Cloning and primary structure of the wide-spectrum amidase from Brevibacterium sp. R312: high homology to the amiE product from Pseudomonas aeruginosa

Soubrier, F; Lévy-Schil, Sophie; Mayaux, Jean‐François; Petre, Dominique; Arnaud, A.; Crouzet, J

doi:10.1016/0378-1119(92)90635-3

Cited by 42 publications

(30 citation statements)

References 18 publications

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“…They are present in phylogenetically distant Gram-positive and Gram-negative organisms. As proposed by Soubrier et al (1992), amiE may therefore have been acquired during evolution by horizontal gene transfer. The GC% of the H. pylori amiE gene is higher than that of the genome overall GC%, consistent with this hypothesis.…”

Section: Discussionmentioning

confidence: 94%

“…The amino acid sequence deduced from this ORF is very similar to that of the amidase from P. aeruginosa (75% identity and 87% similarity; Ambler et al, 1987;Brammar et al, 1987) and from Rhodococcus sp. R312 (75% identity and 85% similarity; Soubrier et al, 1992; Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…The H. pylori amidase is very similar (75% identical residues) to the two other sequenced aliphatic amidases, one from P. aeruginosa (Ambler et al, 1987;Brammar et al, 1987) and one from Rhodococcus sp. R312 (Soubrier et al, 1992), the last two sequences being 81% identical. Other aliphatic amidases have been identified in Methylophilus methylotrophus (Silman et al, 1991), Arthrobacter sp.…”

Section: Discussionmentioning

confidence: 99%

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Identification and characterization of an aliphatic amidase in Helicobacter pylori

Skouloubris

Labigne

Reuse

1997

Molecular Microbiology

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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.

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Section: Discussionmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Identification and characterization of an aliphatic amidase in Helicobacter pylori

Skouloubris

Labigne

Reuse

1997

Molecular Microbiology

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“…strain R312 (enantioselective amidase) (P22984 [44]); and Rhodococcus sp. strain R312 ("wide-spectrum" amidase) (Q01360 [56] …”

Section: Vol 68 2002 Enantioselective Amidase From Rhodococcus Erytmentioning

confidence: 99%

Cloning and Heterologous Expression of an Enantioselective Amidase from Rhodococcus erythropolis Strain MP50

Trott

Bürger

Calaminus

et al. 2002

Appl Environ Microbiol

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The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, ␣-chlorophenylacetamide, and ␣-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.Acylamide amidohydrolases (amidases) are used in biocatalysis for the chemoselective, regioselective, or enantioselective hydrolysis of various amides (17, 59). The chemo-and regioselectivities of amidases are utilized for the production of antibiotics (penicillin acylase), the hydrolysis of C-terminal amide groups in peptides (peptide amidase), the analysis of glycoproteins [peptide-N 4 -(N-acetyl-␤-D-glucosaminyl)asparagine amidase F], or the transformation of cyclic imides (halfamidase, imidase) (5,25,50,55,57,59). Enantioselective amidases are used for the production of optical active D-or L-␣-amino acids, hydroxycarboxylic acids, or ␣-methylarylacetic and ␣-methoxyarylacetic acids. L-specific aminoamidases have been reported for Pseudomonas putida, Mycobacterium neoaurum, and Stenotrophomonas maltophilia, and a D-specific amino acid amidase has been found in Ochrobactrum anthropi. These enzymes usually also convert certain peptides and are therefore referred to as aminopeptidases (3,21,22,41).An evolutionarily different group of amidases has been found which enantioselectively converts 2-methylphenylacetamide (2-phenylpropionamide) and other ␣-methylarylacetamides. This group of amidases has been found in different rhodococci but also in gram-negative organisms, such as Pseudomonas chlororaphis B23 or Agrobacterium tumefaciens d3 (6,31,44,45,49).One of the best-characterized amidases with the ability to enantioselectively hydrolyze various ␣-methylarylacetamides that has been described is from Rhodococcus erythropolis MP50. This isolate was obtained from an enrichment with naproxen nitrile as sole nitrogen s...

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“…3. Aliphatic amidases enable prokaryotes to use acetamides as both carbon and nitrogen source (see Soubrier et al, 1992, and references therein). 4.…”

Section: Cyanide Hydratase Of Pathogenic Fungi Detoxifies Hcnmentioning

confidence: 99%

A new family of carbon‐nitrogen hydrolases

1994

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Using computer methods for database search and multiple alignment, statistically significant sequence similarities were identified between several nitrilases with distinct substrate specificity, cyanide hydratases, aliphatic amidases, @-alanine synthase, and a few other proteins with unknown molecular function. All these proteins appear to be involved in the reduction of organic nitrogen compounds and ammonia production. Sequence conservation over the entire length, as well as the similarity in the reactions catalyzed by the known enzymes in this family, points to a common catalytic mechanism. The new family of enzymes is characterized by several conserved motifs, one of which contains an invariant cysteine that is part of the catalytic site in nitrilases. Another highly conserved motif includes an invariant glutamic acid that might also be involved in catalysis.

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Cloning and primary structure of the wide-spectrum amidase from Brevibacterium sp. R312: high homology to the amiE product from Pseudomonas aeruginosa

Cited by 42 publications

References 18 publications

Identification and characterization of an aliphatic amidase in Helicobacter pylori

Identification and characterization of an aliphatic amidase in Helicobacter pylori

Cloning and Heterologous Expression of an Enantioselective Amidase from Rhodococcus erythropolis Strain MP50

A new family of carbon‐nitrogen hydrolases

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