Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases

Nicholas G.S. McGregor, Marta Artola, Alba Nin-Hill, Daniël Linzel, Mireille Haon, Jos Reijngoud, Arthur Ram, Marie Noëlle Rosso, Gijsbert A. Van Der Marel, Jeroen D.C. Codeé, Gilles P. Van Wezel, Jean Guy Berrin, Carme Rovira*, Herman S. Overkleeft, Gideon J. Davies

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases.

Original languageEnglish
Pages (from-to)4648-4662
Number of pages15
JournalJournal of the American Chemical Society
Issue number10
Early online date13 Feb 2020
Publication statusPublished - 11 Mar 2020

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