Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of additives on the nonenzymatic deamidation of an Asn residue in a peptide and racemization of Asp and/or Asn in lysozyme were investigated at pH 6 and 100 degrees C. These chemical reactions were accelerated by the addition of phosphate ions. Several salts suppressed the deamidation in the presence of phosphate ions, while the salts did not affect the deamidation in the absence of phosphate ion at pH 6 and 100 degrees C. The results indicated that the effect of the salts was due to the suppression of phosphate catalysis. On the other hand, trifluoroethanol (TFE), which induces the conversion of random coiled polypeptides to secondary structured ones, dramatically suppressed the deamidation of an Asn residue in a peptide. The rate of deamidation in the presence of TFE was comparable to that of asparagine (free amino acid), which was very slowly deamidated. Because TFE could not suppress the deamidation of free asparagine, the suppression of the deamidation of an Asn residue in a peptide was attributed to suppression of the catalysis by the peptide bond in the carboxyl terminus. Since the inactivation of lysozyme was caused by multiple chemical reactions such as the deamidation and racemization, it was expected that the inactivation of lysozyme could be prevented by the addition of salts or TFE. Thus, it was confirmed that salts and TFE suppressed the lysozyme inactivation at pH 6 and 100 degrees C.
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PMID:Stabilization of lysozyme against irreversible inactivation by suppression of chemical reactions. 762 34

15N-labeled hen lysozyme has been studied by 2D and 3D NMR in order to characterize its dynamic behavior. The resonances of all main-chain amide nitrogen atoms were assigned, as were resonances of nitrogen atoms in 28 side chains. Relaxation measurements for the main-chain and arginine and tryptophan side-chain 15N nuclei used standard methods, and those for the 15N nuclei of asparagine and glutamine side chains used pulse sequences designed to remove unwanted relaxation pathways in the NH2 groups. The calculated order parameters (S2) show that the majority of main-chain amides undergo only small amplitude librational motions on a fast time scale (S2 > or = 0.8). Increased main-chain motion (0.5 < S2 < 0.8) is observed for a total of 19 residues located at the C-terminus, in loop and turn regions, and in the first strand of the main beta-sheet. Order parameters derived for the side chains range from 0.05 to 0.9; five of the six tryptophan residues have high order parameters (S2 > or = 0.8), consistent with their location in the closely packed core of the protein, whereas the order parameters between 0.05 and 0.3 for arginine residues confirm increased side-chain mobility at the protein surface. Order parameters for the side chains of asparagine and glutamine residues range from 0.2 to 0.8; high values are found for side chains that have low solvent accessible surfaces and well-defined chi 1 values, as measured by 3J alpha beta coupling constants. Many of the main-chain and side-chain groups with low order parameters have higher than average temperature factors in X-ray crystal structures and increased positional uncertainty in NMR solution structures. They also tend to lack persistent hydrogen bond interactions and protection against amide hydrogen exchange. The most significant correlations are found between residues with low order parameters and high surface accessibility in both crystal and solution structures. The results suggest that a lack of van der Waals contacts is a major determinant of side-chain and main-chain mobility in proteins.
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PMID:Structural determinants of protein dynamics: analysis of 15N NMR relaxation measurements for main-chain and side-chain nuclei of hen egg white lysozyme. 769 70

A recently developed multigrid-based Newton method for solving the nonlinear Poisson-Boltzmann equation is applied in an investigation of molecular recognition in the system consisting of the monoclonal antibody HyHEL-5 and hen egg lysozyme. The electrostatic free energy of binding is calculated for the wild-type complex and various mutants in which electrostatic interactions between the two proteins are altered. Mutations which neutralize or reverse the charge of any of the residues involved in salt-links in the native system always yield decreased binding affinities. The stability of the complex can be enhanced through the formation of a new salt-bridge obtained by mutating an asparagine residue of the lysozyme to the negatively-charged aspartate. Ionic strength effects are also examined and found to be significant in some cases.
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PMID:Role of electrostatics in antibody-antigen association: anti-hen egg lysozyme/lysozyme complex (HyHEL-5/HEL). 770 79

