| Summary: | 碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 100 === H. pylori is a gram-negative and microaerophilic bacterium that infects more than 50% of the world population and increases the risk of developing gastric ulcer and stomach cancer. Antibiotics, e.g., amoxicillin and clarithromycin, and a proton-pump inhibitor have been used to treat H. pylori infection, but as H. pylori has become increasingly resistant to antibiotics, treatment often fails. It is important to find a new treatment or antibacterial drug targets to against H. pylori infection. Coenzyme A (CoA) is an essential cofactor and a major intracellular carrier of activated acyl groups in all living organisms. Phosphopantetheine adenylyltransferase (PPAT) from H. pylori is a penultimate, rate-limiting enzyme in the CoA biosynthesis pathway. The enzyme transfer adenylyl group from ATP to phosphorpantetheine, yielding dephospho-CoA and pyrophosphate. Recent study indicated that knock out the PPAT gene in Escherichia coli reducing its CoA levels and preventing bacterial growth. In addition, amino acid sequence and secondary structure between H. pylori and human PPATs are dissimilar. Therefore, PPAT is a potential antibacterial target. We have determined the complex structure of H. pylori PPAT with a CoA molecule (PDB ID: 3OTW). In H. pylori PPAT, the side chains of Thr10, Lys42, Arg88, and Tyr98 form hydrogen bonds with the CoA phosphate group. In contrast, fewer hydrogen bonds between other PPATs and CoA are observed. Interactions between Thr15, Gly17, Ile127, and Arg133 in H. pylori PPAT and CoA alter the orientation of the CoA adenine ring. To investigate the structural and kinetics mechanisms of substrate binding to the phosphopantetheine adenylyltransferase and its mutants from H. pylori, we used alanine-scanning mutagenesis, x-ray crystallography, biophysical, and steady-state kinetics of the reverse reaction approaches to answering these questions.
|
|---|
