Universität zu Köln

Claushuis, Bart, Wojtalla, Fabian ORCID: 0000-0001-7002-8955, Papenhagen, Lisa ORCID: 0009-0000-7580-2356, Cordfunke, Robert A., de Ru, Arnoud H., van Leeuwen, Hans C., Corver, Jeroen, Hensbergen, Paul J. and Baumann, Ulrich ORCID: 0000-0003-0383-0168 (2026). Structural analyses and substrate profiling of PPEP-3 provide new insights into the molecular basis of Pro-Pro endopeptidase specificity. iScience, 29 (1). pp. 1-15. Elsevier. ISSN 2589-0042

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Abstract

[Artikel-Nr.: 114360] 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 in- teractions 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 mech- anisms 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 sub- strate-binding cleft and influence the accommodation of side chains at these positions. Combining compre- hensive substrate profiling with structural analyses offers a powerful approach to uncover the molecular basis of protease function.

Item Type:	Article
Creators:	Creators Email ORCID ORCID Put Code Claushuis, Bart UNSPECIFIED UNSPECIFIED UNSPECIFIED Wojtalla, Fabian UNSPECIFIED https://orcid.org/0000-0001-7002-8955 UNSPECIFIED Papenhagen, Lisa UNSPECIFIED https://orcid.org/0009-0000-7580-2356 UNSPECIFIED Cordfunke, Robert A. UNSPECIFIED UNSPECIFIED UNSPECIFIED de Ru, Arnoud H. UNSPECIFIED UNSPECIFIED UNSPECIFIED van Leeuwen, Hans C. UNSPECIFIED UNSPECIFIED UNSPECIFIED Corver, Jeroen UNSPECIFIED UNSPECIFIED UNSPECIFIED Hensbergen, Paul J. UNSPECIFIED UNSPECIFIED UNSPECIFIED Baumann, Ulrich UNSPECIFIED https://orcid.org/0000-0003-0383-0168 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-812483
Identification Number:	10.1016/j.isci.2025.114360
Journal or Publication Title:	iScience
Volume:	29
Number:	1
Page Range:	pp. 1-15
Number of Pages:	15
Date:	16 January 2026
Publisher:	Elsevier
ISSN:	2589-0042
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Chemistry > Institute of Biochemistry
Subjects:	Chemistry and allied sciences Life sciences
['eprint_fieldname_oa_funders' not defined]:	Publikationsfonds UzK
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/81248