Activity and regulation of an amidase (acylamide amidohydrolase, EC 3.5.1.4) with a wide substrate spectrum from a Brevibacterium sp.

Maestracci, M.; Thiéry, Alain; Bui, K.; Arnaud, A.; Galzy, P.

doi:10.1007/bf00410897

Cited by 47 publications

(21 citation statements)

References 21 publications

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“…R312 (Maestracci et al, 1984) and in P. aeruginosa (Kelly and Clarke, 1962). A complex mechanism of regulation involving transcriptional anti-termination within the amidase operon has been demonstrated in P. aeruginosa (Wilson et al, 1996).…”

Section: Discussionmentioning

confidence: 96%

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: 96%

Identification and characterization of an aliphatic amidase in Helicobacter pylori

Skouloubris

Labigne

Reuse

1997

Molecular Microbiology

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“…This is the first report of an amidase, in culture or in soil, capable of attacking a polymer in this size range (1-2 x 107 MW). Amidase activity has been identified in numerous genera of bacteria, including Rhodococcus (Nawaz et al, 1994), Bacillus (Thalenfeld and Grosswicz, 1976), Mycobacterium (Draper, 1967), Brevibacterium (Maestracci et al, 1984), Alcaligenes (Friedrich and Mitrenga, 1981), and Pseudomonas (Kelly and Clarke, 1962;Clarke, 1970;Ciskanik et al, 1995). In addition, amidase activity has been demonstrated in several fungi, including Aspergillus (Hynes and Pateman, 1970) and Fusarium (Reichel et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…When we provided PAM as an N-source, both PAM-specific activity and amidase activity specific for low MW amides was exhibited, in both enrichment cultures and soils. Amidases that demonstrate broad substrate specificities have been reported (Draper, 1967;Maestracci et al, 1984), although the enzyme preparations used were either crude or only partially purified, consequently could represent a suite of amidase enzymes. There is also a report of multiple amidase isozymes, each with a limited substrate specificity, produced simultaneously by Aspergillus nidulans when induced by a single small amide (Hynes and Pateman, 1970).…”

Section: Discussionmentioning

confidence: 99%

Polyacrylamide as a substrate for microbial amidase in culture and soil

Kay-Shoemake¹,

Watwood²,

Sojka³

et al. 1998

Soil Biology and Biochemistry

102

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Summary-High molecular weight, linear polyacrylamide (PAM) with anionic charge is added to agricultural soils as an anti-erosion additive. Research indicates that soil microorganisms are able to utilize PAM as a source of N and that inorganic N pools are altered in some PAM-treated soils. The potential role of hydrdlytic amidase activity in the microbial utilization of PAM for N was investigated. Intracellular and extracellular amidase activity was measured over time in enrichment cultures which used PAM as sole N source. Enzyme activity increased concomitant with cell growth and N removal from PAM. Cell growth, N removal and amidase production were dependent upon readily-available C in the medium. Amidase activity and substrate specificity were determined for PAM-utilizing enrichment cultures exposed to various N sources. Polyacrylamide-specific amidase activity appears to be inducible, and not constitutive, based on the lack of amidase activity in cultures supplied with only ammonium nitrate for N versus substantial activity when PAM was added as an amendment with or without ammonium nitrate. Cultures amended with propionamide exhibited amidase activity largely specific for this small amide substrate, while cultures supplied with PAM as sole N source exhibited amidase activity specific for formamide, propionamide and PAM. Amidase activity and substrate specificity were determined for PAM-treated and untreated agricultural field soils. Polyacrylamide-specific amidase activity was higher in PAM-treated soil (14.86 ± 14.0 pg NH4 released soil) than in untreated soil (1.02 ± 2.3 pg NH4 released C I soil); activity specific for low molecular weight amides was slightly elevated or unchanged in PAM-treated soil as compared with untreated soil.

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“…strain R312 harbors, as other microorganisms, several different types of amidase activities. For instance, an acylamide amidohydrolase (EC 3.5.1.4) with a wide activity spectrum (wide-spectrum amidase) has been characterized (17). The purified enzyme is apparently a tetramer of four identical subunits (Mr 43,000), is able to hydrolyze a large number of amides into their corresponding organic acids, and also possesses an acyl transferase activity (26).…”

mentioning

confidence: 99%

Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase

Mayaux¹,

Cerebelaud²,

Soubrier³

et al. 1990

J Bacteriol

152

118

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An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp. strain R312. Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amdA. Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans. Moreover, amdA is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon. Our results also showed that Rhodococcus sp. strain N-774 and Brevibacterium sp. strain R312 are probably identical, or at least very similar, microorganisms. The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 imol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide. Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30TC under the control of an E. coli promoter and ribosome-binding site.

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Activity and regulation of an amidase (acylamide amidohydrolase, EC 3.5.1.4) with a wide substrate spectrum from a Brevibacterium sp.

Cited by 47 publications

References 21 publications

Identification and characterization of an aliphatic amidase in Helicobacter pylori

Identification and characterization of an aliphatic amidase in Helicobacter pylori

Polyacrylamide as a substrate for microbial amidase in culture and soil

Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase

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