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Rational design of mechanism-based inhibitors and activity-based probes for the identification of retaining α-l-arabinofuranosidases

DOI: 10.1021/jacs.9b11351 DOI Help

Authors: Nicholas G. S. Mcgregor (The University of York) , Marta Artola (Leiden University) , Alba Nin-hill (Universitat de Barcelona) , Daniel Linzel (Leiden University) , Mireille Haon (INRA, Aix Marseille University, Biodiversité et Biotechnologie Fongiques (BBF)) , Jos Reijngoud (Leiden University) , Arthur F. J. Ram (Leiden University) , Marie-noelle Rosso (INRA, Aix Marseille University, Biodiversité et Biotechnologie Fongiques (BBF)) , Gijsbert A. Van Der Marel (Leiden University) , Jeroen D. C. Codée (Leiden University) , Gilles P. Van Wezel (Leiden University) , Jean-guy Berrin (INRA, Aix Marseille University, Biodiversité et Biotechnologie Fongiques (BBF)) , Carme Rovira (Universitat de Barcelona) , Herman S. Overkleeft (Leiden University) , Gideon J. Davies (The University of York)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of The American Chemical Society

State: Published (Approved)
Published: February 2020
Diamond Proposal Number(s): 18598

Abstract: 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 co-expressed 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.

Journal Keywords: Activity-based protein profiling; cyclophellitol; secretome; glycoside hydrolase; α-L-arabinofuranose

Subject Areas: Chemistry, Biology and Bio-materials

Instruments: I04-Macromolecular Crystallography