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Journal of Bacteriology, February 2003, p. 726-734, Vol. 185, No. 3
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.3.726-734.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of Helicobacter pylori Nickel Metabolism Accessory Proteins Needed for Maturation of both Urease and Hydrogenase

Nalini Mehta,¹ Jonathan W. Olson,^1, and Robert J. Maier^1,2*

Department of Microbiology,¹ Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia²

Received 3 October 2002/ Accepted 4 November 2002

Previous studies demonstrated that two accessory proteins, HypA and HypB, play a role in nickel-dependent maturation of both hydrogenase and urease in Helicobacter pylori. Here, the two proteins were purified and characterized. HypA bound two Ni²⁺ ions per dimer with positive cooperativity (Hill coefficient, approximately 2.0). The dissociation constants K₁ and K₂ for Ni²⁺ were 58 and 1.3 µM, respectively. Studies on purified site-directed mutant proteins in each of the five histidine residues within HypA, revealed that only one histidine residue (His2) is vital for nickel binding. Nuclear magnetic resonance analysis showed that this purified mutant version (H2A) was similar in structure to that of the wild-type HypA protein. A chromosomal site-directed mutant of hypA (in the codon for His2) lacked hydrogenase activity and possessed only 2% of the wild-type urease activity. Purified HypB had a GTPase activity of 5 nmol of GTP hydrolyzed per nmol of HypB per min. Site-directed mutagenesis within the lysine residue in the conserved GTP-binding motif of HypB (Lys59) nearly abolished the GTPase activity of the mutant protein (K59A). In native solution, both HypA and HypB exist as homodimers with molecular masses of 25.8 and 52.4 kDa, respectively. However, a 1:1 molar mixture of HypA plus HypB gave rise to a 43.6-kDa species composed of both proteins. A 43-kDa heterodimeric HypA-HypB complex was also detected by cross-linking. The cross-linked adduct was still observed in the presence of 0.5 mM GTP or 1 µM nickel or when the mutant version of HypA (altered in His2) and HypB (altered in Lys59) were tested. Individually, HypA and HypB formed homodimeric cross-linked adducts. An interaction between HypA and the Hp0868 protein (encoded by the gene downstream of hypA) could not be detected via cross-linking, although such an interaction was predicted by yeast two-hybrid studies. In addition, the phenotype of an insertional mutation within the Hp0868 gene indicated that its presence is not critical for either the urease or the hydrogenase activity.

Present address: Department of Microbiology, North Carolina State University, Raleigh, NC 27695.

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