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)

As one of the final steps in the bacterial growth cycle, daughter cells must be released from one another by cutting the shared peptidoglycan wall that separates them. In Escherichia coli, this delicate operation is performed by several peptidoglycan hydrolases, consisting of multiple amidases, lytic transglycosylases, and endopeptidases. The interactions among these enzymes and the molecular mechanics of how separation occurs without lysis are unknown. We show here that deleting the endopeptidase PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the major peptidoglycan amidases during cell separation. Another endopeptidase, PBP 7, fulfills a secondary role. These functions may be responsible for the contributions of PBPs 4 and 7 to the generation of regular cell shape and the production of normal biofilms. In addition, we find that the E. coli peptidoglycan amidases may have different substrate preferences. When the dd-carboxypeptidase PBP 5 was deleted, thereby producing cells with higher levels of pentapeptides, mutants carrying only AmiC produced a higher percentage of cells in chains, while mutants with active AmiA or AmiB were unaffected. The results suggest that AmiC prefers to remove tetrapeptides from peptidoglycan and that AmiA and AmiB either have no preference or prefer pentapeptides. Muropeptide compositions of the mutants corroborated this latter conclusion. Unexpectedly, amidase mutants lacking PBP 5 grew in long twisted chains instead of straight filaments, indicating that overall septal morphology was also defective in these strains.
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PMID:Daughter cell separation by penicillin-binding proteins and peptidoglycan amidases in Escherichia coli. 1685 23

The Staphylococcus aureus bacteriophage phi11 endolysin has two peptidoglycan hydrolase domains (endopeptidase and amidase) and an SH3b cell wall-binding domain. In turbidity reduction assays, the purified protein can lyse untreated staphylococcal mastitis pathogens, Staphylococcus aureus and coagulase-negative staphylococci (Staphylococcus chronogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus warneri and Staphylococcus xylosus), making it a strong candidate protein antimicrobial. This lytic activity is maintained at the pH (6.7), and the "free" calcium concentration (3 mM) of milk. Truncated endolysin-derived proteins containing only the endopeptidase domain also lyse staphylococci in the absence of the SH3b-binding domain.
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PMID:Lysis of staphylococcal mastitis pathogens by bacteriophage phi11 endolysin. 1705 40

The recombinant phi11 endolysin hydrolyzed heat-killed staphylococci as well as staphylococcal biofilms. Cell wall targeting appeared to be a prerequisite for lysis of whole cells, and the combined action of the endopeptidase and amidase domains was necessary for maximum activity. In contrast, the phi12 endolysin was inactive and caused aggregation of the cells.
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PMID:Lytic activity of recombinant bacteriophage phi11 and phi12 endolysins on whole cells and biofilms of Staphylococcus aureus. 1708 95

Murein endopeptidase A (MepA) from Escherichia coli is a periplasmic peptidoglycan amidase that cleaves d,d amide bonds between d-alanine and meso-2,6-diaminopimelic acid in E. coli peptidoglycan. MepA and its homologues in other proteobacteria share overall structural similarity with d-Ala-d-Ala metallopeptidases and local similarity around the active site with lysostaphin-type enzymes, which has prompted the classification of these enzymes as LAS enzymes. LAS enzymes contain a single divalent cation in the active site, which is tetracoordinated in the crystal structures. Three of the metal ligands are identical in all structures, but the identity of the fourth ligand varies. Two residues in proximity to the metal might act as a general acid/base, but their role is not clear. Here, we report a new MepA expression system, which allows the separation of MepA variants from the endogenous wild-type enzyme, and an HPLC assay with a defined peptidoglycan fragment, which allows assessment of MepA activity without a refolding step. We find that the conserved metal ligands are required for folding (D120) or catalysis (H113, H211). Separate mutations of the candidate catalytic residues H206 or H209 and of the "fourth" metal ligand H110 are tolerated for folding but drastically reduce activity. Mutation of residue W203 to aspartate impairs substrate binding.
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PMID:Mutational analysis of peptidoglycan amidase MepA. 1719 81

