Abstract
Pro-Pro endopeptidases (PPEPs) are secreted bacterial enzymes that uniquely cleave peptide bonds between adjacent proline residues. Their active site accommodates six substrate residues (P3 to P3'), with interactions at these positions determining specificity. In this study, we investigated the substrate specificity of PPEP-3 from Geobacillus thermodenitrificans using synthetic peptide libraries and liquid chromatography-tandem mass spectrometry (LC-MS/MS). We also determined the atomic structures of PPEP-3 in unbound and substrate-bound forms. By correlating substrate profiling with structural data, we identified key mechanisms influencing PPEP-3 specificity. This integrated analysis reveals stark differences in specificity for the P2 and P2' positions compared to other PPEPs, most notably Tyr161 and Phe191, which shape the substrate-binding cleft and influence the accommodation of side chains at these positions. Combining comprehensive substrate profiling with structural analyses offers a powerful approach to uncover the molecular basis of protease function.