Structural Basis for Dual Peptidoglycan Hydrolysis by an E. faecium Minhovirus Tail Spike Lysin

Abstract

Bacteriophages rely on breaching the bacterial cell wall as the first step of infection. We characterise ORF11, a putative tail-spike lysin from the 19 kbp Minhovirus SHEF14, a podovirus infecting Enterococcus faecium. Bioinformatic analyses indicate that ORF11 comprises four domains: a predicted glycosyl hydrolase (D1) a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP, D4), separated by a helical linker (D2) and a CHAP-like domain (D3). This modular organisation is conserved among Enterococcus minhoviruses but differs markedly from analogous proteins in Copernicusvirus phage and related staphylococcal phage. ORF11 2.1 [A] crystal structure reveals an unusual dimeric assembly. The predicted glycosyl hydrolase and CHAP peptidase domains occupy opposite ends of the protein, bridged by the two other domains positioned at the dimer interface. Biochemical assays using recombinant ORF11 and LC-MS/MS confirmed dual peptidoglycan-degrading activity. ORF11 functions as both an N-acetylglucosaminidase and a D, D-endopeptidase, cleaving the bond between the D-alanine in position-4 and the D-aspartate residue at the end of the side chain. Together, these results provide the first structural description of a podovirus tail-spike lysin and demonstrate its bifunctional enzymatic activity. This dual action likely facilitates initial surface recognition and localised peptidoglycan degradation during infection of E. faecium, offering new insights into how minimal-genome phage target this clinically significant antimicrobial-resistant pathogen.