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)

The structures of the choline-dependent pneumococcal murein hydrolases, LYTA amidase and CPL1 lysozyme, and the choline-independent CPL7 lysozyme were analysed by controlled proteolytic digestions. The trypsin cleavage of the CPL1 and CPL7 lysozymes produced two resistant polypeptides, F1 and F7 respectively, corresponding to the N-terminal domain of the enzymes, whereas the amidase LYTA was completely hydrolysed by the protease. Interestingly, the F1 and F7 fragments showed a low, but significant, choline-independent lysozyme activity. Choline reduced the rate of proteolytic hydrolysis of choline-dependent enzymes, suggesting that the C-terminal choline-binding domain adopts a more resistant conformation in the presence of the ligand. On the other hand, the regions encoding the N-terminal domains of the three enzymes have been cloned and expressed in Escherichia coli, showing that these domains adopt an active conformation even in the absence of their C-terminal domains. The lower activity shown by the catalytic domains when compared with that of the complete enzymes suggests that the acquisition of a substrate-binding domain represents a noticeable evolutionary advantage for enzymes that interact with polymeric substrates, allowing them to achieve a higher catalytic efficiency. These results strongly reinforce the hypothesis that the pneumococcal murein hydrolases have been originated by fusion of two structural and functional independent domains, and provide new experimental support to the theory of modular evolution of proteins.
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PMID:Studies on the structure and function of the N-terminal domain of the pneumococcal murein hydrolases. 135 Dec 40

The Ejl amidase is coded by Ej-1, a temperate phage isolated from the atypical pneumococcus strain 101/87. Like all the pneumococcal cell-wall lysins, Ejl has a bimodular organization; the catalytic region is located in the N-terminal module, and the C-terminal module attaches the enzyme to the choline residues of the pneumococcal cell wall. The structural features of the Ejl amidase, its interaction with choline, and the structural changes accompanying the ligand binding have been characterized by CD and IR spectroscopies, differential scanning calorimetry, analytical ultracentrifugation, and FPLC. According to prediction and spectroscopic (CD and IR) results, Ejl would be composed of short beta-strands (ca. 36%) connected by long loops (ca. 17%), presenting only two well-predicted alpha-helices (ca. 12%) in the catalytic module. Its polypeptide chain folds into two cooperative domains, corresponding to the N- and C-terminal modules, and exhibits a monomer <--> dimer self-association equilibrium. Choline binding induces small rearrangements in Ejl secondary structure but enhances the amidase self-association by preferential binding to Ejl dimers and tetramers. Comparison of LytA, the major pneumococcal amidase, with Ejl shows that the sequence differences (15% divergence) strongly influence the amidase stability, the organization of the catalytic module in cooperative domains, and the self-association state induced by choline. Moreover, the ligand affinity for the choline-binding locus involved in regulation of the amidase dimerization is reduced by a factor of 10 in Ejl. Present results evidence that sequence differences resulting from the natural variability found in the cell wall amidases coded by pneumococcus and its bacteriophages may significantly alter the protein structure and its attachment to the cell wall.
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PMID:Characterization of Ejl, the cell-wall amidase coded by the pneumococcal bacteriophage Ej-1. 1207 Mar 31

Pal amidase, encoded by pneumococcal bacteriophage Dp-1, represents one step beyond in the modular evolution of pneumococcal murein hydrolases. It exhibits the choline-binding module attaching pneumococcal lysins to the cell wall, but the catalytic module is different from those present in the amidases coded by the host or other pneumococcal phages. Pal is also an effective antimicrobial agent against Streptococcus pneumoniae that may constitute an alternative to antibiotic prophylaxis. The structural implications of Pal singular structure and their effect on the choline-amidase interactions have been examined by means of several techniques. Pal stability is maximum around pH 8.0 (Tm approximately 50.2 degrees C; DeltaHt = 183 +/- 4 kcal mol(-1)), and its constituting modules fold as two tight interacting cooperative units whose denaturation merges into a single process in the free amidase but may proceed as two well resolved events in the choline-bound state. Choline titration curves reflect low energy ligand-protein interactions and are compatible with two sets of sites. Choline binding strongly stabilizes the cell wall binding module, and the conformational stabilization is transmitted to the catalytic region. Moreover, the high proportion of aggregates formed by the unbound amidase together with choline preferential interaction with Pal dimers suggest the existence of marginally stable regions that would become stabilized through choline-protein interactions without significantly modifying Pal secondary structure. This structural rearrangement may underlie in vitro "conversion" of Pal from the low to the full activity form triggered by choline. The Pal catalytic module secondary structure could denote folding conservation within pneumococcal lytic amidases, but the number of functional choline binding sites is reduced (2-3 sites per monomer) when compared with pneumococcal LytA amidase (4-5 sites per monomer) and displays different intermodular interactions.
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PMID:Structural and thermodynamic characterization of Pal, a phage natural chimeric lysin active against pneumococci. 1524 37

