Crystal structures of a marginally active thymidylate synthase mutant, Arg 126-->Glu

Abstract

Thymidylate synthase (TS) is a long-standing target for anticancer drugs and is of interest for its rich mechanistic features. The enzyme catalyzes the conversion of dUMP to dTMP using the co-enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon-carbon bond formation. Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone. Despite this, when Arg 126 of TS from Escherichia coli was changed to glutamate (R126E), the resulting protein had kcat reduced 2000-fold and Km reduced 600-fold. The crystal structure of R126E was determined under two conditions--in the absence of bound ligand (2.4 A resolution), and with dUMP and the antifolate CB3717 (2.2 A resolution). The first crystals, which did not contain dUMP despite its presence in the crystallization drop, displayed Glu 126 in a position to sterically and electrostatically interfere with binding of the dUMP phosphate. The second crystals contained both dUMP and CB3717 in the active site, but Glu 126 formed three hydrogen bonds to nearby residues (two through water) and was in a position that partially overlapped with the normal phosphate binding site, resulting in a approximately 1 A shift in the phosphate group. Interestingly, the protein displayed the typical ligand-induced conformational change, and the covalent bond to Cys 146 was present in one of the protein's two active sites.