Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our research showed that propionyl acylase gene is on 4.16kb insert DNA fragment originated from S. mycarofaciens in recombinant plasmid pIJM9. For localization of this gene on 4.16kb insert DNA fragment, sub-cloning has been carried out with religation of BamHI digested plasmid pIJM9. Among the transformants, molecular weight of recombinant plasmid pIJM95 harbouring in No. 5 transformant is 5.0kb. Molecular weight of insert DNA fragment is 0.53kb in this plasmid. In bioconversion experiment for spiramycin of No. 5 transformant, the bioconversion product was analysed with TLC, bioautography, HPLC and mass spectrum (FAB). Results showed that the bioconvert product is propionylspiramycin. No. 5 transformant is able to transform spiramycin into propionylspiramycin. Propionyl acylase gene was locolized on 0.53kb insert DNA fragment of recombinant plasmid pIJM95. Analysis result of DNA sequence showed that content of G + C is 68.2% for 0.53kb insert DNA fragment. More than 70 kinds of restriction endonuclease have cut sit on this fragment. From No.54-No.393 nucleotide, there is an open reading frame, which codes a polypeptide consisted of 122 amino acids. Start codon is ATG, stop codon is TGA.
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PMID:[Localization and nucleotide sequence of propionyl acylase gene of Streptomyces mycarofaciens]. 817 38

The vasoactive intestinal peptide (VIP) receptor from the human melanoma cell line IGR39 has been shown to be a 60-kDa glycoprotein. Using serial lectin affinity chromatography, as well as specific glycosidases, we demonstrate that VIP receptor-linked carbohydrates are predominantly tri- or tetraantennary sialylated N-linked oligosaccharides, 27% of which are fucosylated, and some may have terminal galactose residues. Treatment of 125I-VIP receptor complexes with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase revealed the presence of at least three N-linked carbohydrate chains/receptor polypeptide. To investigate the functional role of the carbohydrate moiety, 125I-VIP binding to IGR39 cell membranes was tested in the presence of soluble lectins. Among the lectins tested, only wheat germ agglutinin (WGA) was found to markedly inhibit VIP binding in a dose-dependent manner. Binding data indicated that the presence of the lectin led to a 3-fold increase in Kd value, from 0.15 to 0.44 nM, without any change in the number of available binding sites. The potent inhibitor of WGA binding, N,N',N"-triacetylchitotriose, completely reversed the effect of the lectin. On the other hand, VIP binding inhibition persisted even after neuraminidase treatment, suggesting that sialic acids were not directly involved. Furthermore, WGA inhibition was not abolished although most, if not all, VIP receptor oligosaccharides were converted to high mannose type structures by treating IGR39 cells with deoxymannojirimycin. Finally, whereas the pharmacological profile of VIP receptor was virtually identical, the presence of WGA greatly reduced the VIP-stimulated cAMP in IGR39 cells, indicating that the lectin alters the ability of the receptor to interact with the adenylate cyclase system.
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PMID:Structural and functional analysis of the human vasoactive intestinal peptide receptor glycosylation. Alteration of receptor function by wheat germ agglutinin. 838 3

A simple solid-phase binding assay was used to screen for interactions that the giant myofibrillar protein titin makes with other sarcomeric proteins. The titin used in the tests was purified by a modified procedure that results in isolation of approximately 20 mg relatively undegraded protein in < 24 h. In addition to the approximately 3 MDa polypeptide, bands at approximately 160 kDa and approximately 100 kDa were also consistently seen on gels. Binding of titin to myosin, C-protein, X-protein and AMP-deaminase was observed. The interaction with myosin appears to be with the light meromyosin part of the molecule.
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PMID:A survey of interactions made by the giant protein titin. 844 91

