Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The chemoenzymatic route to 2-deoxy-2-propionamido-D-mannose (1b), 2-butyramido-2-deoxy-D-mannose (2b) and 2-deoxy-2-phenylacetamido-D-mannose (3b) involved N-acylation of 2-amino-2-deoxy-D-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido-D-glucose (1a), 2-butyramido-2-deoxy-D-glucose (2a) and 2-deoxy-2-phenylacetamido-D-glucose (3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy-D-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 degrees C and 8.0-9.0, respectively. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose and 2-acetamido-2-deoxy-6-O-sulfo-D-glucose but not 2-acetamido-2-deoxy-1-O-phospho-D-glucose or 2-acetamido-2-deoxy-D-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al3+, Ca2+, Co2+, Cu2+, Mn2+ or Zn2+ and activated by 1 mM Mg2+. The concentrated enzyme is highly stable at 4 degrees C in the presence of 0.1 M ammonium sulfate.
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PMID:A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups. 1560 34

AMP-deaminase from human liver was purified by two-step phosphocellulose chromatography, and SDS-PAG electrophoresis of the most active enzyme fraction eluted has been performed. The largest of the protein fragments revealed had a size (92 kDa) of an apparent full-size enzyme subunit, and reacted positively with antibodies produced against specific human ampd2 gene product. Three-day storage at cold room temperature modified significantly the electrophoretical pattern of the enzyme, evidencing continuous and progressive degradation of its structure. This is a first report evidencing the presence of apparent full-size form of human liver AMP-deaminase in preparation obtained from endogenous source.
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PMID:Full-size form of human liver AMP-deaminase? 1564 34

The controlled and partial modification of epoxy groups of Eupergit C and EP-Sepabeads with sodium sulfide has permitted the preparation of thiol-epoxy supports. Their use allowed not only the specific immobilization of enzymes through their thiol groups via thiol-disulfide interchange, but also enzyme stabilization via multipoint covalent attachment. Penicillin G acylase (PGA) from Escherichia coli and lipase from Rhizomucor miehei were used as model enzymes. Both enzymes lacked exposed cysteine residues, but were introduced via chemical modification under very mild conditions. In the first moments of the immobilization, a certain percentage of immobilized protein could be released from the support by incubation with DTT; this confirms that the first step was via a thiol-disulfide interchange. Moreover, the promotion of some further epoxy-enzyme bonds was confirmed because no enzyme release was detected after some immobilization time by incubation with DTT. In the case of the heterodimeric PGA, it was possible to demonstrate the formation of at least one epoxy bond per enzyme subunit by analyzing with SDS-PAGE the supernatants obtained after boiling the enzyme derivatives in the presence of mercaptoethanol and SDS. Thermal inactivation studies showed that these multipoint enzyme-support attachments promoted an increase in the stability of the immobilized enzymes. In both cases, the stabilization factor was around 12-15-fold comparing optimal derivatives with their just-thiol immobilized counterparts.
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PMID:Stabilization of enzymes by multipoint immobilization of thiolated proteins on new epoxy-thiol supports. 1581 62

Brevundimonas diminuta TPU 5720 produces an amidase acting L-stereoselectively on phenylalaninamide. The enzyme (LaaA(Bd)) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The final preparation gave a single band on SDS-PAGE with a molecular weight of approximately 53,000. The native molecular weight of the enzyme was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal activity at 50 degrees C and pH 7.5. LaaA(Bd) lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was largely restored by the addition of Co(2+) ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co(2+), suggesting that LaaA(Bd) is a Co(2+)-dependent enzyme. LaaA(Bd) had hydrolyzing activity toward a broad range of L-amino acid amides including L-phenylalaninamide, L-glutaminamide, L-leucinamide, L-methioninamide, L-argininamide, and L-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaA(Bd) was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence of seven open-reading frames (ORFs), one of which (laaA ( Bd )) encodes the amidase. LaaA(Bd) is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA ( Bd ) gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaA(Bd) in cell-free extracts of the E. coli transformant was 25.9 units mg(-1) with L-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720.
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PMID:L: -Stereoselective amino acid amidase with broad substrate specificity from Brevundimonas diminuta: characterization of a new member of the leucine aminopeptidase family. 1600 Dec 51

In this work, we have used supports activated with m-amino-phenylboronic groups to "reversibly" immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.
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PMID:Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports. 1612 5

A successive C-terminal amino acid truncation reaction with acetic anhydride was applied on proteins in polyacrylamide gel. Protein bands separated by conventional SDS-PAGE were excised, partially fixed in the gel with glutaraldehyde ethanol solution, dehydrated with ACN and subjected to the truncation reaction with acetic anhydride formamide solution. Pre-treatment of the gel with pyridine aqueous solution was found to enhance the truncation reaction yields. After the truncation reaction, the products were treated with an aqueous solution of dimethylaminoethanol to hydrolyze oxazolone rings at the C termini of the truncated products and O-acetylated products of serine, threonine and/or tyrosine. Several commercially available proteins of 10-40 kDa, as determined by SDS-PAGE, such as myoglobin, trypsin inhibitor, alpha-hemolysin, cytochrome c, chymotrypsin C chain, elastase, acylase and histone H4, were subjected to the C-terminal analysis. The truncated proteins were in-gel digested with trypsin and the extracted peptides were analyzed by MALDI-TOF MS, giving rise to a series of molecular mass ions of the C-terminal truncated fragments corresponding to the C-terminal amino acid sequence of the relevant protein.
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PMID:C-terminal sequencing method for proteins in polyacrylamide gel by the reaction of acetic anhydride. 1655 87