In a previous comparative proteomic study of Bacillus anthracis examining the influence of the virulence plasmids and of various growth conditions on the composition of the bacterial secretome, we identified 64 abundantly expressed proteins (T. Chitlaru, O. Gat, Y. Gozlan, N. Ariel, and A. Shafferman, J. Bacteriol. 188:3551-3571, 2006). Using a battery of sera from B. anthracis-infected animals, in the present study we demonstrated that 49 of these proteins are immunogenic. Thirty-eight B. anthracis immunogens are documented in this study for the first time. The relative immunogenicities of the 49 secreted proteins appear to span a >10,000-fold range. The proteins eliciting the highest humoral response in the course of infection include, in addition to the well-established immunogens protective antigen (PA), Sap, and EA1, GroEL (BA0267), AhpC (BA0345), MntA (BA3189), HtrA (BA3660), 2,3-cyclic nucleotide diesterase (BA4346), collagen adhesin (BAS5205), an alanine amidase (BA0898), and an endopeptidase (BA1952), as well as three proteins having unknown functions (BA0796, BA0799, and BA0307). Of these 14 highly potent secreted immunogens, 11 are known to be associated with virulence and pathogenicity in B. anthracis or in other bacterial pathogens. Combining the results reported here with the results of a similar study of the membranal proteome of B. anthracis (T. Chitlaru, N. Ariel, A. Zvi, M. Lion, B. Velan, A. Shafferman, and E. Elhanany, Proteomics 4:677-691, 2004) and the results obtained in a functional genomic search for immunogens (O. Gat, H. Grosfeld, N. Ariel, I. Inbar, G. Zaide, Y. Broder, A. Zvi, T. Chitlaru, Z. Altboum, D. Stein, S. Cohen, and A. Shafferman, Infect. Immun. 74:3987-4001, 2006), we generated a list of 84 in vivo-expressed immunogens for future evaluation for vaccine development, diagnostics, and/or therapeutic intervention. In a preliminary study, the efficacies of eight immunogens following DNA immunization of guinea pigs were compared to the efficacy of a PA DNA vaccine. All eight immunogens induced specific high antibody titers comparable to the titers elicited by PA; however, unlike PA, none of them provided protection against a lethal challenge (50 50% lethal doses) of virulent B. anthracis strain Vollum spores.
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PMID:Identification of in vivo-expressed immunogenic proteins by serological proteome analysis of the Bacillus anthracis secretome. 1735 82

Putative N-acetylmuramyl-l-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 lysins exhibited gamma-d-glutaminyl-l-lysine endopeptidase activity. The endopeptidase cleavage specificity of the lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 lysin also displayed beta-d-N-acetylglucosaminidase activity.
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PMID:LambdaSa1 and LambdaSa2 prophage lysins of Streptococcus agalactiae. 1790 88

Functional imaging of subtilisin Carlsberg active center by the idiotypic network yielded a catalytic anti-idiotypic antibody with endopeptidase, amidase, and esterase activities. A monoclonal antibody inhibitory to subtilisin (Ab1 5-H4) was employed as the template for guiding the idiotypic network to produce the catalytic anti-idiotypic Ab2 6B8-E12. Proteolytic activity of 6B8-E12 was demonstrated by zymography using self-quenched fluorescein-BSA conjugate and in a coupled assay detecting Ab2-dependent RNase A inactivation. Cleavage of peptide substrates by 6B8-E12 revealed distinct patterns of hydrolysis with high preference for aromatic residues before or after the scissile bond. Catalytic activity of Ab2 was inhibited by phenylmethylsulfonyl fluoride, a mechanism-based inhibitor of serine hydrolases. 5-H4 and 6B8-E12 were cloned, produced in Escherichia coli as single-chain variable fragments (scFvs), and purified. Kinetic parameters for amidolytic and esterolytic activities were similar in Ab2 and its scFv derivative. Although the antigen-specific portion of 6B8-E12 possesses no primary structure similarity to subtilisin, it mimics proteolytic and amidolytic functions of the parental antigen, albeit with 4 orders of magnitude slower acceleration rates. The lack of detectable endopeptidase activity of 6B8-E12 scFv raises interesting issues concerning general evolution of catalytic activity. The in silico 3D models of Ab1 and Ab2 revealed strong structural similarity to known anti-protease antibodies and to abzymes, respectively. These results indicate that the idiotypic network is capable, to a significant extent, of reproducing catalytic apparatus of serine proteases and further validate the use of imaging of enzyme active centers by the immune system for induction of abzymes accelerating energy-demanding amide bond hydrolysis.
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PMID:Anti-idiotypic antibody mimics proteolytic function of parent antigen. 1802 Apr 54

