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

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  • 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


Publication details

JournalJournal of the American Chemical Society
DateAccepted/In press - 13 Feb 2020
DateE-pub ahead of print - 13 Feb 2020
DatePublished (current) - 11 Mar 2020
Issue number10
Number of pages15
Pages (from-to)4648-4662
Early online date13/02/20
Original languageEnglish


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.

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© 2020 American Chemical Society

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