SEARCH · PubMed Health

Results for “AMIDASES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources

PubMed1996

A mouse amidase specific for N-terminal asparagine. The gene, the enzyme, and their function in the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In both fungi and mammals, the tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose destabilizing activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues. We report the isolation and analysis of a mouse cDNA and the corresponding gene (termed Ntan1) that encode a 310-residue amidohydrolase (termed NtN-amidase) specific for N-terminal asparagine. The approximately 17-kilobase pair Ntan1 gene is located in the proximal region of mouse chromosome 16 and contains 10 exons ranging from 54 to 177 base pairs in length. The approximately 1.4-kilobase pair Ntan1 mRNA is expressed in all of the tested mouse tissues and cell lines and is down-regulated upon the conversion of myoblasts into myotubes. The Ntan1 promoter is located approximately 500 base pairs upstream of the Ntan1 start codon. The deduced amino acid sequence of mouse NtN-amidase is 88% identical to the sequence of its porcine counterpart, but bears no significant similarity to the sequence of the NTA1-encoded N-terminal amidohydrolase of the yeast Saccharomyces cerevisiae, which can deamidate either N-terminal asparagine or glutamine. The expression of mouse NtN-amidase in S. cerevisiae nta1Delta was used to verify that NtN-amidase retains its asparagine selectivity in vivo and can implement the asparagine-specific subset of the N-end rule. Further dissection of mouse Ntan1, including its null phenotype analysis, should illuminate the functions of the N-end rule, most of which are still unknown.

Amidohydrolases↗

PubMed1998

Cloning and nucleotide sequence of amidase gene from Pseudomonas putida.

Amidases are a class of enzymes which convert amides to acids and have potential value in the development of commercial bioprocesses for the production of useful chemicals. A gene encoding an amidase in Pseudomonas putida 5B has been cloned, sequenced, and overexpressed in Escherichia coli. An additional open reading frame (P38K) encoding a putative protein of 38 kDa was found immediately upstream of the amidase gene. This work continues our characterization of a P. putida operon, which now appears to include P38K, amidase, and a stereo-specific nitrile hydratase. This characterization underlies continuing efforts in biocatalyst development.

Amidohydrolases↗

PubMed1987

The amidase regulatory gene (amiR) of Pseudomonas aeruginosa.

Recombinant plasmids carrying the amidase genes of Pseudomonas aeruginosa were used to study the genetic control of amidase synthesis in Escherichia coli and Pseudomonas aeruginosa. The amidase regulator gene, amiR, was found to lie about 2 kbp downstream from the structural gene, amiE. Using plasmids with in vitro-constructed deletions, and plasmids containing subcloned DNA fragments, the amiR gene was located within a 1 kbp ClaI-XhoI DNA fragment. The structural and regulator genes were shown to be transcribed in the same direction. Deletion of DNA sequences between the two genes resulted in increased synthesis of amidase in both E. coli and P. aeruginosa. The intervening sequences showed no repressing effect when tested in trans. The results suggested that the amiR gene could be transcribed from more than one promoter.

Amidohydrolases↗

PubMed1989

Positive control of Pseudomonas aeruginosa amidase synthesis is mediated by a transcription anti-termination mechanism.

The DNA sequence of the region upstream from the amidase structural gene (amiE) of Pseudomonas aeruginosa indicates that amidase (EC 3.5.1.4) is transcribed from an Escherichia coli-like promoter located 150 bp before the amiE translation initiation codon. The sequence between the promoter and the coding sequence includes a single open reading frame followed by an E. coli-like rho-independent transcription terminator. A deletion within the presumed terminator region which disrupts the potential stem/loop formation leads to high constitutive amidase expression which is independent of the product of the regulator gene (amiR). It is proposed that the catabolic aliphatic amidase of P. aeruginosa is regulated by a transcription anti-termination mechanism. The magnoconstitutive mutant PAC433 has promoter and terminator sequences identical to the wild-type PAC1 but contains a single base pair change in the amiE gene ribosome-binding site.

Amidohydrolases↗

PubMed1975

Isolation of an inducible amidase from Pseudomonas acidovorans AE1.

A bacterial strain, AEI, which hydrolysed acetanilide, was isolated from soil and identified as Pseudomonas acidovorans. Numerous amides, esters and enzyme inhibitors were tested as amidase inducers. Phenacetin was chosen as inducer for the large scale cultivation of these organisms because it was less toxic to the bacteria than acetanilide. The induction increased the enzymic activity 250-fold. In comparison, the type culture strain of P. acidovorans, ATTCCI5668, had no amidase activity which could be induced by phenacetin. Optimal growth conditions were established with respect to the concentration of carbon source and inducer so that about 10% of the extractable bacterial protein consisted of the amidase. The organisms were lysed with lysozyme in the presence of EDTA and the enzyme was isolated mainly by column chromatography procedures. A preparation form 60 g (wet wt) bacteria yielded about 100 mg highly purified amidase with a specific activity of 137 mugmol substrate hydrolysed/min/mg protien. In addition to acetanilide, the purified enzyme hydrolysed several other amides and esters. As standard substrate, p-nitroacetanilide was chosen.

