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)

Pneumococcal peptidoglycan amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28) and phage CPL1 lysozyme degrade a common substrate (choline-containing pneumococcal cell walls); the former hydrolyzes the bond between muramic acid and alanine, whereas the latter breaks down the linkage between muramic acid and glucosamine. The amino acid sequences of their C-terminal domains are homologous. Chimeric genes were constructed by site-directed mutagenesis: a unique SnaBI restriction site in the cpl1 gene, coding for the phage lysozyme, was introduced at a location equivalent to the SnaBI site present in the lytA gene, which codes for the pneumococcal amidase. The resulting genes expressed lytic activities at levels similar to those of the parental genes. The gene products, which have been purified to electrophoretical homogeneity, exhibited unusual combined biochemical properties--e.g., by exchange of protein domains, we have switched the regulatory properties of these enzymes without altering their catalytic activities. Chimeric gene construction in Streptococcus pneumoniae and its bacteriophages is an excellent model to study the modular organization of genes and proteins and to help to establish evolutionary relationships between phage and bacteria. These constructions provide an experimental approach to the molecular processes involved in cassette recruitment during evolution and contribute support to the concept of bacteria as adaptable chimeras.
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PMID:Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes. 197 20

In an attempt to simplify the protein folding problem, and also to further investigate the role of alanine as a helix-stabilizing residue, a series of alanines was introduced within the alpha-helix that includes residues 126-134 of T4 lysozyme. In wild-type lysozyme this alpha-helix contains alanine residues at positions 129, 130, and 134. Mutant lysozymes with alanines substituted at positions 128, 131, 132, and 133, either as single substitutions or in selected combinations, were constructed by oligonucleotide-directed mutagenesis. With the exception of the replacement of Leu 133, which is buried within the hydrophobic core of the protein, all the variants were more stable than wild-type lysozyme. The variant with alanines substituted at positions 128, 131, and 132 (E128A/V131A/N132A), which incorporates the sequence Ala 128-Ala 129-Ala 130-Ala 131-Ala 132-Leu 133-Ala 134, has a melting temperature 3.3 degrees C above that of wild-type lysozyme. Determination of the crystal structure of this mutant lysozyme shows that the replacement of Glu 128, Val 131, and Asn 132 with alanine causes alpha-helix 126-134 to rotate 3.4 degrees about an axis parallel to its own axis. This rotation seems to be triggered primarily by the loss of a hydrogen bond between Asn 132 and Ser 117 and is associated with the repacking of several side chains at the interface between alpha-helix 126-134 and the adjacent alpha-helix 115-122.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Toward a simplification of the protein folding problem: a stabilizing polyalanine alpha-helix engineered in T4 lysozyme. 199 63

Our previous results using the Saccharomyces cerevisiae secretion system suggest that intramolecular exchange of disulfide bonds occurs in the folding pathway of human lysozyme in vivo (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 265, 7570-7575). Here we report on the results of introducing an artificial disulfide bond in mutants with 2 cysteine residues substituting for Ala83 and Asp91. The mutant (C83/91) protein was not detected in the culture medium of the yeast, probably because of incorrect folding. Thereupon, 2 cysteine residues Cys77 and Cys95 were replaced with Ala in the mutant C83/91, because a native disulfide bond Cys77-Cys95 was found not necessary for correct folding in vivo (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). The resultant mutant (AC83/91) was secreted as two proteins (AC83/91-a and AC83/91-b) with different specific activities. Amino acid and peptide mapping analyses showed that two glutathiones appeared to be attached to the thiol groups of the cysteine residues introduced into AC83/91-a and that four disulfide bonds including an artificial disulfide bond existed in the AC83/91-b molecule. The presence of cysteine residues modified with glutathione may indicate that the non-native disulfide bond Cys83-Cys91 is not so easily formed as a native disulfide bond. These results suggest that the introduction of Cys83 and Cys91 may act to suppress the process of native disulfide bond formation through disulfide bond interchange in the folding of human lysozyme.
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PMID:Evidence for intramolecular disulfide bond shuffling in the folding of mutant human lysozyme. 200 94

