Drug-resistant variants of Escherichia coli thymidylate synthase: Effects of substitutions at Pro-254

Drug-resistant variants of thymidylate synthase (TS) can potentially be use d in gene therapy applications to decrease the myelosuppressive side effect s of TS-directed anticancer agents or to select genetically modified cells in vivo. Mutations of proline 303 of human TS confer resistance to TS-direc ted 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 resista nce could be achieved with the bacterial enzyme. The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2'-deoxyuridine (FdU rd) compared with wild-type ecTS. Four additional mutants (P254F, P254S, P2 54G, and P254D), each of which complemented growth of a TS-deficient cell l ine, were generated, isolated, and characterized. Steady-state values of K- m for dUMP and k(cat) were not substantially different among the variants a nd were comparable with the wild-type values, but Km for methylenetetrahydr ofolate (CH(2)H(4)PteGlu) was >10-fold higher for P254D. Values of k(on) an d k(off) for nucleotide binding, which were obtained by stopped-flow spectr oscopy, were virtually unchanged among the mutants. Drastic differences wer e observed for CH(2)H(4)PteGlu binding, with K-d values >15-fold higher tha n observed with the wild-type enzyme; surprisingly, the proposed isomerizat ion reaction that is very evident for the wild-type enzyme is not observed with P254S. The decrease in affinity for CH(2)H(4)PteGlu correlates well wi th K-i values obtained for three TS-directed inhibitors. These results show that mutations at Pro-254 specifically affect the initial binding interact ions between enzyme and cofactor and also alter the ability of the mutant e nzymes to undergo conformational changes that occur on ternary complex form ation. The crystal structure of P254S was determined at 1.5 Angstrom resolu tion and is the most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mo stly Asp-253; its carbonyl is rotated approximately 40 degrees, and the sid e chain forms an ion pair with Arg-225.