Adenosine 5'-monophosphate (AMP) deaminase from baker's yeast is an allosteric enzyme containing a single AMP binding site and two ATP regulatory sites per polypeptide [Merkler, D. J., & Schramm, V. L. (1990) J. Biol Chem. 265, 4420-4426]. The enzyme contains 0.98 +/- 0.17 zinc atom per subunit. The X-ray crystal structure for mouse adenosine deaminase shows zinc in contact with the attacking water nucleophile using purine riboside as a transition-state inhibitor [Wilson, D. K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284]. Alignment of the amino acid sequence for yeast AMP deaminase with that for mouse adenosine deaminase demonstrates conservation of the amino acids known from the X-ray crystal structure to bind to the zinc and to a transition-state analogue. On the basis of these similarities, yeast AMP deaminase is also proposed to use a Zn(2+)-activated water molecule to attack C6 of AMP with the displacement of NH3. The pKm and pKi profiles for AMP and a competitive inhibitor overlap in a bell-shaped curve with pKa values of 7.0 and 7.4. This pattern is characteristic of a rapid equilibrium between AMP and the enzyme, thus confirming the rapid equilibrium random kinetic patterns [Merkler, D. J., Wali, A. S., Taylor, J., Schramm, V. L. (1989) J. Biol. Chem. 264, 21422-21430]. The Vmax of the reaction requires one unprotonated and one protonated group with pKa values of 6.4 +/- 0.2 and 7.7 +/- 0.3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytic mechanism of yeast adenosine 5'-monophosphate deaminase. Zinc content, substrate specificity, pH studies, and solvent isotope effects. 850 99

N-Carbamoyl-L-amino acid amidohydrolase was purified to homogeneity for the first time from Alcaligenes xylosoxidans. The enzyme showed high affinity toward N-carbamoyl-L-amino acids with long-chain aliphatic or aromatic substituents, and hydrolyzed those with short-chain substituents quite well. The enzyme hydrolyzed N-formyl- and N-acetylamino acids quickly and very slowly, respectively. The enzyme did not hydrolyze beta-ureidopropionate and ureidosuccinate. The relative molecular mass of the native enzyme was about 135,000 and the enzyme consisted of two identical polypeptide chains. The enzyme activity was significantly inhibited by sulfhydryl reagents and required the following divalent metal ions: Mn2+, Ni2+ and Co2+.
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PMID:Purification and characterization of N-carbamoyl-L-amino acid amidohydrolase with broad substrate specificity from Alcaligenes xylosoxidans. 859 Jun 54

LytA amidase is the best known bacterial autolysin. It breaks down the N-acetylmuramoyl-L-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae and requires the presence of choline residues in the cell-wall teichoic acids for activity. Genetic experiments have supported the hypothesis that its 36-kDa chain has evolved by the fusion of two independent modules: the NH2-terminal module, responsible for the catalytic activity, and the COOH-terminal module, involved in the attachment to the cell wall. The structural organization of LytA amidase and of its isolated COOH-terminal module (C-LytA) and the variations induced by choline binding have been examined by differential scanning calorimetry and analytical ultracentrifugation. Deconvolution of calorimetric curves have revealed a folding of the polypeptide chain in several independent or quasi-independent cooperative domains. Elementary transitions in C-LytA are close but not identical to those assigned to the COOH-terminal module in the complete amidase, particularly in the absence of choline. These results indicate that the NH2-terminal region of the protein is important for attaining the native tertiary fold of the COOH terminus. Analytical ultracentrifugation studies have shown that LytA exhibits a monomer <--> dimer association equilibrium, through the COOH-terminal part of the molecule. Dimerization is regulated by choline interaction and involves the preferential binding of two molecules of choline per dimer. Sedimentation velocity experiments give frictional ratios of 1.1 for C-LytA monomer and 1.4 for C-LytA and LytA dimers; values that deviated from that of globular rigid particles. When considered together, present results give evidence that LytA amidase might be described as an elongated molecule consisting of at least four domains per subunit (two per module) designated here in as N1, N2, C1, and C2. Intersubunit cooperative interactions through the C2 domain in LytA dimer occur under all experimental conditions, while C-LytA requires the saturation of low affinity choline binding sites. The relevance of the structural features deduced here for LytA amidase is examined in connection with its biological function.
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PMID:Structural organization of the major autolysin from Streptococcus pneumoniae. 863 7

