The objective is to understand the several functions of thymidylate synthase, an enzyme that provides the sole de novo pathway for synthesis of a nucleotide that is essential for DNA synthesis, at the level of three dimensional atomic structure. Inhibition of thymidylate synthase causes thymineless death of an organism. Through determining three-dimensional structures of complexes of the enzyme, substrate and cofactor, the reaction mechanisms and conformational dynamics will be understood at atomic resolution, sufficient to eventually design inhibitors, and drugs of higher specificity or tailored function. Modelling and energy calculations are to be developed for eventual design of improved inhibitors, and cancer drugs targeted to the human enzyme. Thymidylate synthase is one of the most conserved proteins throughout all species from bacterial viruses to man. Structure determinations of binding sites of enzymes from other species are to open the way to species specific anti-parasitic drug design, targeted toward Leishmaniasis, malaria and Varicella zoster. Site-directed mutagenesis is coupled to atomic resolution structural analysis and functional assay to define all the determinants of activity, and to understand regions that may provide for regulation in eucaryotes. A powerful selection of functional enzyme (TS) expressed in TS- E. coli is harnessed to cassette mutagenesis of a synthesized gene to produce subtle alteration of function.
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