We are trying to determine the reaction pathway for a key enzyme and drug target, Thymidilate synthase (TS), that provides the sole de novo pathway for the biosynthesis of thymidine 5'-monophosphate (dTMP). As an essential enzyme for DNA synthesis, TS is found in almost all living species and many viruses. Thus, TS is an important drug target for the development of anti-cancer, anti-parasitic, anti-fungal, and anti-viral agents. TS uses methylene tetrahydrofolate as the carbon source for methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) in the reaction. A species that offers great advantage in access to mutagenesis, and for which a wildtype crystal structure has been determined at UCSF, is Lactobacillus casei (LCTS). Many kinetic studies on LCTS and mutants of the protein have been performed in Dr. Santi's lab at UCSF. During the past year we have completed studies on the crystal structures of ternary and binary complexes of the poorly active LCTS mutant E60Q. We are also continuing studies on three other mutants that greatly affect enzyme activity: D221A, D221C, and R218K. We have been able to compare directly the active sites of the wildtype and mutant LCTS ternary complexes in order to deduce crucial changes in important water molecule positions and amino acid side-chain orientations that may affect enzyme activity using the facilities of the Computer Graphics Laboratory.
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