Functional Studies in Comparison of Wild-Type and Mutated Phosphopantetheine adenylyltransferase from Helicobacter pylori

• Main Authors: Luo, Yong-Chun, 羅永淳
• Other Authors: Yin, Hsien-Sheng
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/60918369589074495617

碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 99 === In recent years, many lectures have proved that Helicobacter pylori infection increase the possibility with cancer such as atrophic gastritis and gastric cancer. Nowadays, antibiotic resistance of Helicobacter pylori infection turns a trouble matter to treat patients. This study is to find another biological way to solve these problems. Previous report indicated adequate coenzyme A level was important to bacteria survival. If the CoA pathway has been turned off, coenzyme A level was reduced to prevent bacteria growth. For this reason, we choose the CoA synthesis pathway and focus on phosphopantetheine adenylyltransferase (PPAT) of penultimate catalytic step. Therefore, a series of analytical ultracentrifuge, isothermal titration calorimetry, and circular dichroism experiments with phosphopantetheine adenylyltransferase (HpPPAT) and I4V/N76Y mutant was conducted. Their structures were determined by our laboratory research in 2011. Wild-type HpPPAT was a traditional hexamer like other species, such as E. coli PPAT and M. tuberculosis PPAT. But I4V/N76Y PPAT was a novel domain swapping tetramer. Their oligomer states were proofed again by analytical ultracentrifuge. Surprisingly, fresh preparation and leave for 4℃ overnight samples have different oligomer states. Binding affinity of substrate for HpPPAT was detected by fluorescence spctrophotometry and isothermal titration calorimetry. For ATP, wild-type and I4V/N76Y PPATs showed similar binding affinity and mechanism. But for CoA binding affinity of wild-type PPAT was higher than I4V/N76Y mutant PPAT. Futhermore, secondary structure of wild-type and I4V/N76Y PPATs was similar, but thermo-stability of them purified using high or low salt condition is different slightly. On the basis of this research, preliminary drug screening was conducted and developed new drug to prevent Helicobacter pylori infection in future.