Abstract
The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30 degrees C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4 degrees C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 +/- 0.5 sites/monomer) behave as equivalent (Kd = 2.7 +/- 0.3 mm). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37 degrees C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37 degrees C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile.
Highlights
Together to prevent the rupture of the cell wall and the cell lysis during cell growth and division [1]
LytC is directed to the outer surface by a leader peptide (33 residues), and it remains tightly bound to the cell wall in a mature, active form that comprises a choline-binding module (CBM) made of 11 (p1–p11) repeating units (264 residues) and a catalytic module (CM) belonging to the GH-25 family of glycosyl hydrolases (204 residues)
Attachment of LytC to choline moieties of lipoteichoic and teichoic acids seems to mediate a major reorganization of the polypeptide chain that strongly affects the environment of the aromatic residues present in its CBM
Summary
We have performed a comparative study of LytC and Cpl-1 lysozymes using different approaches. The slow hydrolysis of pneumococcal cultures carried out by LytC contrasts with the fast, uncontrolled lysis of the host cell performed by Cpl-1, the lysozyme encoded by the pneumococcal phage Cp-1, in order to release the infective particles after the virion replication [11]. Cpl-1 and LytC are built of homologous modules (Fig. 1) assembled in opposite locations [7]. Only two choline-binding sites, located at the interfaces of the three first repeats (p1-p2 and p2-p3), seem to be functional, according to the crystal structure of Cpl-1 [9]. The alternation of shorter (17 residues) and longer (21–23 residues) repeats (see Fig. 1) in LytC leads to an uneven distribution of choline-binding sites along the CBM surface [14], previously unseen in other CBMs of known structure (8 –10)
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