Glycosylasparaginase (EC 3.5.1.26) is a lysosomal amidase which hydrolyzes the bond between asparagine and the sugar moiety in N-linked glycoproteins. Deficiency of the enzyme results in aspartylglycosaminuria (AGU), the most common disorder of glycoprotein degradation. Mature enzyme is formed by two proteolytic cleavage steps subsequent to removal of its signal peptide: (1) an activation cleavage in the ER of the initial single-chain 49-kDa polypeptide into a 27-kDa alpha- and 19-kDa beta-subunit; (2) a cleavage in lysosomes which removes 10 amino acids from the C-terminus of the alpha-subunit without affecting enzyme activity. Each subunit of glycosylasparaginase contains one N-linked oligosaccharide (N38, alpha-subunit; N308, beta-subunit). Both oligosaccharides were phosphorylated and releasable by Endo-H digestion, indicating they were of the high-mannose type. These glycosylation sequenons were mutagenized to determine the role of the oligosaccharide at each site in proper folding and transport of glycosylasparaginase. An N38D mutant underwent the lysosomal processing step, indicating that targeting to lysosomes can be via the phosphorylated beta-subunit oligosaccharide alone. Deletion of the beta-subunit oligosaccharide oat N308 by an aspartic acid substitution resulted in very little protein or enzyme activity in the transfected cells, reemphasizing that glycosylation of the beta-subunit site is important for efficient folding and/or targeting. A different mutation to eliminate the same N-glycosylation sequenon (T310A) yielded more protein and enzyme activity, and a double mutant N38D/T310A yielded the same results as the single beta-subunit substitution. Yield of enzyme for all mutants was increased in cells treated with brefeldin A. The N308 glycosylation site of the beta-subunit appears to be more important in maintaining normal transport and stability of human glycosylasparaginase.
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PMID:Glycosylation and phosphorylation of lysosomal glycosylasparaginase. 863 40

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.
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PMID:Acetylpolyamine amidohydrolase from Mycoplana ramosa: gene cloning and characterization of the metal-substituted enzyme. 882 26

The Bacillus subtilis sleB gene, which codes for the enzyme homologous to the germination-specific amidase from Bacillus cereus, was cloned and its nucleotide sequence was determined. Sequence analysis showed that it had an open reading frame of 918 bp, coding for a polypeptide of 305 amino acids with a putative signal sequence of 29 residues. Enzyme activity was not found in germination exudate of B. subtilis spores, which differs from the case of B. cereus enzyme. A B. subtilis mutant with an insertionally inactivated sleB gene revealed normal behavior in growth and sporulation. However, the sleB mutant was unable to complete germination mediated by L-alanine.
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PMID:A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination. 883 Jul 7

The secondary and tertiary structures of the choline-dependent major pneumococcal autolysin LytA amidase and of its COOH-terminal domain, C-LytA, have been investigated by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Deconvolution analysis shows that the far-UV CD spectrum of both proteins is governed by chiral contributions, ascribed to aromatic residue clusters contained in the COOH-terminal module. The secondary structure of LytA, determined from the FTIR spectral features of the amide I' band, results in 19% of alpha-helix and tight loops, 47% of beta-sheets, 23% of turns, and 11% of irregular structures. Similar values are obtained for C-LytA. The addition of choline significantly modifies the far- and near-UV CD spectra of LytA and C-LytA. These changes are attributed to alterations in the environment of their aromatic clusters, since the FTIR spectra indicate that the secondary structure is essentially unaffected. CD choline titration curves at different wavelengths show the existence of two types of binding sites/subunit. Data analysis assuming protein dimerization upon saturation of the high affinity sites reveals positive cooperativity between the low affinity sites. Thermal denaturation of both proteins occurs with the formation of unfolding intermediates and the presence of residual secondary structure in the final denatured state. The irreversibility of the thermal denaturation of LytA and C-LytA results from the collapse of the polypeptide chain into intermolecular extended structures. At saturating concentrations, choline prevents the formation of these structures in the isolated COOH-terminal module.
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PMID:Structural characterization of the unligated and choline-bound forms of the major pneumococcal autolysin LytA amidase. Conformational transitions induced by temperature. 891 May 72


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