A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by... Show moreA group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by its substrate specificity,methods that can extensively characterize substrate specificity are valuabletools for protease research. Previously, we achieved an in-depth characteri-zation of PPEP prime-side specificity. However, PPEP specificity is alsodetermined by the non-prime-side residues in the substrate. To gain a morecomplete insight into the determinants of PPEP specificity, we character-ized the non-prime- and prime-side specificity of various PPEPs using acombination of synthetic combinatorial peptide libraries and mass spec-trometry. With this approach, we deepened our understanding of the P3-P30 specificities of PPEP-1 and PPEP-2, while identifying the endogenoussubstrate of PPEP-2 as the most optimal substrate in our library data. Fur-thermore, by employing the library approach, we investigated the alteredspecificity of mutants of PPEP-1 and PPEP-2. Additionally, we character-ized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displaysa PPEP-1-like prime-side specificity, which was substantiated by the experi-mental data. Intriguingly, another putative PPEP from Clostridioides diffi-cile, CD1597, did not display Pro-Pro endoproteolytic activity.Collectively, we characterized PPEP specificity in detail using our robustpeptide library method and, together with additional structural informa-tion, provide more insight into the intricate mechanisms that govern prote-ase specificity. Show less
