Molecular characterization of two newly recognized lysozymes of the protist Dictyostelium discoideum

The model organism Dictyostelium discoideum functions as a social amoeba that can aggregate, eventually forming a fruiting body composed of a fixed number of cells. This behavior requires a soluble counting factor (CF) complex, which plays a key role in group size determination and has been identified earlier. The CF complex comprises among others the proteins CF45-1 and CF50. Although both proteins share sequence similarities with characterized Chalaropsis- and Entamoeba-type lysozymes, enzymatic activity has not been confirmed until now. CF lysozymes have unusual sequence characteristics consisting of an N-terminal glycoside hydrolase family 25 (GH25) domain and a C-terminal low-complexity region rich in serine, glycine, alanine, and asparagine residues. In this study, we present the production and purification of soluble recombinant CF lysozymes and demonstrate notable enzymatic activity, in particular for CF50. Additionally, a truncated version of CF50, which lacks the C-terminal low-complexity region, displayed significantly enhanced lysozyme activity compared to the entire enzyme. Both CF lysozymes exerted strict pH dependence with maximal activity observed under acidic conditions at pH 3.0-3.5. Moreover, the enzymes displayed highest activity at low ionic strengths and were stable at relatively low temperatures only. Using structural modeling and site-directed mutagenesis, we identified a glutamic acid residue essential for catalysis. Conclusively, we propose a neighboring group catalytic mechanism analogous to that of other GH25 lysozymes.