Figure 2

Specificity of recombinant AEPs. (a) The ability of recombinant AEPs to cyclise the R1 peptide (represented by–) with various flanking sequences was determined. The proportion of cyclic product is displayed relative to the benchmark motif (GL–NGL). The enzymes were added at a nominal concentration of 19.7 µg mL⁻¹ based on quantitation by BCA assay. Incubation times were such that the proportion of cyclic product from the peptide carrying the benchmark motif (GL-NGL) was between 72–81% (OaAEP1_b and OaAEP5, 1 h; OaAEP3, 35 min; OaAEP4, 5 h). The assays were performed in activity buffer (50 mM sodium acetate buffer, pH 5.0, 50 mM NaCl, 1 mM EDTA, 0.5 mM TCEP) at room temperature. The chart shows mean values where n ≥ 3 ± SEM. (b) Schematic describing the nomenclature of N- and C-terminal AEP recognition motifs. The peptide with the extended motif (GLP–STRNGLP) is used as an example (see also Fig. 1a). All residues downstream of the P1 site are released from the final product. (c) MALDI MS spectra of the GL–NGL and GL–NGH peptides 5 hours post-enzyme addition (0.132 µM enzyme, pH 5). Boxed inset zooms in on the region containing the processing product. The first peak is cyclic product and the second peak is likely a + 22 Da sodium adduct. The expected position of linear processing product (monoisotopic mass) is shown by the dashed line.