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:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A series of 24 mutants was made in the buried core of chicken lysozyme at positions 40, 55, and 91. The midpoint temperature of thermal denaturation transition (Tm) values of these core constructs range from 60.9 to 77.3 degrees C, extending an earlier, more limited investigation on thermostability. The Tm values of variants containing conservative replacements for the wild type (WT) (Thr 40-Ile 55-Ser 91) triplet are linearly correlated with hydrophobicity (r = 0.81) and, to a lesser degree, with combined side-chain volume (r = 0.75). The X-ray structures of the S91A (1.9 A) and I55L/S91T/D101S (1.7 A) mutants are presented. The former amino acid change is found in duck and mammalian lysozymes, and the latter contains the most thermostable core triplet. A network of four conserved, buried water molecules is associated with the core. It is postulated that these water molecules significantly influence the mutational tolerance at the individual triplet positions. The pH dependence of Tm for the S91D mutant was compared with that of WT enzyme. The pKa of S91D is 1.2 units higher in the native than in the denatured state, corresponding to delta delta G298 = 1.7 kcal/mol. This is a low value for charge burial and likely reflects the moderating influence of the buried water molecules or a conformational change. Thermal and chemical denaturation and far UV CD spectroscopy were used to characterize the in vitro properties of I55T. This variant, which buries a hydroxyl group, has similar properties to those of the human amyloidogenic variant I56T.
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PMID:Thermal stability determinants of chicken egg-white lysozyme core mutants: hydrophobicity, packing volume, and conserved buried water molecules. 853 41

Adaptive evolution of lysozyme has involved remodelling of amino acid sequences and changes in patterns of gene expression and in gene number. Following an outline of the phenomena likely to be indicative of adaptive evolution and how one can assess them, this chapter focuses on four cases in which lysozyme c has been recruited as a digestive enzyme in the stomachs of creatures needing to retrieve nutrients from microorganisms in fermented food. For each case-ruminant artiodactyls, leaf-eating monkeys, a leaf-eating bird, and fruit flies-the factors likely to be of primary importance in lysozyme's adaptation are examined. Additional examples of apparent adaptation for digestion or antimicrobial defense in animals as diverse as mice, moths, and molluscs are summarized. This chapter considers also the case of three internally clustered residues which among galliform bird lysozymes c occur either as Thr 40, Ile 55, and Ser 91 (TIS) or as Ser 40, Val 55, and Thr 91 (SVT). Reconstruction and testing of six possible intermediate proteins and development of the concept of a neutral corridor of protein traits are described.
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PMID:Adaptive evolution of lysozyme: changes in amino acid sequence, regulation of expression and gene number. 876 7

We designed mutant lysozymes with N-glycosylation signal sequences (Asn48-Gly49-Thr-50 and Asn87-Ile88-Thr89) by substituting Asp to Asn at positions 48 and 87. When these mutant lysozymes were expressed by using yeast (Saccharomyces cerevisiae) in Burkholder minimum medium, N-glycosylation occurred in both lysozymes. The mutant lysozyme with the oligosaccharide at Asn87 showed a similar character to a reported polymannosyl lysozyme [Nakamura, Takasaki, Kobayashi, and Kato (1993) J. Biol. Chem. 268, 12706-12712; Kato, Takasaki, and Ban (1994) FEBS Lett. 355, 76-80]. As judged from the thermodynamic stabilities of the lysozymes obtained by the guanidine hydrochloride denaturation method, the oligosaccharide-bearing mutant lysozymes were more stable by 0.4-1.6 kcal/mol than the corresponding unglycosylated lysozymes. Therefore, it is suggested that the introduction of an N-glycosylation signal sequence into a protein is an effective means to increase the stability of the protein.
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PMID:Stabilization of lysozyme by introducing N-glycosylation signal sequence. 890 90

The paper is investigating the mechanism of stabilization of proteins by polyols at the molecular level. It is addressing the interactions of sorbitol, a polyol commonly used as a protein stabilizing agent, with hen egg white lysozyme, a well studied protein. Differential scanning calorimetry shows an increase in denaturation temperature of lysozyme upon addition of sorbitol at a concentration of 250 mM and above. Increasing sorbitol concentration also caused an increase in signal intensity of the CD spectrum of lysozyme in the wavelength region of 280-300 nm. Two-dimensional nuclear magnetic resonance spectroscopy was used to examine interactions between lysozyme and sorbitol. Most significant changes are manifest in the anomalous relaxation properties of Ala and Thr methyl groups indicating modifications of local motions and possibly compression of the entire structure. This is further corroborated by new intra-protein nuclear Overhauser effects in the presence of sorbitol. There is also evidence that water is displaced from the enzyme surface close to Ile-88 upon addition of sorbitol. In combination these results reveal a complex interplay of different interactions. Comparison to NMR-spectra of lysozyme with a bound inhibitor (tri-N-acetyl-glucosamine) shows that the interaction with sorbitol affects spatially disparate regions of the protein.
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PMID:Towards a molecular level understanding of protein stabilization: the interaction between lysozyme and sorbitol. 923 31

A mutant hen egg white lysozyme, D52E, was designed to correspond to the structure of the mutant T4 lysozyme T26E (Kuroki, R., Weaver, L. H., and Matthews B. W. (1993) Science 262, 2030-2033) to investigate the role of the catalytic residue on the alpha-side of the saccharide in these enzymes. The D52E mutant forms a covalent enzyme-substrate adduct, which was detected by electron ion spray mass spectrometry. X-ray crystallographic analysis showed that the covalent adduct was formed between Glu-52 and the C-1 carbon of the N-acetylglucosamine residue in subsite D of the saccharide binding site. It suggests that the catalytic mechanism of D52E mutant lysozyme proceeds through a covalent enzyme-substrate intermediate indicating a different catalytic mechanism from the wild type hen egg white lysozyme. It was confirmed that the substitution of Asp-52 with Glu is structurally and functionally equivalent to the substitution of Thr-26 with Glu in T4 lysozyme. Although the position of the catalytic residue on the beta-side of the saccharide is quite conserved among hen egg white lysozyme, goose egg white lysozyme, and T4 phage lysozyme, the adaptability of the side chain on the alpha-side of the saccharide is considered to be responsible for the functional variation in their glycosidase and transglycosidase activities.
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PMID:A covalent enzyme-substrate adduct in a mutant hen egg white lysozyme (D52E). 924 66

