Crystallographic studies of thymidylate synthase: Structure of a mammalian enzyme and analyses of invariant non-catalytic residues in a bacterial enzyme

Abstract

Thymidylate synthase (TS, EC 2.1.1.45) is the enzyme responsible for the synthesis of 2'-deoxythymidine 5'-monophosphate (dTMP), using 2'-deoxyuridine 5'-monophosphate (dUMP) as the substrate and 5,10-methylene-5,6,7,8-tetrahydrofolate (CH₂H₄ folate) as both carbon donor and reductant. Inhibition of TS stops growth of rapidly dividing cells, and for decades TS inhibitors such as 5-fluorouracil, and more recently folyl-analogs, have been used as anticancer drugs. However, prior to my studies, there were no structures available of any ligand-bound mammalian TS. In this dissertation I present the crystal structure of rat TS bound to the substrate dUMP and the anticancer drug Tomudex. Unexpectedly, the enzyme has an open conformation, with ligands bound but no covalent adduct between the catalytic cysteine (Cys 189) and the nucleotide, unlike that reported for the same complex with the E. coli enzyme. Three changes in amino acid sequence between the E. coli and rat TS proteins, namely ecT78 → R101, ecW83 → rN106 and ecV262 → rM305, result in loss of van der Waals contacts with Tomudex. These changes coupled with the loss of a hydrogen bond between the Tomudex 2-quinazoline position, which has been changed from the amino group of the cofactor to a methyl group, suggest that Tomudex may inhibit mammalian TS by stabilizing the open conformation. In a second project, I have studied the role of two conserved residues, K48 and R166 in catalysis of E. coli TS. Mutation of each of these residues to glutamine produces nearly inactive proteins. Crystallographic analyses of K48Q and R166Q suggest that the loss of these charged groups reduces binding of the ternary covalent intermediate. Superposition of the mutated structures with a previously determined wild type structure containing a close analog of this intermediate (TS-FdUMP-CH₂H₄ folate), reveals that the mutants are either more open or distorted, and therefore unable to contact the ligands properly. Both K(m) and k(cat) are altered for the two enzymes, with K(m) increasing about 10-fold, and kcat reduced 400-fold (K48Q) and 3,400-fold (R166Q). Taken together, these data suggest that K48Q and R166Q bind weakly one or more of the reaction intermediates, leading to near inactivity.