The overall objectives are to understand the several function of thymidylate synthase (TS), one of the most conserved of all enzymes throughout living organisms, and a paradigm for methyl transfer reactions in pyrimidine biosynthesis. TS is an important drug target since it provides the sole de novo pathway for synthesis of an essential nucleotide for DNA synthesis. The stereochemistry of the carbon-carbon bond forming methyl transfer reaction, the extensive protein structural changes which serve to sequester reactants, and the reorientation of ligands which take place during catalysis are to be defined from atomic structures of complexes of thymidylate synthase which mimic intermediates in the reaction path. Roles of individual residues and water molecules will be determined quantitatively by structure determination of variants generated by mutagenesis, in binary complex with substrate or in ternary complex with substrate and cofactor, or analogs. A highly efficient mutagenesis strategy of a gene synthesized to have optimally placed, unique restriction sites is used to generate substitutions for all other natural amino acids at a site, followed by an initial rapid screen for functional variants. The most informative variants will be purified using a high efficiency expression (-5-30% total protein in E. coli), and two step purification, and assayed quantitatively to define effects on binding and catalytic steps in the reaction. Structures will be determined by difference Fournier methods against one of eight different crystal forms solved for different structural states, highly refined, and related quantitatively to alterations in binding and kinetic rates. The role of TS in transcriptional regulation of the TS gene by its binding of mRNA will be defined at the structural level. Ligand-protein structures and binding affinities for variants will be applied to derive an empirical relationship between binding affinity and solvation, entopic, electrostatic, and van der Waals forces, with the ultimate goal of developing a scheme for accurately predicting binding affinity of modified inhibitors to this or other important drug targets. The structure of a homologous enzymes which catalyzes one-carbon transfer to pyrimidine, deoxyuridylate-hydroxymethylase will be determined. The structural basis for the difference in catalyzing transfer of hydroxide versus hydride in the reaction will be defined.
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