Identification and Characterization of Physiologically Significant Proteins from Helicobacter pylori

• Main Authors: Mon-Juan Lee, 李孟娟
• Other Authors: Haimei Huang
• Format: Others
• Language: en_US
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/69256427772435689739

博士 === 國立清華大學 === 生命科學系 === 94 === Helicobacter pylori is a Gram-negative, microaerophilic human gastric pathogen which infects the gastric mucosa, causes gastritis and peptic ulcer disease, and is also an important risk factor for development of gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma. There was a lack of information on the progression to diseases by H. pylori, which survive at the acidic pH of the gastric environment, a site that few other microbes can colonize. The requirement of a rather stringent culture condition as compared to other bacteria, and the resistance to transformation in many strains, resulted in the lack of adequate systems for genetic manipulation. With the resolution of the complete genome sequence of H. pylori, studies on this etiological agent can be more conveniently implemented through the construction of recombinant proteins. The HP0832 (speE) gene of Helicobacter pylori strain 26695 codes for a putative spermidine synthase, which belongs to the polyamine biosynthetic pathway. Spermidine synthase catalyzes the production of spermidine from putrescine and decarboxylated S-adenosylmethionine (dcSAM), which serves as an aminopropyl donor. The deduced amino acid sequence of the HP0832 gene shares less than 20% sequence identity with most spermidine synthases from mammalian cells, plants and other bacteria. In this study, the HP0832 open reading frame (786 bp) was cloned into the pQE30 vector and overexpressed in Escherichia coli strain SG13009. The resulting N-terminally 6xHis-tagged HP0832 protein (31.9 kDa) was purified by Ni-NTA affinity chromatography at a yield of 15 mg/L of bacteria culture. Spermidine synthase activity of the recombinant protein was confirmed by the production of spermidine after incubating the enzyme with putrescine and dcSAM. Endogenous spermidine synthase of H. pylori was detected with an antiserum raised against the recombinant HP0832 protein. H. pylori strain 26695 contains putrescine and spermidine at a molar ratio of 1:3, but no detectable spermine or norspermidine was observed, suggesting that the spermidine biosynthetic pathway may provide the main polyamines in H. pylori strain 26695. The lack of a complete gatekeeping loop region in H. pylori spermidine synthase was suspected to be responsible for its instability in activity. Mutagenesis on conserved residues in the gatekeeping loop region of E. coli spermidine synthase (EcSDPS) was thus performed to investigate its importance to enzyme catalysis. The inorganic pyrophosphatase of H. pylori (HpPPase) was encoded by the HP0620 (ppa) gene and was a family I PPase. It was a homohexamer consisting of identical 20-kDa subunits. Hydrolysis of PPi by HpPPase relied on the presence of magnesium and followed Michaelis-Menten kinetics, with kcat being 344 s-1 and Km being 83 μM at pH 8.0, which was the optimal pH for catalysis. HpPPase was activated by both thiol and non-thiol reductants, distinct from the previously suggested inactivation/reactivation process involving formation and breakage of disulfide bonds. Substituting Cys15 of HpPPase, which was neither located at the active site nor evolutionarily conserved, resulted in a loss of 50% of activity and a reduction in sensitivity to reductants and oxidized glutathione. In addition, the C15S replacement caused a considerable disruption in thermostability, which exceeded that resulted from active-site mutations such as Y139F HpPPase and those of E. coli. Although Cys15 was not located at the subunit interface of the hexameric HpPPase, several evidences suggested that the C15S substitution destabilized HpPPase through impairing trimer-trimer interactions. These results provided the first evidences that the single cysteine residue of HpPPase was involved in enzyme activation, thermostability, and stabilization of quaternary structure. The Soj and Spo0J proteins, together with one or more parS sequences, constitute the parABS system, which is crucial to chromosome segregation and the progression of cell cycle in many bacteria. A putative oriC region was located at 1,609,162 bp of the circular chromosome of H. pylori 26695. Genes coding for Soj (HP1139) and a plasmid replication-partition related protein containing a Spo0J or ParB conserved domain (HP1138), together with two putative parS sites identified by blasting the parS sequence of Bacillus subtilis against the genome of H. pylori 26695, were found to be located within the origin-proximal 20-30% of the chromosome, similar to several other species. These analyses, together with previous functional studies of recombinant Soj and Spo0J proteins, led to the first implication of the existence of a functional parABS system in H. pylori.