The broad objectives of the work are to understand the functions of thymidylate synthase (TS) and other nucleotide, RNA and DNA-modifying enzymes. TS is an important drug target, since it provides the sole de novo pathway for synthesis of an essential DNA nucleotide. TS is the best characterized of the class of proteins that catalyze methyl transfer reactions in pyrimidine biosynthesis. Insights into the mechanism of TS have been applied to the study of related enzymes, such as the dUMP and dCMP hydroxymethylases, and DNA and RNA cytosine methyltransferases, and have deepened our understanding of general principles of catalysis of two-substrate reactions. The mechanism of TS will be investigated at a very detailed level by determining crystal structures of TS variants, generated by mutagenesis, in binary and ternary complexes with substrate, cofactor, or their analogs. Structures will be related to the results of kinetic and functional assays. A saturation mutagenesis approach will be used to define the roles of the approximately 25 conserved residues in the active site cavity. In this approach, a synthesized L. casei TS gene with strategically placed restriction sites is used to make all substitutions of the natural amino acids at a given site. Mutants of residues shown to have a role in the chemical steps following ternary complex formation will be made in E. coli TS for crystallographic study, since E. coli ternary complex crystal structures can be determined to ad least 2 Angstrom units resolution. Mutations which impair catalysis at different points in the multistep reaction will be used to isolate structures of new reaction intermediates by crystallography. The mechanism for hydride transfer in TS will be studied by determining the structure of SP01 dUMP-hydroxymethylase. This enzyme is structurally and mechanistically closely related to TS, but does not undergo the hydride transfer step. The enzyme will be crystallized and its structure determined by MIR methods. The crystal structure of tRNA pseudouridine synthase I, which modifies a uridine base in an E. Coli tRNA, will be determined from already grown crystals that diffract to 1.35 Angstrom units resolution. MIR and MAD phasing techniques will be used to solve the structure. The protein will also be crystallized as a complex with a tRNA inhibitor. The mechanism of pseudouridine synthase will be investigated by mutagenesis of residues chosen based on the crystal structures.
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