Identification and characterization of an aliphatic amidase in Helicobacter pylori

Abstract

SummaryWe report, for the first time, the presence in Helicobacter pylori of an aliphatic amidase that, like urease, contributes to ammonia production. Aliphatic amidases are cytoplasmic acylamide amidohydrolases (EC 3.5.1.4) hydrolysing short-chain aliphatic amides to produce ammonia and the corresponding organic acid. The finding of an aliphatic amidase in H. pylori was unexpected as this enzyme has only previously been described in bacteria of environmental (soil or water) origin. The H. pylori amidase gene amiE (1017 bp) was sequenced, and the deduced amino acid sequence of AmiE (37 746 Da) is very similar (75% identity) to the other two sequenced aliphatic amidases, one from Pseudomonas aeruginosa and one from Rhodococcus sp. R312. Amidase activity was measured as the release of ammonia by sonicated crude extracts from H. pylori strains and from recombinant Escherichia coli strains overproducing the H. pylori amidase. The substrate specificity was analysed with crude extracts from H. pylori cells grown in vitro; the best substrates were propionamide, acrylamide and acetamide. Polymerase chain reaction (PCR) amplification of an internal amiE sequence was obtained with each of 45 different H. pylori clinical isolates, suggesting that amidase is common to all H. pylori strains. A H. pylori mutant (N6-836) carrying an interrupted amiE gene was constructed by allelic exchange. No amidase activity could be detected in N6-836. In a N6-urease negative mutant, amidase activity was two-to threefold higher than in the parental strain N6. Crude extracts of strain N6 slowly hydrolysed formamide. This activity was affected in neither the amidase negative strain (N6-836) nor a double mutant strain deficient in both amidase and urease activities, suggesting the presence of an independent discrete formamidase in H. pylori. The existence of an aliphatic amidase, a correlation between the urease and amidase activities and the possible presence of a formamidase indicates that H. pylori has a large range of possibilities for intracellular ammonia production.

Differential Regulation of Amidase- and Formamidase-mediated Ammonia Production by the Helicobacter pylori Fur Repressor

“…Amidase, Formamidase, and Urease Enzyme Assays-The enzymatic activity of urease, amidase, and formamidase were determined in fresh H. pylori lysates by measuring ammonia production from hydrolysis of urea, acrylamide, or formamide, respectively, by using the Berthelot reaction as described previously (13,14,25). The concentration of ammonia present in the samples was inferred from a standard NH 4 Cl concentration curve.…”

“…The presence of nitrogen regulatory systems is likely though, because the activity of the different ammonia-producing enzymes seems to be balanced. The absence of urease activity leads to higher amidase activity (13), whereas the combined absence of urease and arginase led to higher formamidase activity (14). Conversely, the absence of arginase also led to alterations in the activity of the amino acid deaminases (12), and thus, it is thought that the intracellular nitrogen status of H. pylori is controlled through yet unidentified regulatory systems.…”

“…Urea is thought to be the main source of ammonia in the gastric environment, but H. pylori does have alternative pathways for the production of ammonia via amino acid catabolism (12) and via the activity of its two paralogous amidases, AmiE 1 and AmiF (13, 14). Aliphatic amidase (AmiE, EC 3.5.1.4) and formamidase (AmiF, EC 3.5.1.49) catalyze the conversion of amide substrates to the corresponding carboxylic acid and ammonia (13,14).The control of the intracellular nitrogen status is important for living organisms, and this can be mediated by several different nitrogen regulatory systems. These include the P II (GlnB) signal transduction protein and NtrBC two-component regulatory system, which are widespread throughout the bacterial kingdom (15), but alternative nitrogen regulatory systems exist (16 -18).…”

“…Ammonia plays a central role in pathogenesis and metabolism of the important human pathogen H. pylori, because it not only serves as nitrogen source (8) but also contributes to epithelial cell damage and apoptosis (9,10), is involved in chemotactic motility (11), and is required for acid resistance (2,3). Urea is thought to be the main source of ammonia in the gastric environment, but H. pylori does have alternative pathways for the production of ammonia via amino acid catabolism (12) and via the activity of its two paralogous amidases, AmiE 1 and AmiF (13,14). Aliphatic amidase (AmiE, EC 3.5.1.4) and formamidase (AmiF, EC 3.5.1.49) catalyze the conversion of amide substrates to the corresponding carboxylic acid and ammonia (13,14).…”

“…Urea is thought to be the main source of ammonia in the gastric environment, but H. pylori does have alternative pathways for the production of ammonia via amino acid catabolism (12) and via the activity of its two paralogous amidases, AmiE 1 and AmiF (13, 14). Aliphatic amidase (AmiE, EC 3.5.1.4) and formamidase (AmiF, EC 3.5.1.49) catalyze the conversion of amide substrates to the corresponding carboxylic acid and ammonia (13,14).…”