Acetamides↗

PubMed2004

S-stereoselective piperazine-2-tert-butylcarboxamide hydrolase from Pseudomonas azotoformans IAM 1603 is a novel L-amino acid amidase.

An amidase acting on (R,S)-piperazine-2-tert-butylcarboxamide was purified from Pseudomonas azotoformans IAM 1603 and characterized. The enzyme acted S-stereoselectively on (R,S)-piperazine-2-tert-butylcarboxamide to yield (S)-piperazine-2-carboxylic acid. N-terminal and internal amino acid sequences of the enzyme were determined. The gene encoding the S-stereoselective piperazine-2-tert-butylcarboxamide amidase was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 2.1 kb of genomic DNA revealed the presence of two ORFs, one of which (laaA) encodes the amidase. This enzyme, LaaA is composed of 310 amino acid residues (molecular mass 34 514 Da), and the deduced amino acid sequence exhibits significant similarity to hypothetical and functionally characterized proline iminopeptidases from several bacteria. The laaA gene modified in the nucleotide sequence upstream from its start codon was overexpressed in Escherichia coli. The activity of the recombinant LaaA enzyme in cell-free extracts of E. coli was 13.1 units.mg(-1) with l-prolinamide as substrate. This enzyme was purified to electrophoretic homogeneity by ammonium sulfate fractionation and two column chromatography steps. On gel-filtration chromatography, the enzyme appeared to be a monomer with a molecular mass of 32 kDa. It had maximal activity at 45 degrees C and pH 9.0, and was completely inactivated in the presence of phenylhydrazine, Zn2+, Ag+, Cd2+ or Hg2+. LaaA had hydrolyzing activity toward L-amino acid amides such as L-prolinamide, L-proline-p-nitroanilide, L-alaninamide and L-methioninamide, but did not act on the peptide substrates for the proline iminopeptidases despite their sequence similarity to LaaA. The enzyme also acted S-stereoselectively on (R,S)-piperidine-2-carboxamide, (R,S)-piperazine-2-carboxamide and (R,S)-piperazine-2-tert-butylcarboxamide. Based on its specificity towards L-amino acid amides, the enzyme was named L-amino acid amidase. E. coli transformants overexpressing the laaA gene could be used for the S-stereoselective hydrolysis of (R,S)-piperazine-2-tert-butylcarboxamide.

Amides↗

PubMed2004

Construction, characterization, and use of small-insert gene banks of DNA isolated from soil and enrichment cultures for the recovery of novel amidases.

To obtain new amidases of biocatalytic relevance, we used microorganisms indigenous to different types of soil and sediment as a source of DNA for the construction of environmental gene banks, following two different strategies. In one case, DNA was isolated from soil without preceding cultivation to preserve a high degree of (phylo)genetic diversity. Alternatively, DNA samples were obtained from enrichment cultures, which is thought to reduce the number of clones required to find a target enzyme. To selectively sustain the growth of organisms exhibiting amidase activity, cultures were supplied with a single amide or a mixture of different aromatic and non-aromatic acetamide and glycine amide derivatives as the only nitrogen source. Metagenomic DNA was cloned into a high-copy plasmid vector and transferred to E. coli, and the resulting gene banks were searched for positives by growth selection. In this way, we isolated a number of recombinant E. coli strains with a stable phenotype, each expressing an amidase with a distinct substrate profile. One of these clones was found to produce a new and highly active penicillin amidase, a promising biocatalyst that may allow higher yields in the enzymatic synthesis of beta-lactam antibiotics.

Acetamides↗

PubMed1972

N-acetylmuramyl-L-alanine amidase of Bacillus licheniformis and its L-form.

A cell wall lytic enzyme has been demonstrated to be a component of the membrane of Bacillus licheniformis NCTC 6346 and an l-form derived from it. The lytic enzyme, characterized as an N-acetylmuramyl-l-alanine amidase, is solubilized from membranes by nonionic detergents. Ionic detergents inactivate the enzyme. In the bacterium the specific activities of amidase and d-alanine carboxypeptidase in mesosomes are approximately 65% of those in membranes. Selective transfer of lytic enzyme from nongrowing L-forms, L-form membranes, and protoplasts to added walls occurred after mixing, and 31 to 77% of the enzyme lost from L-form membranes was recovered on the walls. Membranes isolated from L-forms growing in the presence of added walls contained as little as 13% of the amidase found in membranes of a control culture. These results have been interpreted as showing that in vivo the amidase is "bound" to the surface of the bacterial cell membrane in such a location that it can be readily accessible to the cell wall.

