Structure-function studies on Dgt triphosphohydrolase

National Institute Of Environmental Health Sciences

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Abstract

This project is concerned with the structural and functional aspects of a dGTPase of the bacterium E. coli, which has the unusual specificity: dGTP-> dG + PPPi. Although this activity was reported a number of years ago, its function in the cell has been unknown. Recently, we discovered a novel mutator activity in E. coli resulting from a defect in the dgt gene, encoding the above activity. Thus, insight into the function of the Dgt dGTPase may be gleaned from studies of the dgt mutator effect. As one hypothesis, we are considering the possibility that Dgt may be an activity functioning in regulating the canonical dGTP pool in the cell. A second function pertains to antiviral activities. Here, reduction in dGTP levels may impede the ability of bacteriophages to replicate their DNA, thus protecting the bacterium against phage-mediated killing. Our studies of the Dgt protein and function include several approaches: (i) a genetic analysis of the dgt mutator effect in the bacterium E. coli, (ii) a biochemical analysis of the Dgt protein, including study of its regulation and substrate specificity, and (iii) structural analysis of Dgt protein by various methods. The crystal structure of Dgt has revealed a hexameric structure containing two allosterically active ssDNA molecules, and kinetic experiments have shown that ssDNA binding leads to Dgt activation, primarily by lowering the km for the dGTP substrate. The project is now being extended to corresponding enzymes from other bacterial species. Interestingly, these proteins, while all triphosphohydrolases, display different specificities for both dNTP substrates and allosteric activation.