Carboxypeptidase in prolyl oligopeptidase family: Unique enzyme activation and substrate-screening mechanisms

Abstract

Edited by Wolfgang PetiSerine 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.

The acid-base-nucleophile catalytic triad in ABH-fold enzymes is coordinated by a set of structural elements

“…The low rate of amino-acid conservation at position IV hints that residue X IV could affect the enzymatic activity, given that its side chain has a suitable conformation that does not interfere with the catalytic triad, with amino acids of variable side-chain lengths occurring at position IV, including glutamine [33] and tryptophan [34]. Indeed, residue X IV is suggested to be involved in the optimal configuration of the catalytic triad [32,35] and the enzymatic activity [36][37][38], to participate in substrate binding [15,30,[38][39][40][41] and to affect the thermostability [35] of ABH fold enzymes. Site-directed mutagenesis of residue X IV in different ABH fold enzymes has confirmed its significance in the enzymatic activity, resulting in a wide range of effects upon its replacement: from decreased [32,[35][36][37] and acute loss [38] of enzymatic activity, to improved activity [36,38,41] and increased thermostability [35].…”

“…The tripeptide residue X IV -residue X IV+1 -residue X IV+2 retains its amino-acid conservation in some ABH fold enzyme families, and thus, it is suggested that it can be used as an additional sequence identifier of different ABH fold enzyme families [32,37]. Residue X IV+2 is also extensively studied for the formation of the hydrophobic substrate pocket [22,25,32,[37][38][39][46][47][48] and its role in the enzymatic function [32,49,50].…”

Phosphonic Analogs of Alanine as Acylpeptide Hydrolase Inhibitors

“…Acylpeptide hydrolase (APH; EC 3.4.19.1), also known as acylaminoacyl‐peptidase (AAP), is a serine protease, which, together with prolyl oligopeptidase (POP), dipeptidyl peptidase IV (DPPIV) and oligopeptidase B (OB), belongs to the prolyl oligopeptidase family (clan SC, family S9) [1–3] . APH primarily removes N‐acetylated amino acids residues and thus regulates the cellular level of N‐terminal acetylation of proteins [4] .…”

Intrinsic basis of thermostability of prolyl oligopeptidase from Pyrococcus furiosus

“…Prolyl oligopeptidase is a Ser type protease located in the cytoplasm that is twice the size of conventional proteases like trypsin, pepsin, etc. 31 , 32 . This enzyme acts on peptide hormones and neuropeptides to make them functional.…”