Alanine↗

PubMed1990

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

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 mumol 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 30 degrees C under the control of an E. coli promoter and ribosome-binding site.

Amides↗

PubMed1991

Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product.

Using in vitro-constructed deletions and subcloned DNA fragments, we have identified a new gene, amiC, which regulates expression of the inducible Pseudomonas aeruginosa aliphatic amidase activity. The DNA sequence of the gene has been determined, and an open reading frame encoding a polypeptide of 385 amino acids (molecular mass, 42,834 Da) has been identified. A search of sequence libraries has failed to find homologies with other published sequences. The amiC translation termination codon (A)TGA overlaps the initiation codon for the downstream amiR transcription antitermination factor gene, implying that the amiCR operon is coordinately regulated. Disruption of the amiC open reading frame by insertion and deletion leads to constitutive amidase synthesis, suggesting that AmiC is a negative regulator. This is confirmed by the finding that a broad-host-range expression vector carrying amiC (pSW41) represses amidase expression in a series of previously characterized P. aeruginosa amidase-constitutive mutants. The AmiC polypeptide has been purified from PAC452(pSW41), and N-terminal amino acid sequencing has confirmed the gene identification.

Amidohydrolases↗

PubMed2000

Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (Nt(N)-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1(-/-) mouse strains lacked the Nt(N)-amidase activity but retained glutamine-specific Nt(Q)-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1(-/-) fibroblasts. The Ntan1(-/-) mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.

Amidohydrolases↗

PubMed1999

Effect of dextran derivatives on arginine amidase activities released from isolated rabbit arteries.

In the present research we examined the levels and types of arginine amidase activities that were released from isolated rabbit arteries treated with heparin or chondroitin sulfate. Heparin accelerated the release of arginine amidase activity from the isolated rabbit ear artery, the induction was not significant; a slight increase in activity was observed in the level of arginine amidase released from isolated rabbit aorta, but no significant difference was observed. On the other hand, it was revealed that the addition of chondroitin sulfate, accelerated this release from isolated rabbit ear artery with 5% significant differences. After the addition of chondroitin sulfate, the arginine amidase activity released from isolated rabbit arteries was analyzed using various affinity adsorption methods. This analysis confirmed the presence of two types of fibrinolytic enzymes: plasminogen/plasmin activity and plasminogen activators, but no thrombin was detected.

Animals↗

PubMed1979

The structure of lipopolysaccharide from an Escherichia coli heptose-less mutant. III. Two fatty acyl amidases from Dictyostelium discoideum and their action on lipopolysaccharide derivatives.

Two fatty acyl amidases have been partially purified from the slime mold, Dictyostelium discoideum. Their action on lipopolysaccharide derivatives, especially Compound I, has been studied. Amidase I removes specifically the beta-hydroxymyristyl group, which is present on the amino group adjacent to the C-1 phosphate. The product, Compound V, is then a substrate for Amidase II, which removes the remaining beta-hydroxymyristyl group from the amino group in the distal glucosamine ring to give Compound VI. Compound I itself is resistant to Amidase II. Thus, the two enzymes show a high degree of structural specificity. The structure of lipopolysaccharide from the E. coli K-12 mutant is concluded in the light of studies reported in this and the accompanying papers, and this structure is discussed in relation to other bacterial lipopolysaccharides.

Amidohydrolases↗

PubMed1999

Beta-lactamase-free penicillin amidase from Alcaligenes sp.: isolation strategy, strain characteristics, and enzyme immobilization.

Isolation and characterization of a beta-lactamase (EC 3.5.2.6)-free, penicillin amidase (penicillin amidohydrolase, EC 3.5.1. 11)-producing organism is reported. The test strain was isolated by an enrichment technique with a substrate other than penicillins. The isolated strain belongs to the genus Alcaligenes. Phenylacetic acid was found to be the inducer of penicillin amidase. The amidase has a broad substrate spectrum. It is very active against penicillin G and semisynthetic cephalosporins, whereas penicillin V and semisynthetic penicillins acted moderately as a substrate. Immobilized cells of Alcaligenes sp. were shown to act as a reversible enzyme.

Alcaligenes↗

PubMed1980

Substrate specificity of penicillin amidase from E. coli.

