The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogues

Aliphatic amidases (EC 3.5.1.4) are enzymes catalysing the hydrolysis of sh ort-chain amides to produce ammonia and the corresponding organic acid. Suc h an amidase, AmiE, has been detected previously in Helicobacter pylori. An alysis of the complete H. pylori genome sequence revealed the existence of a duplicated amidase gene that we named amiF. The corresponding AmiF protei n is 34% identical to its AmiE paralogue. Because gene duplication is widel y considered to be a fundamental process in the acquisition of novel enzyma tic functions, we decided to study and compare the functions of the paralog ous amidases of H. pylori. AmiE and AmiF proteins were overproduced in Esch erichia coli and purified by a two-step chromatographic procedure. The two H. pylori amidases could be distinguished by different biochemical characte ristics such as optimum pH or temperature. AmiE hydrolysed propionamide, ac etamide and acrylamide and had no activity with formamide. AmiF presented a n unexpected substrate specificity: it only hydrolysed formamide. AmiF is t hus 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 ca rrying single and double mutations of amiE and amiF were constructed. The s ubstrate specificities of these enzymes were confirmed in H. pylori. Produc tion of AmiE and AmiF proteins is dependent on the activity of other enzyme s involved in the nitrogen metabolism of H. pylori (urease and arginase res pectively). Our results strongly suggest that (i) the H. pylori paralogous amidases have evolved to achieve enzymatic specialization after ancestral g ene duplication; and (ii) the production of these enzymes is regulated to m aintain intracellular nitrogen balance in H. pylori.