The method given earlier for predicting the thermodynamics of protein unfolding from the x-ray structure of a protein is applied here to the poly(L-alanine) helix. First, the fitting parameters derived earlier from a data base of 10 proteins were used to predict the unfolding thermodynamics of 4 other proteins. The agreement between the observed and predicted values is comparable to that found for the 10 proteins studied initially. Next, the temperature dependences of the Gibbs energy and enthalpy changes for unfolding of bacteriophage T4 lysozyme were predicted and compared with data in the literature. The predicted and observed temperature dependences are similar and the predicted results indicate that cold denaturation should be observed at low temperatures, as observed recently for a T4 lysozyme mutant. The fitting parameters derived from thermodynamic data for protein unfolding and for hydration of model compounds were used to predict the unfolding thermodynamics of the poly(L-alanine) helix. The results predict that helix formation is enthalpy-driven, and the predicted enthalpy change for unfolding (0.86 kcal per mol per residue) is close to the value found in a recent calorimetric study of a 50-residue alanine-rich helix.
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PMID:Prediction of the thermodynamics of protein unfolding: the helix-coil transition of poly(L-alanine). 201 95

To investigate the mechanism of disulfide-bond-coupled de novo folding of human lysozyme, we have constructed 23 mutant enzymes in which cysteine residue(s) were replaced by alanine(s). The mutant genes were translated in vitro in a system composed of rabbit reticulocyte lysate, canine pancreatic microsomal vesicles and oxidized glutathione. This system allows the formation of intramolecular disulfide bonds in translation products translocated into the microsomal lumen. The mobilities of the translation products were analyzed by SDS/PAGE in nonreducing conditions. Some mutant lysozymes were found to form a compact conformation with native-like mobility in the presence of SDS. The de novo formation of the SDS-resistant compact conformation of each mutant correlated well with its efficiency of secretion by Saccharomyces cerevisiae. Our results suggest that the de novo synthesized products reflect the conformational states in vivo to some extent, and that the formation of SDS-resistant compact conformation can be regarded as a necessary condition for allowing lysozyme to be secreted. In addition, the analysis of a mutant C116A (Cys116----Ala) under different oxidative conditions suggests two distinct pathways for the disulfide-bond-coupled formation of the compact conformation.
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PMID:Behavior of cysteine mutants of human lysozyme in de novo synthesis and in vivo secretion. 204 Mar 7

Lysis of Escherichia coli induced by either D-cycloserine, moenomycin, or penicillin G was monitored by studying murein metabolism. The levels of the soluble murein precursor UDP-N-acetylmuramyl-L-alanyl-D-glutamyl-m-diaminopimelyl-D-alanyl- D-alanine (UDP-MurNAc-pentapeptide) and the carrier-linked MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol as well as N-acetylglucosamine-beta-1,4-MurNAc-(pentapeptide)-pyrophosphoryl- undecaprenol varied in a specific way. In the presence of penicillin, which is known to interfere with the cross-linking of murein, the concentration of the lipid-linked precursors unexpectedly decreased before the onset of lysis, although the level of UDP-MurNAc-pentapeptide remained normal. In the case of moenomycin, which specifically blocks the formation of the murein polysaccharide strands, the lipid-linked precursors as well as UDP-MurNAc-pentapeptide accumulated as was expected. D-Cycloserine, which inhibits the biosynthesis of UDP-MurNAc-pentapeptide, consequently caused a decrease in all three precursors. The muropeptide composition of the murein showed general changes such as an increase in the unusual DL-cross bridge between two neighboring meso-diaminopimelic acid residues and, as a result of uncontrolled DL- and DD-carboxypeptidase activity, an increase in tripeptidyl and a decrease in tetrapeptidyl and pentapeptidyl moieties. The average length of the glycan strands decreased. When the glycan strands were fractionated according to length, a dramatic increase in the amount of single disaccharide units was observed not only in the presence of penicillin but also in the presence of moenomycin. This result is explained by the action of an exo-muramidase, such as the lytic transglycosylases present in E. coli. It is proposed that antibiotic-induced bacteriolysis is the result of a zipperlike splitting of the murein net by exo-muramidases locally restricted to the equatorial zone of the cell.
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PMID:Analysis of murein and murein precursors during antibiotic-induced lysis of Escherichia coli. 204 64

