Noncatalytic residues P160 and K287 contribute to the enhanced catalytic activity of catalase from a clinical Helicobacter pylori strain

Helicobacter pylori encounters oxidative stress during gastric colonization, and catalase KatA is a major defense against hydrogen peroxide. Although KatA is highly conserved among H. pylori strains, the functional consequences of naturally occurring sequence variation remain incompletely understood. We identified a clinical isolate, G272, that showed higher hydrogen peroxide tolerance than the reference strain 26695 and investigated whether differences in KatA sequence and enzymatic properties were associated with this phenotype. Hydrogen peroxide tolerance was compared between H. pylori G272 and H. pylori 26695. A katA -disrupted G272 mutant and a complemented strain were constructed to assess the contribution of KatA to detectable whole-cell catalase activity. KatA proteins from G272 and 26695 were expressed in Escherichia coli and purified for biochemical analysis. Sequence comparison and structure-guided mapping were used to prioritize candidate residues, and two G272-derived mutants, KatA G272-P160H and KatA G272-K287Q , were generated by site-directed mutagenesis. The four recombinant KatA proteins were then compared for specific catalytic activity, pH and temperature responses, and time-dependent activity retention after pre-incubation. H. pylori G272 showed higher tolerance to hydrogen peroxide than H. pylori 26695 under the assay conditions used here. Disruption of katA abolished detectable whole-cell catalase activity in G272, and chromosomal complementation restored detectable catalase activity. Recombinant KatA G272 showed higher specific activity than KatA 26695 under the standard assay conditions (3,593 ± 207 vs. 2,918 ± 130 U/mg protein). Sequence comparison identified five amino acid differences between the two enzymes. Analysis of a published H. pylori KatA sequence dataset showed that P160 and K287 were common natural variants, and structure-guided mapping prioritized these residues for functional testing. Both the P160H and K287Q substitutions reduced KatA G272 catalytic activity under multiple pH and temperature conditions and at selected pre-incubation time points. The noncatalytic residues P160 and K287 contributed to the higher catalytic activity of KatA G272 , supporting the functional relevance of naturally occurring sequence variation outside the KatA catalytic center. These enzyme-level differences may contribute to strain-level variation in peroxide tolerance, but direct chromosomal replacement experiments are required to determine whether P160 and K287 influence this phenotype in H. pylori .