PMID:11359566

Citation	Skouloubris, S, Labigne, A and De Reuse, H (2001) The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogues. Mol. Microbiol. 40:596-609
Abstract	Aliphatic amidases (EC 3.5.1.4) are enzymes catalysing the hydrolysis of short-chain amides to produce ammonia and the corresponding organic acid. Such an amidase, AmiE, has been detected previously in Helicobacter pylori. Analysis of the complete H. pylori genome sequence revealed the existence of a duplicated amidase gene that we named amiF. The corresponding AmiF protein is 34% identical to its AmiE paralogue. Because gene duplication is widely considered to be a fundamental process in the acquisition of novel enzymatic functions, we decided to study and compare the functions of the paralogous amidases of H. pylori. AmiE and AmiF proteins were overproduced in Escherichia coli and purified by a two-step chromatographic procedure. The two H. pylori amidases could be distinguished by different biochemical characteristics such as optimum pH or temperature. AmiE hydrolysed propionamide, acetamide and acrylamide and had no activity with formamide. AmiF presented an unexpected substrate specificity: it only hydrolysed formamide. AmiF is thus the first formamidase (EC 3.5.1.49) related to aliphatic amidases to be described. Cys-165 in AmiE and Cys-166 in AmiF were identified as residues essential for catalysis of the corresponding enzymes. H. pylori strains carrying single and double mutations of amiE and amiF were constructed. The substrate specificities of these enzymes were confirmed in H. pylori. Production of AmiE and AmiF proteins is dependent on the activity of other enzymes involved in the nitrogen metabolism of H. pylori (urease and arginase respectively). Our results strongly suggest that (i) the H. pylori paralogous amidases have evolved to achieve enzymatic specialization after ancestral gene duplication; and (ii) the production of these enzymes is regulated to maintain intracellular nitrogen balance in H. pylori.
Links	PubMed
Keywords	Amidohydrolases/genetics; Amidohydrolases/isolation & purification; Amidohydrolases/metabolism; Amino Acid Sequence; Cloning, Molecular; Escherichia coli; Evolution, Molecular; Gene Expression; Genes, Bacterial; Helicobacter pylori/enzymology; Helicobacter pylori/genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Sequence Homology, Amino Acid
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HELPY:AMIF		GO:0006807: nitrogen compound metabolic process	ECO:0000315:		P		Table 3: An amiF-deficient mutant strain showed decreased formamidase activity, which was measured by the rate of formation of ammonia. The residual formamidase activity was accounted for by the urease genes ureA and ureB. A mutant strain that was deficient in ureA, ureB, and amiF showed no formamidase acitivty.	complete CACAO 6194

References

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