Incubation of lysozyme with acetaldehyde (0.44 M) at room temperature for 2 h produces a 62% inhibition of enzymic activity. Because the active site cleft contains tryptophyls, asparagine, glutamine, and an arginine residue, and because acetaldehyde reacts with indoles, amides, and guanidines, it is suggested that these sites are likely ones for alkylation. The epsilon-amino groups of lysines on the surface of the molecule are also susceptible to covalent modification. Total acetylation of lysozyme has been reported to inactivate the enzyme. These results suggest the possibility that inactivation of a fraction of the lysozyme activity by acetaldehyde may decrease the effectiveness of the enzyme in chronic alcoholics, thereby leading to an increased potential for susceptibility to bacterial infection.
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PMID:Acetaldehyde-modified lysozyme function: its potential implication in the promotion of infection in alcoholics. 777 70

A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.
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PMID:Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation. 788 12

To investigate the immunogenicity of glycopeptides, a peptide fragment from hen egg lysozyme, HEL(81-96)-Y (here named 1) which is immunogenic in H-2k mice and known to bind to the murine major histocompatibility complex (MHC) class II molecule Ek, was synthesized in five different glycosylated forms. The N-terminal serine of HEL(81-96)-Y was derivatized with D-glucose (2), maltotriose (3), and a branched D-glucose pentasaccharide (4). Furthermore, 1 was prepared with a central serine or asparagine derivatized with the branched D-glucose pentasaccharide (5) and GlcNAc (6), respectively. The ability of the five glycopeptides and the non-glycosylated peptide, labeled with 125I, to bind to the two MHC class II molecules, Ak and Ek, was studied using a gel filtration assay. None of them could bind to Ak. Neither 5 nor 6 were able to bind to Ek. Surprisingly 2, 3 and 4 bound better to Ek than did the non-glycosylated peptide 1. The increased binding varied depending on the type of oligosaccharide attached to the N terminus of the peptide. The better binding to Ek of glycopeptide 4 was found to be due to an increased association rate. The binding of 1 as well as 4 was optimal at pH 5.0. Functional studies showed that 4 was able to elicit a heteroclitic proliferative response from T cells of mice immunized with the native non-glycosylated peptide. Circular dichroism studies of 1 and 4 indicated a more unordered structure of 4 and a predominant alpha-helical conformation of 1, suggesting that the MHC class II molecule may bind to peptides which are in a non-alpha-helical conformation. These results demonstrate that glycosylation has considerable influence on peptide immunogenicity for T lymphocytes.
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PMID:Attachment of oligosaccharides to peptide antigen profoundly affects binding to major histocompatibility complex class II molecules and peptide immunogenicity. 818 18

The large molecular size of N-glycosylated lysozyme with a polymannose chain was predominantly expressed in the yeast carrying the lysozyme expression plasmid in 9-fold greater secretion compared with the wild type. Complementary DNA encoding hen egg white lysozyme was subjected to site-directed mutagenesis to obtain the Asn-X-Ser/Thr sequence that is the signal for asparagine-linked (N-linked) glycosylation. At positions 49, 67, 70, and 103, the signal for N-linked glycosylation was created. Only the mutant lysozyme whose glycine 49 was substituted with asparagine was expressed in the two types of glycosylated forms, a small oligomannose chain (Man18GlcNAc2)-linked form and a large polymannose chain (Man310GlcNAc2)-linked form, whereas other mutants were not glycosylated. The secreted amount of polymannosyl lysozyme was much higher than that of the oligomannosyl lysozyme. Both types of glycosylated lysozymes were susceptible to endo-beta-N-acetylglucosaminidase cleavage of their carbohydrate chains. The average molecular masses of oligomannosyl and polymannosyl lysozymes were 18 and 71 kDa, respectively. The length of the polymannose chain was found to be 200-350 residues/molecule of lysozyme according to the estimation of the molecular mass distribution by low angle laser light scattering measurements. The protein conformation estimated by CD analysis was completely conserved in these glycosylated lysozymes. The enzymatic activities of oligomannosyl and polymannosyl lysozymes were 100 and 91%, respectively, of wild-type protein when glycol chitin was used as a substrate. In addition, the polymannosyl lysozyme revealed remarkable heat stability in that no coagulation was observed under conditions in which the wild-type lysozyme coagulated. Thus, this novel glycoprotein can be used as a reporter in studies of the processing and sorting of glycoproteins and as a model of the expression of foreign genes in yeast for the construction of stable enzymes.
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PMID:Hyperglycosylation of hen egg white lysozyme in yeast. 850 5

