Structural and biochemical characterization of a new phage-encoded muramidase, KTN6 Gp46

Background: Endolysins are phage-encoded lytic enzymes that degrade bacterial peptidoglycan at the end of phage lytic cycles to release new phage particles. These enzymes are being explored as an alternative to small-molecule antibiotics.

Methods: The crystal structure of KTN6 Gp46 was determined and compared with a ColabFold model. Cleavage specificity was examined using a peptidoglycan digest and reversed-phase high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS).

Results: The structure of KTN6 Gp46 could be determined at 1.4 Å resolution, and key differences in loops of the putative peptidoglycan binding domain were identified in comparison with its closest known homologue, the endolysin of phage SPN1S. Reversed-phase HPLC/MS analysis of the reaction products following peptidoglycan digestion confirmed the muramidase activity of Gp46, consistent with structural predictions.

Conclusion: These insights into the structure and function of endolysins further expand the toolbox for endolysin engineering and explore their potential in enzyme-based antibacterial design strategies.