Helicobacter pylori nickel metabolism accessory proteins needed for the maturation of both urease and hydrogenase

Abstract

Helicobacter pylori is a causative agent of chronic gastritis and peptic ulcer disease. Prolonged infection may lead to gastric cancer. The bacterium has an unique ability to invade and persist in the extremely acidic stomach. H. pylori possesses structural and accessory genes for the synthesis and maturation of two nickel containing enzymes, hydrogenase and urease. Insertional mutagenesis of two hydrogenase accessory genes, hypA and hypB, showed their involvement in the activation of both hydrogenase and urease. The role played by these genes in hydrogenase maturation was not unexpected, however their involvement in urease activation was quite surprising, as the bacterium appeared to have a complete set of urease maturation genes. To understand the role of HypA and HypB further, both HypA and HypB proteins were purified from H. pylori. In some bacteria these accessory proteins have been shown to either bind nickel or hydrolyze GTP. HypA from H. pylori bound to two nickel ions per dimer with positive cooperativity, but lacked GTPase activity. A mutant HypA protein (H2A) failed to bind nickel. HypB possessed GTPase activity, but did not bind nickel. A mutant HypB protein (K59A) showed negligible GTP hydrolyzing activity. Alleles for these two mutant proteins were introduced into H. pylori. The resulting mutant strains lacked hydrogenase activity, and had only 2% (hypA mutant) and 1.4% (hypB mutant) of the urease activity shown by the wild type strain. Crosslinking studies between HypA and HypB in the presence of dimethyl suberimidate showed formation of a 43-kDa heterodimeric complex, composed of both proteins in a 1:1 molar ratio. A similar complex was seen when a mixture of the two proteins was subjected to gel filtration chromatography. These data suggest that HypA is involved in nickel sequestration and HypB in nickel incorporation via its GTP hydrolyzing activity during the maturation of urease and hydrogenase.