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A group of bacterial proteases, the Pro‐Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline‐proline bonds in proteins. Since a protease's function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in‐depth characterization of PPEP prime‐side specificity. However, PPEP specificity is also determined by the non‐prime‐side residues in the substrate. To gain a more complete insight into the determinants of PPEP specificity, we characterized the non‐prime‐ and prime‐side specificity of various PPEPs using a combination of synthetic combinatorial peptide libraries and mass spectrometry. With this approach, we deepened our understanding of the P3‐P3′ specificities of PPEP‐1 and PPEP‐2, while identifying the endogenous substrate of PPEP‐2 as the most optimal substrate in our library data. Furthermore, by employing the library approach, we investigated the altered specificity of mutants of PPEP‐1 and PPEP‐2. Additionally, we characterized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP‐4. Based on structural comparisons, we hypothesized that PPEP‐4 displays a PPEP‐1‐like prime‐side specificity, which was substantiated by the experimental data. Intriguingly, another putative PPEP from Clostridioides difficile, CD1597, did not display Pro‐Pro endoproteolytic activity. Collectively, we characterized PPEP specificity in detail using our robust peptide library method and, together with additional structural information, provide more insight into the intricate mechanisms that govern protease specificity. [ABSTRACT FROM AUTHOR]

Flagella are essential for motility and pathogenicity in many bacteria. The main component of the flagellar filament, flagellin (FliC), often undergoes post-translational modifications, with glycosylation being a common occurrence. In Pseudomonas aeruginosa PAO1, the b-type flagellin is O -glycosylated with a structure that includes a deoxyhexose, a phospho-group, and a previous unknown moiety. This structure resembles the well-characterized glycan (Type A) in Clostridioides difficile strain 630, which features an N -acetylglucosamine linked to an N -methylthreonine via a phosphodiester bond. This study aimed to characterize the b-type glycan structure in Pseudomonas aeruginosa PAO1 using a set of mass spectrometry experiments. For this purpose, we used wild-type P. aeruginosa PAO1 and several gene mutants from the b-type glycan biosynthetic cluster. Moreover, we compared the mass spectrometry characteristics of the b-type glycan with those of in vitro modified Type A-peptides from C. difficile strain 630Δ erm . Our results demonstrate that the thus far unknown moiety of the b-type glycan in P. aeruginosa consists of an N,N -dimethylthreonine. These data allowed us to refine our model of the flagellin glycan biosynthetic pathway in both P. aeruginosa PAO1 and C. difficile strain 630.