An arachidonic acid-stimulated Ser/Thr phosphatase activity was detected in soluble extracts prepared from rat pituitary clonal GH4C1 cells, rat or bovine brain, and bovine heart. The enzyme activity was purified to homogeneity from bovine brain as a monomer with a Mr of 63,000 and a specific activity of 32 nmol of Pi released per min/mg of protein when assayed in the presence of 10 microM phosphocasein in the absence of lipid. Arachidonic acid stimulated activity 4-14-fold, with half-maximal stimulation at 50-100 microM, when assayed in the presence of a variety of phosphosubstrates including casein, reduced carboxamidomethylated and maleylated lysozyme, myelin basic protein, and histone. Oleic acid, linoleic acid, and palmitoleic acid also stimulated activity; however, saturated fatty acids and alcohol or methyl ester derivatives of fatty acids did not significantly affect activity. The lipid-stimulated phosphatase was identified as the bovine equivalent of protein phosphatase 5 or a closely related homolog by sequence analysis of proteolytic fragments generated from the purified enzyme. When recombinant rat protein phosphatase 5 was expressed as a cleavable glutathione S-transferase fusion protein, the affinity-purified thrombin-cleaved enzyme exhibited a specific activity and sensitivity to arachidonic acid similar to those of the purified bovine brain enzyme. These results suggest that protein phosphatase 5 may be regulated in vivo by a lipid second messenger or another endogenous activator.
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PMID:Purification of a fatty acid-stimulated protein-serine/threonine phosphatase from bovine brain and its identification as a homolog of protein phosphatase 5. 927 97

The cyanogen bromide (CNBr)/formic acid cleavage reactions of wild-type and trifluoromethionine (TFM)-containing recombinant lambda lysozyme were studied utilizing ESI and MALDI mass spectrometry. Detailed analysis of the mass spectra of reverse-phase HPLC-purified cleavage fragments produced from treatment of the wild-type and labeled proteins with CNBr indicated cleavage solely of methionyl peptide bonds with no observation of cleavage at TFM. N-Acetyl-TFM was also found to be resistant to reaction with CNBr, in contrast to N-acetyl-methionine. The analysis also indicated differential reactivity among the three methionine positions in the wild-type enzyme. Additionally, formylation of intact enzyme as well as peptide fragments were observed and characterized and indicated that serine, threonine, as well as C-terminal homoserine side chains are partially formylated under standard cleavage protocols.
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PMID:CNBr/formic acid reactions of methionine- and trifluoromethionine-containing lambda lysozyme: probing chemical and positional reactivity and formylation side reactions by mass spectrometry. 961 87

Live T. cruzi trypomastigotes and amastigotes possess ecto-protein tyrosine phosphatase activity as indicated by the ability of intact cells to catalyze dephosphorylation of tyrosine phosphorylated myelin basic protein, [32P]TyrRaytide, phosphotyrosine, or the phosphotyrosine analog p-nitrophenylphosphate (p-NPP). The dephosphorylation of myelin basic protein (MBP) and p-NPP was inhibited by sodium o-vanadate, zinc chloride and NaF, while dephosphorylation of [32P]TyrRaytide was insensitive to zinc chloride but sensitive to o-vanadate and NaF. In contrast, live cells were not able to dephosphorylate serine or threonine phosphorylated peptides ([32P]Kemptide) or proteins ([32P]RCM-lysozyme and [32P]MBP).
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PMID:Ecto-protein tyrosine phosphatase activity in Trypanosoma cruzi infective stages. 965 37

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 insertion of a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) into the C-terminus in hen egg white lysozyme by genetic modification resulted in an unstable structure which caused little secretion in a yeast expression system, although this modification is useful to enhance bactericidal action to gram-negative bacteria [Ibrahim et al. (1994) J. Biol. Chem. 269, 5059-5063]. To enhance the secretion of the unstable hydrophobic pentapeptide fused lysozymes (H5-Lz), we attempted to introduce the signal sequence (Asn-X-Ser/Thr) of N-linked glycosylation into lysozyme and to suppress the quality control of the unstable mutant in the yeast expression system. The polymannosyl hydrophobic fused lysozyme (H5/G49N-Lz) having the N-glycosylation signal sequence was expressed in the medium at 3.4 times that of unglycosylated lysozyme. Further, the secretion of the unstable mutant lysozyme was done in the Saccharomyces cerevisiae disrupted calnexin gene to avoid the degradation of the unstable mutant by the quality control. Although disruption of the calnexin gene did not lead to gross effects on the levels of growth of S. cerevisiae (W303-1b), the secretion amount of H5/G49N-Lz in calnexin disrupted S. cerevisiae was 2.5 times larger than that in wild type S. cerevisiae. These results suggest that the secretion of unstable glycosylated lysozyme (H5/G49N) was suppressed by the quality control function of calnexin and that the disruption of calnexin is effective to increase the secretion of unstable glycosylated protein.
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PMID:Enhanced secretion of hydrophobic peptide fused lysozyme by the introduction of N-glycosylation signal and the disruption of calnexin gene in Saccharomyces cerevisiae. 986 32


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