Structure And Function Of Small Molecular Weight Adenosine Succinate Synthase And Pharmacological Study Of Its Products

Adenylosuccinate synthase (AdSS) presents in all organisms, which catalyze IMP with L-Asp to form adenylosuccinate, in a reaction driven by the hydrolysis of GTP to GDP. Genome research results show that almost all of AdSS from the mesophilic organism contain430-460amino acid residues, while, amino acid sequences of AdSS from thermophilic archaea or viruses are90-120amino acids shorter than those of AdSS isolated from mesophilic organisms.These small molecular weight AdSS can be classified as a class. So far, the research about structure and function of mesophilic organism AdSS is rich, however, the structure and function of small molecular weight AdSS is unknown yet.My mentor, Professor Xie had successfully measured2.5A resolution crystal structure of PhAdSS which is a kind of thermophilic biological sources AdSS in previous studies, and this is the first crystal structure of thermophilic biological sources AdSS, this result lays the foundation for the study of the structure and function of small molecular weight AdSS. Under the guidance of instructors, I assume the study of the relationship between spatial structure and function particularity of PhAdSS. Enzymatic kinetics assaies and crystal structure analysis was carried out to determine the Michaelis constant and the crystal structure of PhAdSS co-crystallized with GTP and IMP with2.7A resolution. We found that PhAdSS has a good thermal stability at room temperature, its biological activity is almost equal to the mesophilic AdSS in a323K. There is a discussion about the unique spatial structural features and catalytic mechanism based on the results of the enzyme kinetics parameters and the crystal structure analysis. As a product of AdSS, s-AMP is one of the structural analogs of AMP. This means that it may activate AMPK so as to improve glucose and lipid metabolism. However, there was no related research reports about the pharmacological activity of s-AMP. To confirm this conjecture, I launched a study about s-AMP’s pharmacology. The results confirmed that s-AMP has a better lipid-lowering activity and lower cytotoxicity. s-AMP can act as a lead compound to conduct the study about mechanism of action and metabolism studies of lipid-lowering drugs PhAdSS can serve as a new catalyst for large-scale synthesis of s-AMP to provide raw material security.Meanwhile, I also bear the crystallographic studies of C-terminal SH3domain of human Tks4and successful implementated the expression, purification in E.coli and the crystallization of the C-terminal SH3domain of human TKs4. X-ray diffraction data of2.3A resolution was collected on beamline BL17U at the Shanghai Synchrotron Radiation Facility(SSRF), the crystallographic parameters of C-terminal SH3domain of human Tks4was calculated. From the arrangement of the molecules in the crystal, we predict that C-terminal SH3domain Tks4has a unique interaction.