Biochemical and structural characterization of PPEP-3 from Geobacillus thermodenitrificans

ABSTRACT: The members of the group of Pro-Pro endopeptidases (PPEPs) are secreted bacterial endoproteases that display a unique preference for hydrolyzing their substrates between two proline residues. The active site cleft of PPEPs accommodates the six substrate residues P3 to P3’, and the interactions between these and the protease sites S3 to S3’ determine PPEP specificity beyond the scissile Pro-Pro peptide bond. In this study, we have characterized the substrate specificity of PPEP-3 from the thermophilic bacterium Geobacillus thermodenitrificans in detail using synthetic combinatorial peptide libraries in combination with LC-MS/MS analyses. There is a stark difference for the P2 and P2’ positions compared to PPEP-1: instead of Asn and Val at P2, either Ser or basic residues are preferred, while Pro is almost exclusively tolerated at P2’. In the crystal structure of substrate-bound PPEP-3, most notably Tyr161 and Phe191, which differ from the corresponding residues in PPEP-1 (histidine and leucine, respectively), greatly influence the P2 and P2’ specificity. By correlating the substrate specificity profile to the structure, we explore the various mechanisms that determine PPEP-3 specificity and highlight differences with other PPEPs.

INSTRUMENT(S):

ORGANISM(S): Geobacillus Thermodenitrificans

SUBMITTER: Bart Claushuis  

LAB HEAD: Peter A. van Veelen

PROVIDER: PXD061585 | Pride | 2025-11-11

REPOSITORIES: Pride