PMID:9364923

Citation	Skouloubris, S, Labigne, A and De Reuse, H (1997) Identification and characterization of an aliphatic amidase in Helicobacter pylori. Mol. Microbiol. 25:989-98
Abstract	We 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 (37746Da) 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.
Links	PubMed
Keywords	Amidohydrolases/analysis; Amino Acid Sequence; Cloning, Molecular; DNA, Recombinant; Escherichia coli/enzymology; Escherichia coli/genetics; Genes, Bacterial/genetics; Helicobacter pylori/chemistry; Helicobacter pylori/enzymology; Helicobacter pylori/genetics; Molecular Sequence Data; Mutation/genetics; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Sequence Homology, Amino Acid; Substrate Specificity
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HELPY:AMIE		GO:0006807: nitrogen compound metabolic process	ECO:0000315:		P		Table 2: No amidase activity was detected on propionamide, acrylamide, acetamide, or isobutyramide in the amiE mutant. Amidase activity was measured by the rate of release of ammonia.	complete CACAO 6288
HELPY:AMIE	involved_in	GO:0006807: nitrogen compound metabolic process	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete

References

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