer resistance. In addition, because ecTS is intrinsically resistant to several antifolates when compared with human TS, we suspected that greater resistance could be achieved with the bacterial enzyme. The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2'-deoxyuridine (FdUrd) compared with wild-type ecTS. Four additional mutants (P254F, P254S, P254G, and P254D), each of which complemented growth of a TS-deficient cell line, were generated, isolated, and characterized. Steady-state values of K_m for dUMP and k_cat were not substantially different among the variants and were comparable with the wild-type values, but K_m for methylenetetrahydrofolate (CH₂H₄PteGlu) was >10-fold higher for P254D. Values of k_on and k_off for nucleotide binding, which were obtained by stopped-flow spectroscopy, were virtually unchanged among the mutants. Drastic differences were observed for CH₂H₄PteGlu binding, with K_d values >15-fold higher than observed with the wild-type enzyme; surprisingly, the proposed isomerization reaction that is very evident for the wild-type enzyme is not observed with P254S. The decrease in affinity for CH₂H₄PteGlu correlates well with K_i values obtained for three TS-directed inhibitors. These results show that mutations at Pro-254 specifically affect the initial binding interactions between enzyme and cofactor and also alter the ability of the mutant enzymes to undergo conformational changes that occur on ternary complex formation. The crystal structure of P254S was determined at 1.5 Å resolution and is the most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253; its carbonyl is rotated approximately 40°, and the side chain forms an ion pair with Arg-225.