Bardaweel, Sanaa2012-01-172012-01-172011-12https://hdl.handle.net/11299/119747University of Minnesota Ph.D. dissertation. December 2011. Major: Medicinal Chemistry. Advisor: Dr. Carston R. Wagner. 1 computer file (PDF); xvii, 178 pages.Histidine triad nucleotide binding proteins (Hints) are members of the histidine triad (HIT) protein superfamily of nucleotidyl transferases and hydrolyases. It has been recently demonstrated that Hints are efficient phosphoramidases and therefore activators of potent antiviral and anticancer pronucleotides. In spite of their high evolutionary conservation among all kingdoms of life, and the several regulatory functions in which Hints have been implicated, a clear connection between their observed function and their catalytic efficiency has not been elucidated. To gain a comprehensive understanding of the essential role of these ubiquitous enzymes, our laboratory has devoted a considerable effort toward the delineation of the principles governing Hints catalysis and cellular function. Such understanding will provide an unprecedented ability to assess the role of these highly conserved, but functionally unknown enzymes. Since Hints are found in both prokaryotes and eukaryotes, we have attempted to understand their function, mechanism, and structural determinants in prokaryotes, under the assumption that their role may be at least partially conserved among members of the tree of life. Recently, we have demonstrated by E. coli gene disruption studies that the bacterial Hint enzyme is necessary for growth under high salt conditions, and when alanine is a carbon and nitrogen source. Through a combination of phenotypic screening and complementation experiments with wild-type and ecHinT knock-out E. coli strains, we have shown that catalytically-active ecHinT is required for growth on D-alanine. In addition, using Hint-inhibitors and active-site mutants, we have demonstrated that expression of catalytically-active ecHinT is essential for the activity of the enzyme D-amino acid dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-amino acids metabolic pathway. These results are considered as the first report in literature that shows a successful connection between a discovered Hint-related phenotype and the catalytic activity of Hint. Previously, we have demonstrated that lysyl-AMP generated by LysRS is a substrate for both human and E. coli Hints. In addition, we have shown that the ability of Hint to hydrolyze lysyl-AMP depends on its enzymatic activity. Here, we demonstrate that the molecular determinants governing this regulation appear to reside in the C-terminus region of Hint. Interestingly, the ecHinT-DadA interaction appears also to be governed by both ecHinT-activity and the C-terminus loop. We have also expanded our scope to look at possible toxicity of D-alanine in E. coli strains lacking dadA or hinT. Our results demonstrate that E. coli mutants lacking dadA or hinT are highly susceptible to D-alanine toxicity and that the catalytic activity of Hint is an essential requirement to protect E. coli from the observed toxicity of D-alanine. Based on careful analysis of the combined results from the ecHinT-LysRS and ecHinT-DadA potential interactions, and comprehensive understanding of the D-alanine metabolic pathway in bacteria, we proposed a possible regulatory mechanism of Hint, LysRS and DadA on global protein translational processes to prevent D-amino acids toxicity in E. coli.en-USBiocatalysisHistidine triad nucleotide binding proteinPhenotypeMedicinal ChemistryThesis or Dissertation