The amidase of Nocardia sp. is one of important industrial enzymes. Based on DNA and protein sequence alignment from different strains, a new gene of amidase was successfully cloned from Nocardia YS-2002, which is widely used for industrial production of acrylamide in China. DNA sequence analyses showed that the 1466bp cloned-fragment contains promoter, open reading frame and terminating-palindrome. Protein sequence alignment and phylogenetic tree analyses showed that the amidase coming from Nocardia sp. YS-2002 is a kind of specialamidase, without the typical conserved sequence of the amidases. Enzymatic characteristics predictions indicated that the molecular weight and pI of the new amidase is approximately 38.05 kD and 4.88, respectively, and it would be stable when heterogeneously expressed in E. coli. By inserting the ORF of the amidase into plasmid pET-28a(+), a recombinant strain, pEAB, was selected using E. coli BL21(DE3) as the host. SDS-PAGE analyses of both the whole cells and ultrasonic-treated cells confirmed the feasibility of the heterogeneous expression of amidase in the recombinant E. coli. But the activity of amidase in E. coli BL21(DE3) not more than 0.5 u/mg, because most of the enzymes expressed were formed as inclusion bodies.
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PMID:[Molecular cloning of an amidase gene from Nocardia sp. and its expressionin Escherichia coli]. 1689 10

A N-carbamoyl-D-amino acid amidohydrolase gene (hyuC) from Sinorhizobium morelens S-5 was cloned by LA PCR, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the hyuC gene exhibited high homology to the amino acid sequences of D-carbamoylase from other sources. The gene could be highly expressed in Escherichia coli, and the recombinant enzyme was purified 16.1-fold to homogeneity with a yield of 21.2% by heat treatment and three steps of column chromatography. The results of gel filtration on Superdex 200 HR and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 38-kDa subunits. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl alpha-amino acid to the corresponding free amino acid, and it was strictly D-specific. The enzyme showed broad substrate specificity, and exhibited high activity in the hydrolysis of N-carbamoyl-D-p-hydroxyphenylglycine as substrate. The enzyme did not hydrolyze N-carbamoyl-beta-alanine. The optimum pH and temperature of the enzyme were pH 7.0 and 60 degrees C, respectively. Enzyme activity was slightly improved by Ca2+ and Fe2+, and nearly not affected by metal chelators and sulfhydryl reagents. The enzyme showed high thermal and oxidative stability. These results show that the enzyme has great potential for industrial application.
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PMID:[Cloning, expression and purification of D-carbamoylase from Sinorhizobium morelens S-5]. 1703 56

An amidase (EC 3.5.1.4) in branch 2 of the nitrilase superfamily, from the thermophilic strain Geobacillus pallidus RAPc8, was produced at high expression levels (20 U/mg) in small-scale fermentations of Escherichia coli. The enzyme was purified to 90% homogeneity with specific activity of 1,800 U/mg in just two steps, namely, heat-treatment and gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopic (EM) analysis of the homogenous enzyme showed the native enzyme to be a homohexamer of 38 kDa subunits. Analysis of the biochemical properties of the amidase showed that the optimal temperature and pH for activity were 50 and 7.0 degrees C, respectively. The amidase exhibited high thermal stability at 50 and 60 degrees C, with half-lives greater than 5 h at both temperatures. At 70 and 80 degrees C, the half-life values were 43 and 10 min, respectively. The amidase catalyzed the hydrolysis of low molecular weight aliphatic amides, with D: -selectivity towards lactamide. Inhibition studies showed activation/inhibition data consistent with the presence of a catalytically active thiol group. Acyl transfer reactions were demonstrated with acetamide, propionamide, isobutyramide, and acrylamide as substrates and hydroxylamine as the acyl acceptor; the highest reaction rate being with isobutyramide. Immobilization by entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4 degrees C and on Amberlite-XAD57 resulted in low protein binding and low activity, but immobilization on Eupergit C beads at 25 degrees C with cross-linking resulted in high protein binding yield and high immobilized specific activity (80% of non-immobilized activity). Characterization of Eupergit C-immobilized preparations showed that the optimum reaction temperature was unchanged, the pH range was somewhat broadened, and stability was enhanced giving half-lives of 52 min at 70 degrees C and 30 min at 80 degrees C. The amidase has potential for application under high temperature conditions as a biocatalyst for D: -selective amide hydrolysis producing enantiomerically pure carboxylic acids and for production of novel amides by acyl transfer.
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PMID:A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity. 1734 19

Rhodobacter sphaeroides OU5 utilized l-phenylalanine as sole source of nitrogen for growth. The metabolites of l-phenylalanine catabolism, i.e. 4-hydroxy phenylalanine (l-tyrosine), 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenyl-pyruvic acid (DOPP), 3,4-dihydroxyphenyllactic acid (DOPLA), 3,4-dihydroxyphenyl-acetic acid (DOPAc) and 3,4-dihydroxybenzoic acid (PC), were identified using liquid chromatography-mass spectroscopy (LC-MS). With 2-oxoglutarate as an amino acceptor, DOPA aminotransferase activity was observed with cell-free extracts and the product DOPP was confirmed through mass analysis. Reductive deamination of DOPA also occurred in the absence of 2-oxoglutarate, whose products were 3,4-dihydroxyphenylpropionic acid (DPPA) and ammonia. The enzyme DOPA-reductive deaminase (DOPARDA) was purified to its homogeneity and characterized. DOPARDA has an obligate requirement for NADH and is functional at low concentrations of the substrate (<150 microM). The molecular mass of the purified enzyme was approximately 274kD and the enzyme could be a heterotetramer of 110, 82, 43 and 39kD subunits as determined by SDS-PAGE.
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PMID:Catabolism of L-phenylalanine and L-tyrosine by Rhodobacter sphaeroides OU5 occurs through 3,4-dihydroxyphenylalanine. 1761 48


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