Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily

Abstract

The urea cycle enzyme arginase (EC 3.5.3.1) hydrolyzes l‐arginine to l‐ornithine and urea. Mammalian arginases require manganese, have a highly alkaline pH optimum and are resistant to reducing agents. The gastric human pathogen, Helicobacter pylori, also has a complete urea cycle and contains the rocF gene encoding arginase (RocF), which is involved in the pathogenesis of H. pylori infection. Its arginase is specifically involved in acid resistance and inhibits host nitric oxide production. The rocF gene was found to confer arginase activity to Escherichia coli; disruption of plasmid‐borne rocF abolished arginase activity. A translationally fused His6–RocF was purified from E. coli under nondenaturing conditions and had catalytic activity. Remarkably, the purified enzyme had an acidic pH optimum of 6.1. Both purified arginase and arginase‐containing H. pylori extracts exhibited optimal catalytic activity with cobalt as a metal cofactor; manganese and nickel were significantly less efficient in catalyzing the hydrolysis of arginine. Viable H. pylori or E. coli containing rocF had significantly more arginase activity when grown with cobalt in the culture medium than when grown with manganese or no divalent metal. His6–RocF arginase activity was inhibited by low concentrations of reducing agents. Antibodies raised to purified His6–RocF reacted with both H. pylori and E. coli extracts containing arginase, but not with extracts from rocF mutants of H. pylori or E. coli lacking the rocF gene. The results indicate that H. pylori RocF is necessary and sufficient for arginase activity and has unparalleled features among the arginase superfamily, which may reflect the unique gastric ecological niche of this organism.

Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

“…The ratio of the k cat /K 0.5 can provide an estimate of the apparent catalytic efficiency. The ratio for the Co 21 -enzyme is approximately fourfold higher than the Mn 21 -enzyme, which is consistent with the earlier report (11). This clearly suggests that the rate of reaction in the H. pylori enzyme depends on the nature of the divalent metal ions.…”

“…For holo proteins, the experiments were carried out after heat activation. The heat activation is reported to be essential for full activation of the arginases including H. pylori enzyme (10,11) and it has been suggested that one of the metal ions can go deep into the active site and form a bimetallic cluster. The heat-activated metal reconstituted protein showed approximately twofold higher activity than without heat activated (data not shown), consistent with other arginases that a bimetallic cluster is essential for full activation of the enzyme.…”

“…For Co 21 -protein, the concentration of the metal was kept 20-fold higher than the apoenzyme but for Mn 21 , it was kept 500-fold higher than the apoenzyme. The mixture was heat activated at 50 8C for about 30 min as reported earlier (11) and then passed through Sephadex G-25 columns equilibrated with 20 mM Tris, pH 7.4. The flow through was used for the metal analysis following reported PAR [4-(2-pyridylazo) resorcinol] assays with 6M guanidine hydrochloride (21,22).…”

“…H. pylori, a human pathogen infects the stomach of half of the population worldwide and causes chronic active gastritis, which can lead to peptic ulcer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma (7)(8)(9). H. pylori has a complete urea cycle and contains the rocF gene encoding arginase (10,11). This enzyme produces endogenous urea that can be utilized by urease to generate ammonia and carbon dioxide, and thus neutralizes the surrounding acidic environment so that the bacterium can survive.…”

Enzymatic Activity of Xyloglucan Xylosyltransferase 5

“…1D). In addition, XXT2 was eluted from the Ni-NTA column using a buffer containing imidazole, which has been shown to affect the activity of other enzymes (Cotovio et al, 1996;McGee et al, 2004;Zhang et al, 2011). To investigate the effect of imidazole on XXT2, activity assays were carried out in the presence of 250 mM imidazole and without imidazole in the reaction mixture.…”