Monday 6th, November 2023 11:00

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Activity-based profiling of retaining glycosidases

Herman Overkleeft

Leiden Institute of Chemistry, Leiden University


Keywords: mechanism-based inhibitors, activity-based protein profiling, biomass polysaccharides, GH discovery

Activity-based protein profiling (ABPP) is a rapidly emerging field in chemical biology research. Enzymes that employ a mechanism in processing their substrate that involves formation of a covalent enzyme-intermediate adduct can be blocked by mechanism-based suicide inhibitors: compounds that react within the enzyme active site to form a covalent and irreversible adduct. Introduction of a reporter moiety (the purple bulb in the below picture) yields an activity-based probe (ABP) through which enzyme activities are discovered (comparative ABPP) and the efficacy of enzyme inhibitors analysed (competitive ABPP).

Figure 1.    Activity-based profiling of GH10 xylanases.

Our work on ABPP development focuses on retaining glycosidases: hydrolytic enzymes able to cleave interglycosidic linkages and that do so through the formation of covalent enzyme-substrate intermediates. Configurational and functional analogues of the natural product and mechanism-based retaining beta-glucosidase inhibitor, cyclophellitol, prove to be highly versatile tools to study retaining glycosidases of various nature and origin in relation to human health and disease, but also in the field of biotechnology. In this lecture our results in studying mammalian1 and microbial2,3 retaining endoglycosidases will be presented.

1) C. de Boer, Z. Armstrong, V. A. J. Lit, U. Barash, G. Ruijgrok, I. Boyango, M. M. Weitzenberg, S. P. Schröder, A. J. C. Sarris, N. J. Meeuwenoord, P. Bule, Y. Kayal, N. Ilan, J. D. C. Codée, I. Vlodavsky, H. S. Overkleeft, G. J. Davies and L. Wu, Mechanism based heparanase inhibitors reduce cancer metastasis in vivo, Proc. Natl. Acad. Sci. USA 2022, 119, e2203167119.

2) Y. Chen, Z. Armstrong, M. Artola, B. I. Florea, C.-L. Kuo, C. de Boer, M. S. Rasmussen, M. A. Hachem, G. A. van der Marel, J. D. C. Codée, J. M. F. G. Aerts, G. J. Davies and H. S. Overkleeft, Activity-based protein profiling of retaining  a-amylases in complex biological samples, J. Am. Chem. Soc. 2021, 143, 2423-2432.

3) S. P. Schröder, C. de Boer, N. G. S. McGregor, R. J. Rowland, O. Moroz, E. Blagova, J. Reijngoud, M. Arentshorst, D. Osborn, M. D. Morant, E. Abbate, M. A. Stringer, K. B. R. M. Krogh, L. Raich, C. Rovira, J.-G. Berrin, G. P. van Wezel, A. F. J. Ram, B. I. Florea, G. A. van der Marel, J. D. C. Codée, K. S. Wilson, L. Wu, G. J. Davies and H. S. Overkleeft, Dynamic and functional profiling of xylan-degrading enzymes in Aspergillus secretomes using activity-based probes, ACS Cent. Sci. 2019, 5, 1067-1078.

Published on October 9, 2023