1. The kinetic parameters of 12 substrates of penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11) from E. coli have been determined. Most of the penicillin amidase amide substrates containing a phenylacetyl group in the acyl moiety have been shown to have similar catalytic constants of 50 s-1. Substitution of the phenylacetyl group b 2-thienylacetyl group (cephalothin, cephaloridine) having a similar structure leads to a slight decrease in kcat. 2. Nonspecific penicillin amidase substrates, which contain a free amino group in their acyl moiety, are characterized by a strong dependence of kcat, on the structure of the leaving group with Km being constant. To investigate the free amino group influence on the reaction kinetics, pH-dependences of kcat/Km of enzymatic hydrolysis of phenylacetic and D-(-)-alpha-aminophenylacetic acid p-nitroanilides have been studied. It has been shown that enzyme binds the deprotonated form of the substrate only. 3. Under thermodynamically favourable conditions for the synthesis of beta-lactam antibiotics (at low pH), a concentration of the deprotonated substrate form is very low, and the reaction proceeds in the bimolecular regime. The value of the second-order rate constant for the substrate having a free amino group is small even at pH 7.5, and sharply decreases as does the pH. Hence, despite the favourable thermodynamic conditions for the production of all beta-lactam antibiotics, low reaction rate is the basic hindrance for enzymatic synthesis of penicillins and cephalosporins having a free amino group in the acyl moiety.

Amidohydrolases↗

PubMed1987

Nature of the interactions involved in the lipid-protein complexes of the Escherichia coli N-acetylmuramoyl-L-alanine amidase.

Depending on its concentration, phosphatidylglycerol, one of the three main Escherichia coli phospholipid species, is able to activate or inactivate the E. coli murein amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28) (Vanderwinkel, E. and De Vlieghere, M. (1985) Biochim. Biophys. Acta 838, 54-59). The mechanisms underlying the modulation of this enzyme activity were studied by analyzing the effects of cations, polycationic molecules, various surfactants and amphiphilic water-soluble compounds. K+, Mg2+ and polyamines were all able to prevent completely the enzyme inactivation produced by millimolar order concentration of phosphatidylglycerol. The efficiencies of the ionic species tested were in the order K+ less than Mg2+ = putrescine less than spermidine less than spermine. The kinetics of the counteraction processes were all sigmoidal. By contrast, the activation of the murein amidase produced by phosphatidylglycerol in micromolar concentration appeared to be insensitive to the ionic strength of the medium. Surfactants and amphiphilic molecules differing in their polar head and hydrophobic tail were found to activate the enzyme at various degrees for concentrations below their critical micellar concentration. The non-ionic surfactants were the most potent activators and remarkably mimicked the phosphatidylglycerol activation. The enzyme activation process appeared to require only a hydrophobic solvation shell around the protein. All kinetic data supported our previous interpretation of the phosphatidylglycerol-enzyme interactions in terms of multisite non-allosteric theory.

Amidohydrolases↗

PubMed2003

The Escherichia coli amidase AmiC is a periplasmic septal ring component exported via the twin-arginine transport pathway.

The N-acetylmuramoyl-l-alanine amidases of Escherichia coli (AmiA, B and C) are periplasmic enzymes that remove murein cross-links by cleaving the peptide moiety from N-acetylmuramic acid. Ami- cells form chains, indicating that the amidases help to split the septal murein. Interestingly, cells defective in the twin-arginine protein transport (Tat) pathway show a similar division defect. We find that both AmiA and AmiC are routed to the periplasm via Tat, providing an explanation for the Tat- division phenotype. Taking advantage of the ability of Tat to export prefolded (fluorescent) green fluorescent protein (GFP) to the periplasm, we sublocalized AmiA and AmiC in live cells using functional fusions to GFP. Interestingly, the periplasmic localization of the fusions differed markedly. AmiA-GFP appeared to be dispersed throughout the periplasm in all cells. AmiC-GFP similarly appeared throughout the periplasm in small cells, but was concentrated almost exclusively at the septal ring in constricting cells. Recruitment of AmiC to the ring was mediated by an N-terminal non-amidase targeting domain and required the septal ring component FtsN. AmiC therefore replaces FtsN as the latest known recruit to the septal ring and is the first entirely periplasmic component to be localized.

Amino Acid Sequence↗

PubMed1982

Purification and characterization of two phage PBSX-induced lytic enzymes of Bacillus subtilis 168: an N-acetylmuramoyl-L-alanine amidase and an N-acetylmuramidase.

After heat-induction of the defective phage PBSX in a xhi-1479 mutant of Bacillus subtilis 168, the culture lysed rapidly even if the lyt-2 mutation was present (which greatly reduces the amount of the bacterial autolysins). Two lytic enzymes, an N-acetylmuramoyl-L-alanine amidase and an endo-N-acetylmuramidase, were purified from the culture supernatant. The amidase was readily distinguished from the bacterial amidase by its low molecular weight. In addition, it was not inhibited by antibody directed against the bacterial enzyme. These results indicate that PBSX does not rely on the bacterial autolysins to accomplish lysis.

Amidohydrolases↗