The three-dimensional structure of a mutant human lysozyme, C77/95A, in which residues Cys77 and Cys95 were replaced by alanine, was determined at 1.8-A resolution by x-ray crystallography. The properties of this mutant protein have been well characterized with respect to its thermal stability and secretion efficiency in a yeast expression system. The overall three-dimensional structure of C77/95A was found to be essentially identical to that of the wild-type human lysozyme, although the coordinates were shifted by more than 0.5 A and the thermal factors of the main-chain atoms were increased in the vicinity of residue 77. The reduction in thermal stability of this mutant has been previously explained by an increase in entropy of the unfolded state. In addition, a packing defect (cavity) produced by the removal of the disulfide bond was detected in the three-dimensional structure of C77/95A. This cavity can also be a reason why the stability of the protein is reduced because the free energy of the folded state could be expected to increase. The increased secretion efficiency cannot be due mainly to the three-dimensional structure, but may possibly be related to some event in the pathway of protein secretion. One of the possibilities might involve molecular flexibilities in the secondary or tertiary structure for lack of one of the disulfide bonds.
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PMID:The crystal structure of a mutant human lysozyme C77/95A with increased secretion efficiency in yeast. 206 30

The partial molar volumes of various compounds that model protein constituent groups, such as tripeptides (Gly-X-Gly, where X = Gly, Ala, Val, Leu, Ile, Pro, Met, His, Ser), homopeptides (Glyn, n = 3,4,5), and simple organic analogues of amino acid side chains (methanol, acetamide, propanamide, acetic acid, propanoic acid, n-butanamine, n-butanamine nitrate, n-propylguanidine nitrate, 4-methylphenol), have been determined in aqueous solution with a vibrational densimeter in the temperature range of 5-85 degrees C. The partial molar volumes of amino acid side chains and the peptide unit were estimated from the data obtained. Assuming additivity of component groups, the partial molar volumes of polypeptide chains of several proteins over a broad temperature range were calculated. The partial molar volume functions of four proteins (myoglobin, cytochrome C, ribonuclease A, lysozyme) were compared with those determined experimentally for the unfolded and native forms of these proteins. It has been shown that the average deviation of the calculated functions from the experimental ones does not exceed 3% over the temperature range studied.
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PMID:Partial molar volumes of polypeptides and their constituent groups in aqueous solution over a broad temperature range. 208 Dec 62

Hydrolysis of Staphylococcus aureus 209 P cell wall peptidoglycan was accompanied by the liberation of 1.3 mol of C-terminal and 1.2 mol of N-terminal glycine per mole of Glu as well as of 0.5 mol of N-terminal and 0.3 mol of C-terminal alanine. Gel chromatography on Sephadex G-25, ion-exchange chromatography on QAE-Sephadex A-50 and paper electrophoresis of S. aureus peptidoglycan hydrolysates gave seven homogeneous fractions; these fractions were structurally defined. Lysoamidase hydrolyzed bonds Mur-Ala, Gly-Gly and Mur-GlcN in the peptidoglycan molecule. Hydrolysis of glycan chains was accompanied by the formation of large fragments, (GlcN-Mur)9 and (GlcN-Mur)28. The lytic effect of lysoamidase on S. aureus peptidoglycan is coupled with bacteriolytic enzymes of lysoamidase: acetmuramyl amidase, glycyl--glycine endopeptidase and acetyl--muramidase.
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PMID:[Hydrolysis of a Staphylococcus aureus cell wall peptidoglycan by 209 P lysoamidase]. 208 20

Human lysozyme is made up of 130 amino acid residues and has four disulfide bonds at Cys6-Cys128, Cys30-Cys116, Cys65-Cys81, and Cys77-Cys95. Our previous results using the Saccharomyces cerevisiae secretion system indicate that the individual disulfide bonds of human lysozyme have different functions in the correct in vivo folding and enzymatic activity of the protein (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). In this paper, we report the results of experiments that were focused on the roles of Cys65 and Cys81 in the folding of human lysozyme protein in yeast. A mutant protein (C81A), in which Cys81 was replaced with Ala, had almost the same enzymatic activity and conformation as those of the native enzyme. On the other hand, another mutant (C65A), in which Cys65 was replaced with Ala, was not found to fold correctly. These results indicate that Cys81 is not a requisite for both correct folding and activity, whereas Cys65 is indispensable. The mutant protein C81A is seen to contain a new, non-native disulfide bond at Cys65-Cys77. The possible occurrence of disulfide bond interchange during our mapping experiments cannot be ruled out by the experimental techniques presently available, but characterization of other mutant proteins and computer analysis suggest that the intramolecular exchange of disulfide bonds is present in the folding pathway of human lysozyme in vivo.
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PMID:Evidence for difference in the roles of two cysteine residues involved in disulfide bond formation in the folding of human lysozyme. 211 Jan 67


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