The transepithelial flux of cydiastatin 4 and analogs across flat sheet preparations of the anterior midgut of larvae of the tobacco hawkmoth moth, Manduca sexta, was investigated using a combination of reversed-phase high-performance liquid chromatography (RP-HPLC), enzyme-linked immunosorbent assay (ELISA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The lumen to hemolymph (L-H) flux of cydiastatin 4 was dose and time-dependent, with a maximum rate of flux of c. 178 pmol/cm2/h) measured after a 60-min incubation with 100 micromol/l of peptide in the lumen bathing fluid. The rates of flux, L-H and H-L, across the isolated gut preparations were not significantly different. These data suggest that uptake across the anterior midgut of larval M. sexta is via a paracellular route. Cydiastatin 4 was modified to incorporate a hexanoic acid (Hex) moiety at the N-terminus, the N-terminus extended with 5 P residues and/or the substitution of G7 with Fmoc-1-amino-cyclopropylcarboxylic acid (Acpc). The incorporation of hexanoic acid enhanced the uptake of these amphiphilic analogs compared to the native peptide. Analogs were also more resistant to enzymes in hemolymph and gut preparations from larval M. sexta. A modified N-terminus gave protection against aminopeptidase-like activity and incorporation of Acpc inhibited endopeptidase-like activity. Although analogs were stable in the hemolymph, they were susceptible to amidase-like activity in the gut, which appears to convert the C-terminal amide group to a free carboxylic acid, identified by an increase in 1 mass unit of the peptide analog.
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PMID:Transepithelial flux of an allatostatin and analogs across the anterior midgut of Manduca sexta larvae in vitro. 1820 64

Lysostaphin is a zinc metalloenzyme which has a specific lytic action against Staphylococcus aureus. Lysostaphin has activities of three enzymes namely, glycylglycine endopeptidase, endo-beta-N-acetyl glucosamidase and N-acteyl muramyl-L-alanine amidase. Glycylglycine endopeptidase specifically cleaves the glycine-glycine bonds, unique to the interpeptide cross-bridge of the S. aureus cell wall. Due to its unique specificity, lysostaphin could have high potential in the treatment of antibiotic-resistant staphylococcal infections. This review article presents a current understanding of the lysostaphin and its applications in therapeutic agent as a treatment against antibiotic-resistant S. aureus and methicillin-resistant S. aureus (MRSA) infections, either alone or in combination with other antibiotics.
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PMID:Lysostaphin: an antistaphylococcal agent. 1860 87

Streptococcal pathogens contribute to a wide variety of human and livestock diseases. The routine use of antibiotics to battle these pathogens has produced a new class of multidrug-resistant streptococci. Thus, there is a need for new antimicrobials. Bacteriophage endolysins (peptidoglycan hydrolases) comprise one group of new antimicrobials that are reportedly refractory to resistance development. The LambdaSa2 prophage endolysin gene was recently isolated from a Group B streptococcal genome, expressed on an Escherichia coli plasmid, and shown by homology screening and biochemical analysis to harbor an amidase-5 (endopeptidase) domain, an amidase-4 (glycosidase) domain, and two Cpl-7 cell wall-binding domains. In this study, turbidity reduction and plate lysis assays indicate that this hydrolase shows strong lytic activity toward Streptococcus pyogenes, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus equi, GES, and GGS. Deletion analysis indicates that the N-terminal endopeptidase domain with both Cpl-7 domains can lyse with a higher specific activity than the full-length protein (against some strains). This dual Cpl-7 domain truncated version also shows weak lytic activity against methicillin-resistant Staphylococcus aureus (MRSA) and the coagulase negative staphylococci, Staphylococcus xylosus. The truncated constructs harboring the glycosidase domain are virtually inactive, showing only minimal activity on plate lysis assays.
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PMID:LambdaSa2 prophage endolysin requires Cpl-7-binding domains and amidase-5 domain for antimicrobial lysis of streptococci. 1867 93


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