Biochemical properties of the superoxide dismutase from the pathogenic bacterium Helicobacter pylori.

Helicobacter pylori, a pathogenic aerotolerant bacterium colonizing the gastric mucosa, causes gastritis and ulcer and, if not eradicated, may lead to the development of gastric tumors. H. pylori resistance to moderate oxygen concentrations is mainly due to the key anti-oxidant role played by superoxide dismutase (HpSOD), the enzyme scavenging the toxic superoxide anions formed during oxygen consumption. The 3D structure of a recombinant form of HpSOD showed that this enzyme, belonging to the Fe-SOD family, contains an extended C-terminal tail, which is missing in other bacterial SODs and whose role remains obscure. Furthermore, in some cultures of H. pylori the endogenous HpSOD was found anchored to the flagellar sheath of the bacterium. In the present study the biochemical properties of the recombinant HpSOD were investigated to improve the knowledge on the enzyme functions, which could explain the H. pylori survival in the harsh conditions of the stomach. The research is also focused on the possible role played by the unusual C-terminal extension of HpSOD. The high specific activity (5000 U/mg) of the recombinant enzyme and its discrete heat resistance (T1/2 = 64C) ensure its functional efficiency. The effect of typical inhibitors and inactivators of SODs was investigated; while sodium azide caused only a low inhibition, hydrogen peroxide and peroxynitrite provoked a significant inactivation of HpSOD. It is known that the genes involved in glutathione biosynthesis are missing in the H. pylori genome; however, HpSOD undergoes a glutathionylation reaction by the oxidized form of glutathione. A mutagenic analysis aimed at the replacement of the two cysteine residues possessed by HpSOD allowed the identification of C79 as the target residue of the S-glutathionylation reaction. The production of a deleted form of HpSOD lacking the C-terminal extension of the enzyme is on the way, to check the effect on the biochemical properties of the enzyme. P26.33