Characterization of Ejl, the cell-wall amidase coded by the pneumococcal bacteriophage Ej-1.
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.
Sáiz
José L
JL
Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.
López-Zumel
Consuelo
C
Monterroso
Begoña
B
Varea
Julio
J
Arrondo
José Luis R
JL
Iloro
Ibon
I
García
José L
JL
Laynez
José
J
Menéndez
Margarita
M
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
D004798:Enzymes; C076332:EJL amidase; D009238:N-Acetylmuramoyl-L-alanine Amidase; D002794:Choline