Abstact

The inherent resistance of pseudopeptidoglycan to lysozyme and antibiotics has drawn considerable research attention to archaeal cell wall hydrolases from methanogenic archaeal phages. The C39 peptidases derived from methanogenic archaeal phages exhibit cleavage activity toward synthetic isopeptide substrates that mimic pseudopeptidoglycan cross-links, distinguishing them functionally from bacterial C39 peptidases, which process double glycine motifs in the ABC transport system I. However, the precise cleavage mechanism remains unclear. In this study, we systematically characterized the substrate specificities and catalytic properties of three phage-derived C39 peptidases-C39-PeiR, C39-PhiF1, and C39-PhiF3-using synthetic isopeptides and p-nitroaniline-modified peptides. X-ray crystallography and site-directed mutagenesis assays further revealed that the loop region immediately adjacent to alpha-helix 7 in C39-PeiR constitutes a critical structural determinant distinguishing its functional repertoire from bacterial orthologs. Additionally, C39 peptidases were classified into 16 distinct clades based on structural variations, leading to the identification of novel pseudopeptidoglycan endoisopeptidases.