The peptidoglycan-binding domain of a mycobacterial prophage-encoded LysinB reveals diverse approaches for domain conservation in hydrolases

Mycobacteriophage-encoded LysinB enzymes target mycolyl ester linkages in mycolyl-arabinogalactan-peptidoglycan of mycobacterium hosts and generally exhibit a globular architecture. Here, we present the structural and functional characterization of a novel Mycobacterium fortuitum prophage-encoded modular LysinB (LysinB_MF), which contains the α/β hydrolase domain and a distinct peptidoglycan-binding domain (PGBD). The enzyme's active site features the conserved Ser-Asp-His catalytic triad common to esterases and forms a funnel-like topology. We demonstrate that although both the full-length LysinB_MF and its PGBD-truncated variant possessed intrinsic antimicrobial properties against M. smegmatis and pathogenic M. fortuitum, LysinB_MFΔPGBD exhibited a notable three-fold increase in esterase activity compared to the full-length enzyme. Structural analysis reveals that the PGBD shares similarity with the cell-wall binding domain of an amidase from Clostridium acetobutylicum, suggesting its acquisition due to domain mobility. Phylogenetic analysis of PGBD sequences similar to LysinB_MF shows a widespread occurrence and conservation across diverse hydrolases, with varying copy numbers ranging from one to ten per enzyme, exhibiting remarkable structural diversity. Further analysis of the protein family identified PGBDs as integral components of enzymes with diverse functional properties conserved across kingdoms, highlighting their evolutionary and functional significance.