The efficacy of bacteriophage-encoded endolysin relies on active residues: A molecular perspective

Abstract Bacteriophage-encoded endolysins have been recognized as promising antibacterial specialists for combating antibiotic-resistant bacteria. Endolysins have been restricted in the case of treatment of Gram-negative bacteria exogenously, where the outer membrane prohibits access to peptidoglycan. Therefore, this study mainly focuses on the structural and functional mechanism of endolysins of phage EcSw (ΦEcSw), responsible for glycosidase degradation of bacterial peptidoglycans. We conducted a computational study to analyze the protein folding, active domains and active residues of endolysin. The structural similarity-based docking identified the active residues (Glu19 and Asp34) of endolysin involved in the recognition and transglycosylase cleavage of the bacterial peptidoglycan. The enzymatic activities of the computationally predicted and highly conserved catalytic residues were validated through site-directed mutagenesis. The antibacterial assay suggesting that the wild type endolysin have significant lytic activity compare with its mutants including E19D, E19K, D34E, D34H and K19/H34 that can modulate the function of endolysins. Overall, our study utilized protein engineering, gene mutations, and computational models to identify the active residues of functional endolysin. Furthermore, the mutational study corroborated the application of phage endolysins as an antimicrobial therapy for combating multidrug-resistant pathogenic diseases and future pandemics.