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

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. The ORF11 2.5 Angstrom crystal structure reveals that ORF11 comprises four domains: a predicted glycosyl hydrolase (D1) a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP, D4), separated by a helical linker (D2) and a CHAP like domain (D3). The crystal form reveals a dimeric assembly with the predicted glycosyl hydrolase (D1) and CHAP peptidase (D4) domains located adjacent to each other (but at opposite ends of the dimer) within one subunit and separated by domains D2 and D3- positioned at the dimer interface. The presence of the D3 CHAP-like domain is unusual among related bacteriophage tail-lysins from related small podoviruses but conserved in E. faecium Minhoviruses. 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 an Enterococcus podovirus tail-spike lysin and demonstrate 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 small-genome phages target this clinically significant antimicrobial-resistant pathogen.