title: Characterization of Ejl, the cell-wall amidase coded by the pneumococcal bacteriophage Ej-1.
abstract: 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.
authors.affiliation: Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
authors.forename: José L
authors.initials: JL
authors.lastname: Sáiz
authors.forename: Consuelo
authors.initials: C
authors.lastname: López-Zumel
authors.forename: Begoña
authors.initials: B
authors.lastname: Monterroso
authors.forename: Julio
authors.initials: J
authors.lastname: Varea
authors.forename: José Luis R
authors.initials: JL
authors.lastname: Arrondo
authors.forename: Ibon
authors.initials: I
authors.lastname: Iloro
authors.forename: José L
authors.initials: JL
authors.lastname: García
authors.forename: José
authors.initials: J
authors.lastname: Laynez
authors.forename: Margarita
authors.initials: M
authors.lastname: Menéndez
mesh_terms: D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000737:chemistry / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002794:Choline / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013055:Spectrophotometry, Infrared; D017108:Streptococcus Phages / Q000201:enzymology / Q000235:genetics; D013816:Thermodynamics; D014461:Ultracentrifugation
chemical_list: D004798:Enzymes; C076332:EJL amidase; D009238:N-Acetylmuramoyl-L-alanine Amidase; D002794:Choline