Engineering of a T7 Bacteriophage Endolysin Variant with Enhanced Amidase Activity

Abstract

The therapeutic use of bacteriophage-encoded endolysins as enzybiotics has increased significantly in recent years due to the emergence of antibiotic resistant bacteria. Phage endolysins lyse the bacteria by targeting their cell wall. Various engineering strategies are commonly used to modulate or enhance the utility of therapeutic enzymes. This study employed a structure-guided mutagenesis approach to engineer a T7 bacteriophage endolysin (T7L) with enhanced amidase activity and lysis potency via replacement of a noncatalytic gating residue (His 37). Two H37 variants (H37A and H37K) were designed and characterized comprehensively using integrated biophysical and biochemical techniques to provide mechanistic insights into their structure−stability−dynamics−activity paradigms. Among the studied proteins, cell lysis data suggested that the obtained H37A variant exhibits amidase activity (∼35%) enhanced compared to that of wild-type T7 endolysin (T7L-WT). In contrast to this, the H37K variant is highly unstable, prone to aggregation, and less active. Comparison of the structure and dynamics of the H37A variant to those of T7L-WT evidenced that the alteration at the site of H37 resulted in long-range structural perturbations, attenuated the conformational heterogeneity, and quenched the microsecond to millisecond time scale motions. Stability analysis confirmed the altered stability of H37A compared to that of its WT counterpart. All of the obtained results established that the H37A variant enhances the lysis activity by regulating the stability−activity trade-off. This study provided deeper atomic level insights into the structure−function relationships of endolysin proteins, thus aiding researchers in the rational design of engineered endolysins with enhanced therapeutic properties.

Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against Staphylococcus aureus Strains

“…Endolysins are peptidoglycan-lysing enzymes and expressed at the end of the lytic cycle of bacteriophages for release of assembled progeny. Exogenous treatment has demonstrated that recombinant endolysins are novel antimicrobials, and various recombinant endolysins are being assessed in clinical trials [ 18 , 19 , 20 ]. Modular endolysins are composed of various enzymatic domains and cell-wall-binding domains that have independent activities [ 21 ].…”