Hen egg white lysozyme was genetically modified to have extreme heat stability and strong antimicrobial activity against Gram negative bacteria and the modified lysozymes were secreted in yeast and tobacco. Complementary DNA encoding lysozyme was subjected to site-directed mutagenesis to have the Asn-X-Thr(Ser) sequence that is the signal for asparagine-linked glycosylation at the positions 49. The glycosyl lysozyme enhanced heat stability was expressed in the yeast carrying the modified lysozyme cDNA. The expression amount of glycosyl lysozyme was about 10 mg/l of yeast culture medium. Using the same yeast expression system, the lysozyme enhanced antimicrobial action by inserting hydrophobic penta-peptide at the C-terminus were secreted in a small amount (less than 100 micrograms/l in the yeast culture medium). These cDNA constructs of modified lysozymes were engineered into tabacco through Agrobacterium-mediated transformation in order to construct antimicrobial plant. The expression of lysozymes was confirmed by the reverse transcriptional PCR, SDS-PAGE analysis and lytic activity of transformants of tobacco. The transformant having the highest lytic activity expressed about 40 micrograms of lysozyme per g of leaf tissue.
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PMID:Production of genetically modified lysozymes having extreme heat stability and antimicrobial activity against gram negative bacteria in yeast and in plant. 973 52

The expression of 21 novel genes located in the region from dnaA to abrB of the Bacillus subtilis chromosome was analyzed. One of the genes, yaaH, had a predicted promoter sequence conserved among SigE-dependent genes. Northern blot analysis revealed that yaaH mRNA was first detected from 2 h after the cessation of logarithmic growth (T(2)) of sporulation in wild-type cells and in spoIIIG (SigG(-)) and spoIVCB (SigK(-)) mutants but not in spoIIAC (SigF(-)) and spoIIGAB (SigE(-)) mutants. The transcription start point was determined by primer extension analysis; the -10 and -35 regions are very similar to the consensus sequences recognized by SigE-containing RNA polymerase. A YaaH-His tag fusion encoded by a plasmid with a predicted promoter for the yaaH gene was produced from T(2) of sporulation in a B. subtilis transformant and extracted from mature spores, indicating that the yaaH gene product is a spore protein. Inactivation of the yaaH gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yaaH mutant spores in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was almost the same as that of wild-type spores, but the mutant spores were defective in L-alanine-stimulated germination. These results suggest that yaaH is a novel gene encoding a spore protein produced in the mother cell compartment from T(2) of sporulation and that it is required for the L-alanine-stimulated germination pathway.
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PMID:The Bacillus subtilis yaaH gene is transcribed by SigE RNA polymerase during sporulation, and its product is involved in germination of spores. 1041 57

Insertional inactivation of the yrbA gene of Bacillus subtilis reduced the resistance of the mutant spores to lysozyme. The yrbA mutant spores lost their optical density at the same rate as the wild-type spores upon incubation with L-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this medium yrbA spores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis of yrbA transcripts in various sig mutants indicated that yrbA was transcribed by RNA polymerase with sigma(E) beginning at 2 h after the start of sporulation. The yrbA promoter was localized by primer extension analysis, and the sequences of the -35 (TCATAAC) and -10 (CATATGT) regions were similar to the consensus sequences of genes recognized by sigma(E). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intact yrbA mutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination of yrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of the yrbA mutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination.
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PMID:Characterization of the yrbA gene of Bacillus subtilis, involved in resistance and germination of spores. 1043 71


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