Three-dimensional structural determination combined with comprehensive comparisons with the homologs is a straightforward strategy to decipher the molecular function of an enzyme. However, in many cases it's difficult to obtain the complex structure with the substrate/ligand. Structure-based molecular simulation provides an alternative solution to predict the binding pattern of a substrate/ligand to the enzyme. The Streptococcus pneumoniae LytB is a peptidoglycan hydrolase that cleaves the glycosidic bond and therefore involves the cell division; however, the details of catalytic mechanism remain unknown. Based on the crystal structure of the catalytic domain of LytB (termed LytBCAT), we describe here how to assign the molecular functions of three LytBCAT modules: SH3b, WW, and GH73, using structural comparisons. Moreover, we dock a putative tetrasaccharide-pentapeptide substrate of peptidoglycan onto LytBCAT to provide the details of substrate binding pattern. The tetrasaccharide-pentapeptide is well accommodated in a T-shaped substrate binding pocket formed by the three modules. The conclusions deduced from structural comparison and simulation are further proved by the hydrolytic activity assays in combination with site-directed mutagenesis.
Keywords: Hydrolytic activity assay; LytB; Peptidoglycan; Peptidoglycan hydrolase; Simulation; Streptococcus pneumoniae; Structural comparison.