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)

We have cloned a gene (aphA) encoding acetylpolyamine amidohydrolase from Mycoplana ramosa ATCC 49678, (previously named Mycoplana bullata). A genomic library of M. ramosa was screened with an oligonucleotide probe designed from a N-terminal amino acid sequence of the enzyme purified from M. ramosa. Nucleotide sequence analysis revealed an open reading frame of 1,023 bp which encodes a polypeptide with a molecular mass of 36,337 Da. This is the first report of the structure of acetylpolyamine amidohydrolase. The aphA gene was subcloned under the control of the trc promoter and was expressed in Escherichia coli MM294. The recombinant enzyme was purified, and the enzymatic properties were characterized. Substrate specificities, Km values, and Vmax values were identical to those of the native enzyme purified from M. ramosa. In the analysis of the metal-substituted enzymes, we found that the acid limb of pH rate profiles shifts from 7.2 for the original zinc enzyme to 6.6 for the cobalt enzyme. This change suggests that the zinc atom is essential for the catalytic activity of the enzyme similarly to the zinc atom in carboxypeptidase A.
 ...
PMID:Acetylpolyamine amidohydrolase from Mycoplana ramosa: gene cloning and characterization of the metal-substituted enzyme. 882 26

A thermostable aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) from Bacillus stearothermophilus was overexpressed in E. coli and characterized with respect to metal content, metal dependence, heat stability, and quaternary structure. Like other enzymes of the aminoacylase family, native aminoacylase contains one Zn2+ ion per subunit. Several other transition metal ions (Co2+, Mn2+ and Cd2+) also sustain aminoacylase activity toward N-acetyl L-alanine with Cd2+ giving the highest turnover number. The stability constants of the respective metal complexes were estimated by activity measurements in metal buffer systems. Co2+ also acts as an activator mainly by lowering the Km for the substrate. These data and CD spectra obtained with the native and the metal-free enzyme suggest a predominantly structural role for the intrinsic metal ion of thermostable aminoacylase. In contrast to previous reports the enzyme behaved as a dimer in analytical gel filtration.
 ...
PMID:Thermostable aminoacylase from Bacillus stearothermophilus: significance of the metal center for catalysis and protein stability. 896 73

Cobalt is an essential component of a low molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1. We have found a new gene, nhlF, in the DNA region sandwiched between nhlBA encoding L-NHase and amdA encoding amidase, which are involved in the degradation of nitriles. The product of nhlF, NhlF, shows a significant sequence similarity with those of hoxN from Alcaligenes eutrophus, hupN from Bradyrhizobium japonicum, nixA from Helicobacter pylori, and ureH from Bacillus sp., which are considered to be involved in nickel uptake into these cells. Sequence and hydropathy plot analyses have shown that NhlF encodes a 352-amino acid (aa) protein with eight hydrophobic putative membrane-spanning domains. nhlF expression in R. rhodochrous ATCC 12674 and Escherichia coli JM109 confers uptake of 57Co in their cells, but not of 63Ni. The expression of both nhlF and nhlBA in R. rhodochrous ATCC 12674 exhibited higher NHase activity than nhlBA expression. These findings together with the inhibitory effect by uncouplers (CCCP and SF6847) for the cobalt uptake suggest that NhlF mediates the cobalt transport into the cell energy-dependently finally to provide L-NHase.
 ...
PMID:A novel transporter involved in cobalt uptake. 899 Jan 57

A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S)-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8 and 50 degrees C, respectively. The enzyme was stable in the range of pH 7-9 and at temperatures up to 30 degrees C. The enzyme activity was inhibited by Cu2+, Co2+, Ni2+, and Zn2+, and this inhibition was reversed by EDTA.M.
 ...
PMID:Isolation and characterization of highly (R)-specific N-acetyl-1-phenylethylamine amidohydrolase, a new enzyme from Arthrobacter aurescens AcR5b. 923 88

The soluble acylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) from hog intestinal mucosa was 11,000-fold purified for the first time using a new four-step procedure involving an immunoaffinity chromatography. The resulting protein, which had an isoelectric point of 5.2 and a M(r) of 90,000 was composed of two apparently identical N-acylated polypeptide chains. Its amino acid composition was comparable to that of hog kidney acylase I. The enzyme had a pH optimum at 8.0 and required Zn2+ or Co2+. The optimal temperature for the acylase reaction was 40 degrees C and the activation energy of thermodenaturation was estimated at 260 kJ mol-1. The enzyme was strongly inhibited when preincubated with chelating agents, by diethyl pyrocarbonate under histidine-modifying conditions as well as by sulfhydryl compounds. The reaction of the purified enzyme with the synthetic substrate furylacryloyl-L-methionine was partly characterized as follows: Km = 0.22 +/- 0.03 mM, kcat = 128.0 +/- 17.8 s-1 and kcat/Km = 5.8 +/- 1.6 x 10(5) M-1 s-1. The L-stereoisomer of methionine competitively inhibited the enzyme reaction with a Ki of 3.4 +/- 0.2 mM. It is suggested that acylase I might not only be involved in the catabolism of intracellular N-acylated protein but also be responsible for the biological utilization of N-acylated food proteins.
 ...
PMID:The hog intestinal mucosa acylase I: subcellular localization, isolation, kinetic studies and biological function. 925 35

