Kinetic Landscape of a Peptide Bond-Forming Prolyl Oligopeptidase

Abstract

Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising druglike molecules because of their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we propose a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where the cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is among the fastest macrocyclases described to date, and this work is a necessary step toward designing broad-specificity efficient macrocyclases.

Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates

“…Binding of the inactive mutant S577A to the 25mer, 35mer, a series of truncated substrates (10mer–14mer), as well as the recognition sequence (17mer) WTAEHVDQTLASGNDIC, the truncated recognition sequences VDQTLASGNDIC and TLASGNDIC, and the leader peptide MFDTNATRLP were evaluated by isothermal titration calorimetry (ITC). The results of S577A with both the 25mer and recognition sequence have previously been reported 21 . Binding of H698A to the 25mer was also measured.…”

“…3 ). The kinetic data for expressed protein with the 25mer but not 35mer have been previously reported 15 , 21 . The 25mer then rebinds (in competition with the 35mer) for macrocyclization.…”

“…The only peptide showing no detectable binding at concentrations up to 1 mM was the 10-residue leader peptide MFDTNATRLP. The full-length substrates and products displayed tight binding ( K d-25mer–S577A = 67 ± 14 nM 21 , K d-25mer–H698A = 47 ± 11 nM, K d-35mer = 120 ± 30 nM, K d-recognition = 430 ± 10 nM 21 ; binding is dominated by enthalpic contributions (Supplementary Fig. 6b ) (Fig.…”

“…The enzymes that catalyze close to traceless peptide bond formation regardless of the peptide sequence—, i.e., only one residue from the precursor peptide recognition tag is carried over to the final cyclic product—are PatGmac family members, butelase, and POP macrocyclases. The POPB from Basidiomycete fungi such as Amanita bisporigera and Galerina marginata (GmPOPB) species have been reported as having k cat values comparable to butelase, the fastest rate observed for peptide macrocyclisation 15 , 21 . GmPOPB is the macrocyclase responsible for macrocyclization of amatoxins, eight amino-acid ribosomal peptides with the core sequence IWGIGC(N/D)P. Amatoxins are cyclic peptides further modified by a characteristic sulfoxide cross-link between tryptophan and a cysteine (Fig.…”

Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants

“…S5D). This contrasts with binding studies completed with GmPOPB, in which the cyclized region of the peptide enhances binding affinity (36).…”