Chandravanshi_2025_Biochemistry__

Peptidases of the prolyl oligopeptidase (S9 MEROPS) family play a pivotal role in various physiological processes. Among the S9 family, the S9C subfamily is remarkably diverse in exhibiting enzymatic activities such as acylaminoacyl peptidase, dipeptidyl peptidase, endopeptidase, and carboxypeptidase activity. Predicting enzymatic activity for putative peptidase of the S9C subfamily remains a significant challenge. Here, we report the biophysical and biochemical characterization of a putative peptidase from Mycolicibacterium phlei (S9mp; UniProt: A0A5N5URA7) from the S9C subfamily. Our findings establish S9mp as the first known member of this family to predominantly exhibit endopeptidase activity, which requires a peptide substrate with a free C-terminal for efficient binding and catalysis. Arg443 was identified as a critical residue for substrate binding and stabilization, particularly for smaller peptide substrates. Arg443Ala mutagenesis leads to a several-fold reduction in the enzymatic activity, underscoring its crucial role. Structural analyses using SAXS and AlphaFold confirmed a tetrameric assembly featuring a central oligomeric pore, which may influence substrate accessibility and limit the cleavage of peptides up to nine amino acids in length. These findings deepen our understanding of S9mp's enzymatic mechanisms and provide valuable insights into the molecular basis of its substrate specificity.