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 ␤-strands (ca. 36%) connected by long loops (ca. 17%), presenting only two well-predicted ␣-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.Keywords: Pneumococcal cell-wall amidases; pneumococcal bacteriophages; Ejl; Ej-1; choline binding proteins; DSC; CD; IR The cell-wall lytic enzymes encoded by pneumococcus are involved in a variety of physiological functions associated with cell-wall growth, wall turnover, and cell separation (Tomasz 1984). Their polypeptide chains have evolved from the fusion of two independent modules, which are responsible for either the catalytic activity or the attachment to the choline residues of pneumococcal cell wall, which act as an antenna where lysins dock by the so-called cholinebinding module, ChBM (García et al. 1990;López et al. 1995López et al. , 2000. The cell-wall lytic enzymes coded by many Reprint requests to: M. Menéndez, Instituto de Química-Física "Rocasolano," CSIC, Serrano 119, 28006 Madrid, Spain; e-mail: mmenendez@ iqfr.csic.es; fax: 34-91-5642431.Abbreviations: ChBM, choline-binding module; Ejl, amidase encoded by the Ejl gene from Ej-1 phage; LytA, major autolysin from S. pneumoniae; IR, Fourier transform infrared spectroscopy; X, aromatic residue; DSC, differential scanning calorimetry; ⌬H, calorimetric enthalpy change; ⌬H vH , van't Hoff enthalpy change; 〈⌬H i (T)〉, excess enthalpy chan...

Insights into the Structure-Function Relationships of Pneumococcal Cell Wall Lysozymes, LytC and Cpl-1

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“…The far UV region of the CD spectrum of murein hydrolases encoded by pneumococcus and its phages is characterized by the presence of a maximum in the region of 224 -230 nm (15,17,24,25), which is sensitive to the interaction of choline with the tryptophan residues present at the binding site (8 -10). Interestingly, the presence of this band in the spectrum of LytC strictly depends on choline binding ( Fig.…”

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