Abstract
Helicobacter pylori, a microaerophilic, Gram-negative and spiral-shaped organism. It is the major cause of gastric ulcers and an early risk factor for gastric cancer. Since the whole genome sequences of H. pylori strain 26695 have been reported by Tomb, J. F in 1997. We aim on structural genomics study of H. pylori strain 26695 based on database search, gene annotation, sequence prediction and alignments. In this thesis, one of our targets (HP0291) had been successfully cloned, characterized and confirmed its function: The chorismate mutase in H. pylori. HP0291, encoding hypothetical protein from H. pylori strain 26695, was purified to homogeneity on a SDS polyacrylamide gel with a deduced molecular mass of 11.3 kDa and yield about 2 mg native protein per liter Luria-Bertani Medium. Circular dichroism spectra indicate HP0291 is a typical α-helix structure protein with the helicity about 40 %, and the melting temperature is higher than 75°C. Analytical ultracentrifuge and cross-linking experiments on the purified HP0291 protein reveal that it has oligomer phenomenon, and the major form is dimer. By cysteine modification, the S-Carboxymethylated HP0291 (HP0291-m) shows similar secondary structures and thermal stabilities to HP0291 but contain better enzyme activities compare to HP0291 especially when the reaction buffer containing no effectors. The kinetic data show Michaelis-Menten constant (Km) values of HP0291 and HP0291-m are 355 µM; 284 µM, and a Kcat/Km values of 30.24 sec-1 mM-1; 39.61 sec-1 mM-1 respectively. The phylogenetic tree of chorismate mutases characterized so far indicated that HP0291 shows similarities to the AroQ-type enzyme.