High-molecular-mass nitrile hydratase (H-NHase, 530 kDa) is a cobalt-containing enzyme produced by Rhodococcus rhodochrous J1. For efficient production of H-NHase in R. rhodochrous ATCC12674, several plasmids were constructed. The enzyme was produced in the recombinant Rhodococcus cells only in the presence of an upstream region (approximately 4 kb) of the H-NHase gene under the control of the promoter for the amidase-NHase gene cluster from Rhodococcus sp. N-774. Although H-NHase was produced as a soluble protein in the cells, the protein did not show NHase activity. However, when the recombinant R. rhodochrous ATCC12674 cells were cultured in the presence of amide compounds, such as crotonamide and methacrylamide, markedly high NHase activity was detected, Gel-filtration chromatography revealed that the NHases produced by the cells grown in the presence and absence of the amide compounds had a molecular mass of more than 500 kDa and 50-80 kDa respectively. These results suggest that the amide compounds are essential for subunit assembly to form an enzymatically active multimer. By the use of the recombinant expression system, NHase activity 1.7 times higher than that of the original strain, R. rhodochrous J1, was achieved.
 ...
PMID:Overexpression of high-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1 in recombinant Rhodococcus cells. 965 Feb 55

A poly-His tag was fused in the glutaryl acylase (GA) from Acinetobacter sp. strain YS114 cloned in E. coli yielding a fully active enzyme. Biochemical analyses showed that the tag did not alter the maturation of the chimeric GA (poly-His GA) that undergoes a complex post-translational processing from an inactive monomeric precursor to the active heterodimeric enzyme. This enzyme has been used as a model to develop a novel and very simple procedure for one-step purification of poly-His proteins via immobilized metal-ion affinity chromatography on tailor-made supports. It was intended to improve the selectivity of adsorption of the target protein on tailor-made chelate supports instead of performing a selective desorption. The rate and extent of the adsorption of proteins from a crude extract from E. coli and of pure poly-His tagged GA on different metal chelate supports was studied. Up to 90% of proteins from E. coli were adsorbed on commercial chelate supports having a high density of ligands attached to the support through long spacer arms, while this adsorption becomes almost negligible when using low ligand densities, short spacer arms and Zn2+ or Co2+ as cations. On the contrary, poly-His GA adsorbs strongly enough on all supports. A strong affinity interaction between the poly-His tail and a single chelate moiety seems to be the responsible for the adsorption of poly-His GA. By contrast, multipoint weak interactions involving a number of chelate moieties seem to be mainly responsible for adsorption of natural proteins. By using tailor-made affinity supports, a very simple procedure for one-step purification of GA with minimal adsorption of host proteins could be performed. Up to 20 mg of GA were adsorbed on each ml of chelate support while most of accompanying proteins were hardly adsorbed on such supports. Following few washing steps, the target enzyme was finally recovered (80% yield) by elution with 50 mM imidazole with a very high increment of specific activity (up to a 120 purification factor).
 ...
PMID:Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports. 1042 52

A moderate thermophile, Bacillus sp. BR449 was previously shown to exhibit a high level of nitrile hydratase (NHase) activity when growing on high levels of acrylonitrile at 55 degrees C. In this report, we describe the cloning of a 6.1 kb SalI DNA fragment encoding the NHase gene cluster of BR449 into Escherichia coli. Nucleotide sequencing revealed six ORFs encoding (in order), two unidentified putative proteins, amidase, NHase beta- and alpha-subunits and a small putative protein of 101 amino acids designated P12K. Spacings and orientation of the coding regions as well as their gene expression in E. coli suggest that the beta-subunit, alpha-subunit, and P12K genes are co-transcribed. Analysis of deduced amino acid sequences indicate that the amidase (348 aa, MW 38.6 kDa) belongs to the nitrilase-related aliphatic amidase family, and that the NHase beta- (229 aa, MW 26.5 kDa) and alpha- (214 aa, MW 24.5 kDa) subunits comprise a cobalt-containing member of the NHase family, which includes Rhodococcus rhodochrous J1 and Pseudomonas putida 5B NHases. The amidase/NHase gene cluster differs both in arrangement and composition from those described for other NHase-producing strains. When expressed in Escherichia coli DH5alpha, the subcloned NHase genes produced significant levels of active NHase enzyme when cobalt ion was added either to the culture medium or cell extracts. Presence of the P12K gene and addition of amide compounds as inducers were not required for this expression.
 ...
PMID:Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp. BR449 into Escherichia coli. 1097 71

The Bacillus subtilis CwlC and the Bacillus polymyxa var. colistinus CwlV are the cell wall lytic N-acetylmuramoyl-l-alanine amidases in the CwlB (LytC) family. Deletion in the CwlC amidase from the C terminus to residue 177 did not change the amidase activity. However, when the deletion was extended slightly toward the N terminus, the amidase activity was entirely lost. Further, the N-terminal deletion mutant without the first 19 amino acids did not have the amidase activity. These results indicate that the N-terminal half (residues 1-176) of the CwlC amidase, the region homologous to the truncated CwlV (CwlVt), is a catalytic domain. Site-directed mutagenesis was performed on 20 highly conserved amino acid residues within the catalytic domain of CwlC. The amidase activity was lost completely on single amino acid substitutions at two residues (Glu-24 and Glu-141). Similarly, the substitution of the two glutamic acid residues (E26Q and E142Q) of the truncated CwlV (CwlV1), which corresponded to Glu-24 and Glu-141 of CwlC, was critical to the amidase activity. The EDTA-treated CwlV1 did not have amidase activity. The amidase activity of the EDTA-treated CwlV1 was restored by the addition of Zn2+, Mn2+, and Co2+ but not by the addition of Mg2+ and Ca2+. These results suggest that the amidases in the CwlB family are zinc amidases containing two glutamic acids as catalytic residues.
 ...
PMID:Mutational analysis of catalytic sites of the cell wall lytic N-acetylmuramoyl-L-alanine amidases CwlC and CwlV. 1137 3

The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A. tumefaciens. The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass. The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron. The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 mol per min per mg and a Km of 7.9 microM.
 ...
PMID:Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium. 1160 11


<< Previous 1 2 3 4 5 6 7 Next >>