Structural adaptations for carboxypeptidase activity in putative S9 acylaminoacyl peptidase from Bacillus subtilis.

Prolyl oligopeptidase, or S9 (MEROPS) family enzymes are crucial drug targets due to their association with various diseases, neurological disorders, cell growth, and survival. These implications render them an exceptionally fascinating field of research. Despite sharing similar structural features, they exhibit diverse enzyme activities, including endopeptidase, dipeptidyl peptidase, and acylaminoacyl peptidase. Additionally, a few members of the S9 family demonstrate carboxypeptidase activity. A recent study showed that the S9 peptidase of Bacillus subtilis (S9bs) possesses the conserved sequence feature necessary for carboxypeptidase activity despite being annotated as an acylaminoacyl peptidase in the UniProt database. However, the mechanism of action and identity of S9bs as carboxypeptidase remain unclear. Consequently, we focused our studies on thoroughly investigating S9bs for its carboxypeptidase activity. In the present study, we report biochemical and biophysical analyses of S9bs, confirming its identity as a carboxypeptidase. Further, structural analysis reveals the molecular basis of S9bs' carboxypeptidase activity, highlighting the crucial structural elements like the "cavity loop" and the "two-arginine" residues essential for this activity. Additionally, our studies confirmed that S9bs forms a stable tetrameric assembly and established its quaternary molecular arrangement, which reveals the presence of an oligomeric pore. Altogether, these structural features play a crucial role in substrate selection for S9 carboxypeptidases. Overall, our findings reveal a distinct carboxypeptidase within the S9 family and significantly enhance our understanding of these enzymes. Moreover, this study sheds light on the mechanisms underlying carboxypeptidase activity, offering valuable insights that could contribute to therapeutic and drug design.