Choline-binding modules are present in some virulence factors and many other proteins of Streptococcus pneumoniae (Pneumococcus). The most extensively studied choline-binding module is C-LytA, the C-terminal moiety of the pneumococcal cell-wall amidase LytA. The three-dimensional structure of C-LytA is built up from six loop-hairpin structures forming a left-handed beta-solenoid with four choline-binding sites. The affinity of C-LytA for choline and other structural analogues allows its use as an efficient fusion tag for single-step purification of hybrid proteins. In the present study, we characterize the folding and stability of C-LytA by chemical and thermal equilibrium denaturation experiments. Unfolding experiments using guanidinium chloride at pH 7.0 and 20 degrees C suggest the existence of two partly folded states (I1 and I2) in the following model: N (native)-->I1<=>I2. The N-->I1 transition is non-co-operative and irreversible, and is significant even in the absence of a denaturant. In contrast, the I1<=>I2 transition is co-operative and reversible, with an associated freeenergy change (DeltaG(0)) of 30.9+/-0.8 kJ x mol(-1). The residual structure in the I2 state is unusually stable even in 7.4 M guanidinium chloride. Binding of choline stabilizes the structure of the native state, induces its dimerization and prevents the accumulation of the I1 species ([N]2<=>[I2]2, DeltaG(0)=50.1+/-0.8 kJ x mol(-1)). Fluorescence and CD measurements, gel-filtration chromatography and limited proteolysis suggest that I1 differs from N in the local unfolding of the N-terminal beta-hairpins, and that I2 has a residual structure in the C-terminal region. Thermal denaturation of C-LytA suggests the accumulation of at least the I1 species. These results might pave the way for an effective improvement of its biotechnological applications by protein engineering.
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PMID:Accumulation of partly folded states in the equilibrium unfolding of the pneumococcal choline-binding module C-LytA. 1557 22

Streptococcus pneumoniae colonizes the human upper respiratory tract, and this asymptomatic colonization is known to precede pneumococcal disease. In this report, chemically defined and semisynthetic media were used to identify the initial steps of biofilm formation by pneumococcus during growth on abiotic surfaces such as polystyrene or glass. Unencapsulated pneumococci adhered to abiotic surfaces and formed a three-dimensional structure about 25 microm deep, as observed by confocal laser scanning microscopy and low-temperature scanning electron microscopy. Choline residues of cell wall teichoic acids were found to play a fundamental role in pneumococcal biofilm development. The role in biofilm formation of choline-binding proteins, which anchor to the teichoic acids of the cell envelope, was determined using unambiguously characterized mutants. The results showed that LytA amidase, LytC lysozyme, LytB glucosaminidase, CbpA adhesin, PcpA putative adhesin, and PspA (pneumococcal surface protein A) mutants had a decreased capacity to form biofilms, whereas no such reduction was observed in Pce phosphocholinesterase or CbpD putative amidase mutants. Moreover, encapsulated, clinical pneumococcal isolates were impaired in their capacity to form biofilms. In addition, a role for extracellular DNA and proteins in the establishment of S. pneumoniae biofilms was demonstrated. Taken together, these observations provide information on conditions that favor the sessile mode of growth by S. pneumoniae. The experimental approach described here should facilitate the study of bacterial genes that are required for biofilm formation. Those results, in turn, may provide insight into strategies to prevent pneumococcal colonization of its human host.
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PMID:Biofilm formation by Streptococcus pneumoniae: role of choline, extracellular DNA, and capsular polysaccharide in microbial accretion. 1693 41

Phosphocholine moieties decorating the pneumococcal surface are used as a docking station for a family of modular proteins, the so-called choline binding proteins or CBPs. Choline recognition is essential for CBPs function and may also be a determinant for their quaternary structure. There is little knowledge about modular arrangement or oligomeric structures in this family. Therefore, we have used the small angle X-ray scattering (SAXS) technique combined with analytical ultracentrifugation in order to model the three-dimensional envelope of two highly different CBPs: the phage encoded Cpl-1 lysozyme and the pneumococcal phosphorylcholine esterase Pce. Both enzymes have an N-terminal catalytic module and a C-terminal choline-binding module (CBM) that attaches them to the bacterial surface and comprises six and ten sequence repeats in Cpl-1 and Pce, respectively. SAXS experiments have shown an inherent conformational plasticity in Cpl-1 that accounts for the different relative position of these regions in the solution and crystal structures. Dimerization of Cpl-1 upon choline binding has been also visualised for the first time, and monomer-monomer interactions take place through the first CBR where a non-canonical choline binding site has now been identified. This mode of association seems to be independent of the absence or presence of the Cpl-1 catalytic module and reveals that the arrangement of the monomers differs from that previously found in the isolated CBM dimer of pneumococcal LytA amidase. In contrast, Pce displays the same modular disposition in the solution and crystal structures, and remains almost invariant upon choline binding. The present results suggest that protein dimerization and duplication of CBRs may be alternative but not equivalent ways of improving cell wall recognition by CBPs, since they provide different interaction geometries for choline residues present in (lipo)teichoic acids.
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PMID:Insights into molecular plasticity of choline binding proteins (pneumococcal surface proteins) by SAXS. 1706 29

The bacterial cell wall is in part composed of the peptidoglycan (PG) layer that maintains the cell shape and sustains the basic cellular processes of growth and division. The cell wall of Gram-positive bacteria also carries teichoic acids (TAs). In this work, we investigated how TAs contribute to the structuration of the PG network through the modulation of PG hydrolytic enzymes in the context of the Gram-positive Streptococcus pneumoniae bacterium. Pneumococcal TAs are decorated by phosphorylcholine residues which serve as anchors for the Choline-Binding Proteins, some of them acting as PG hydrolases, like the major autolysin LytA. Their binding is non covalent and reversible, a property that allows easy manipulation of the system. In this work, we show that the release of LytA occurs independently from its amidase activity. Furthermore, LytA fused to GFP was expressed in pneumococcal cells and showed different localization patterns according to the growth phase. Importantly, we demonstrate that TAs modulate the enzymatic activity of LytA since a low level of TAs present at the cell surface triggers LytA sensitivity in growing pneumococcal cells. We previously developed a method to label nascent TAs in live cells revealing that the insertion of TAs into the cell wall occurs at the mid-cell. In conclusion, we demonstrate that nascent TAs inserted in the cell wall at the division site are the specific receptors of LytA, tuning in this way the positioning of LytA at the appropriate place at the cell surface.
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PMID:Nascent teichoic acids insertion into the cell wall directs the localization and activity of the major pneumococcal autolysin LytA. 3274 29

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