Radiation-resistant Key Protein Drrra And Deformation Of Streptococcus Smu.595 Protein, Structural And Functional Studies

This thesis contains two parts:the structural and functional studies of DrRRA from D.radiodurans R1 and the structural studies of a putative dihydroorotate dehydrogenase (DHOD) from S. mutans.Two-component systems are predominant signal transduction pathways in prokaryotes, and also exist in many archaea as well as some eukaryotes. A typical TCS consists of a histidine kinase and a cognate response regulator. DrRRA has been identified to be essential for the extreme radioresistance of D.radiodurans R1 and is a member of the OmpR/PhoB family of response regulators. In this thesis, we report the crystal structures of DrRRA (1-215) truncation from Dr. and its effector domain truncation at resolutions of 2.3 A and 1.6 A, respectively. DrRRA possesses an N-terminal regulatory domain and a C-terminal winged helix-turn-helix DNA-binding domain, with phosphorylation of the regulatory domain modulating the activity of the protein. Small angle X-ray scattering (SAXS) technique was applied to elucidate a solution structure of DrRRA. From the SAXS measurements, molecular size and shape of DrRRA could be estimated.On the basis of these analytical estimation, it was found that the domain orientation of DrRRA suggests a mechanism of mutual inhibition by the two domains. Activation of DrRRA would require a disruption of this interface to allow the regulatory domain to form the intra- or inter- molecule interactions that are associated with the active state and to allow the recognition helix to interact with DNA. This effects may form a switch, regulating the activity of DrRRA. The domain orientation exhibited by DrRRA also provides a rationale for the variation in linker length that is observed within the OmpR/PhoB family of response regulators.Streptococcus mutans is one of the pathogenic species involved in the dental caries, especially in the initiation and development stage. Here, we report the crystal structure of smu.595, a putative dihydroorotate dehydrogenase (DHOD) from S. mutans solved at 2.4A resolution. DHOD is a flavin mononucleotide containing enzyme, which catalyzes the oxidation of L-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. The reductive lysine methylation procedure was applied in order to improve the diffraction-quality of the crystal. The analysis of the S. mutans DHOD crystal structure depicts that this enzyme is class 1A DHOD and also suggests the potential sites, which are further exploited for the design of highly specific inhibitors through the technology of structure-based vaccine design.