Abstract
Serine peptidases of the prolyl oligopeptidase (POP) family are of substantial therapeutic importance because of their involvement in diseases such as diabetes, cancer, neurological diseases, and autoimmune disorders. Proper annotation and knowledge of substrate specificity mechanisms in this family are highly valuable. Although endopeptidase, dipeptidyl peptidase, tripeptidyl peptidase, and acylaminoacyl peptidase activities have been reported previously, here we report the first instance of carboxypeptidase activity in a POP family member. We determined the crystal structures of this carboxypeptidase, an S9C subfamily member from Deinococcus radiodurans, in its active and inactive states at 2.3-Å resolution, providing an unprecedented view of assembly and disassembly of the active site mediated by an arginine residue. We observed that this residue is poised to bind substrate in the active structure and disrupts the catalytic triad in the inactive structure. The assembly of the active site is accompanied by the ordering of gating loops, which reduces the effective size of the oligomeric pore. This prevents the entry of larger peptides and constitutes a novel mechanism for substrate screening. Furthermore, we observed structural adaptations that enable its carboxypeptidase activity, with a unique loop and two arginine residues in the active site cavity orienting the peptide substrate for catalysis. Using these structural features, we identified homologs of this enzyme in the POP family and confirmed the presence of carboxypeptidase activity in one of them. In conclusion, we have identified a new type within POP enzymes that exhibits not only unique activity but also a novel substrate-screening mechanism.
Highlights
Serine peptidases of the prolyl oligopeptidase (POP) family are of substantial therapeutic importance because of their involvement in diseases such as diabetes, cancer, neurological diseases, and autoimmune disorders
Dipeptidyl peptidase, tripeptidyl peptidase, and acylaminoacyl peptidase activities have been reported previously, here we report the first instance of carboxypeptidase activity in a POP family member
We provide an unprecedented view of the assembly shii; ApAAP, acylaminoacyl peptidase from A. pernix; S9C enzyme from Geobacillus stearothermophilus (S9Cgs), serine carboxypeptidase from G. stearothermophilus; PDB, Protein Data Bank; pNA, para-nitroanilide; r.m.s.d., root mean square deviation
Summary
Serine peptidases of the prolyl oligopeptidase (POP) family are of substantial therapeutic importance because of their involvement in diseases such as diabetes, cancer, neurological diseases, and autoimmune disorders. We determined the crystal structures of this carboxypeptidase, an S9C subfamily member from Deinococcus radiodurans, in its active and inactive states at 2.3-Å resolution, providing an unprecedented view of assembly and disassembly of the active site mediated by an arginine residue. AAP, is an acylaminoacyl peptidase that cleaves N-acetylated peptides to generate N-acetylated amino acids and peptides with free N termini [14, 15] Despite this variation, the members of this family share a common structural fold comprising two domains, a catalytic ␣/-hydrolase domain, which harbors residues of the active site, and a cylindrical -propeller domain, which buries the active site at the domain interface and forms a large dome-shaped cavity over it (16 –19). Our study adds a new dimension to the POP family of peptidases in terms of both enzyme activity and mechanism
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