Bacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer at the end of the lytic infection cycle to allow the viral progeny release. The specific activity and structure of these proteins have boosted their study as new antimicrobials against pathogens including multidrug resistant bacteria as well as the development of new biotechnological tools for bacterial diagnostics and detection, among others. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in case of endolysins of bacteriophages infecting Gram-negative species. Only a limited number of studies have analyzed the peptidoglycan bond cleaved by endolysins and most annotations of enzymatic specificity only rely on sequence similarity. As a result, available databases contain inaccurate descriptions of biochemical specificities.
In this study, we report the functional and biochemical characterization of the modular Salmonella phage endolysin Gp110 which comprises an uncharacterized Domain of Unknown Function (DUF3380; pfam11860) in its C-terminus and shows the highest specific activity (34,240 U/µM) compared to fourteen previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to a 1.7- to 364-fold higher activity). This endolysin showed an optimal enzymatic activity at pH 8 and an elevated thermal resistance. Reversed phase-HPLC analysis coupled to mass spectrometry showed that DUF3380 has N-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycan with various peptide stem compositions, making it an attractive enzyme to develop novel antimicrobial agents.