Structural and functional studies on cad, the anti-tumoral target protein leading de novo biosynthesis of pyrimidines

Abstract

CAD is a large multifunctional protein catalyzing the initial three steps in de novo biosynthesis of pyrimidine nucleotides in animals. Since pyrimidines are the building blocks of nucleic acids and the precursors of other key macromolecular substances, the up-regulation of CAD’s activity is essential for cell growth and proliferation, especially in neoplastic cells. Thus, CAD has been considered an attractive target for the development of anti-tumoral compounds. However, despite the central metabolic role and its therapeutic potential, due to the lack of knowledge about its organization and the structure and function of its different enzymatic domains, no robust inhibitor that could be used as anti-proliferative drug has been designed thus far. In this bachelor thesis, a research study on CAD’s DHOase domain was conducted, particularly focused in understanding the role of a flexible loop that appears to play a conserved catalytic role from E.coli to humans. To examine the catalytic mechanism of this loop, a cloning approach was designed to generate a human DHOase construct bearing the equivalent flexible loop of the E.coli enzyme. This chimeric protein was expressed either in mammalian cells or in bacteria cultures and purified using different chromatographic techniques. Activity assays on both the forward and reverse directions of the reaction were then performed in the chimeric huDHOase to estimate the turnover rate of the mutant. With a negligible enzymatic activity (less than 2% of the wild type) the experiments here presented prove that, despite having a conserved functional role, the flexible loop of E.coli and human DHOases are not interchangeable. Overall, the results confirmed the implication of the flexible loop in oligomerization and in the catalytic mechanism of DHOases, highlighting key differences in the functioning of both mammalian and bacterial enzymes that will be exploited in future work for the rational design of specific inhibitors against CAD.