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

Autor: Jean-Guy Berrin, Jos Reijngoud, Marie-Noëlle Rosso, D. Linzel, Herman S. Overkleeft, Alba Nin-Hill, Gideon J. Davies, G. A. Van Der Marel, G.P. van Wezel, Jeroen D. C. Codée, Marta Artola, Mireille Haon, Carme Rovira, Arthur F. J. Ram, Nicholas McGregor
Přispěvatelé: York Structural Biology Laboratory Department of Chemistry, The University of York, Leiden Institute of Chemistry, Universiteit Leiden [Leiden], Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona (UB), Biodiversité et Biotechnologie Fongiques (BBF), École Centrale de Marseille (ECM)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), eMolecular Microbiology and Biotechnology, Institute of Biology Leiden, Leiden University, fInstitució Catalana de Recerca, Institucio Catalana de Recerca i Estudis Avancats (ICREA), EPSRC (EP/K039660/1, EP/M028127/1), Trametes gibbosa BRFM 1770, Abortiporus biennis BRFM 1778, Hexagonia nitida BRFM 1802, Trametes ljubarskyi BRFM 1659, Leiotrametes menziesii BRFM 1781, Fomes fomentarius BRFM 1823 and Trametes meyenii BRFM 1810., University of Barcelona, Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Dpt of Molecular Cell Biology [Leiden], Leiden University Medical Center (LUMC), Institute Biology Leiden (IBL), Institució Catalana de Recerca i Estudis Avançats (ICREA), University of York [York, UK], Universiteit Leiden, Universiteit Leiden-Universiteit Leiden
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Zdroj: Journal of the American Chemical Society
Journal of the American Chemical Society, American Chemical Society, 2020, ⟨10.1021/jacs.9b11351⟩
Journal of the American Chemical Society, 142(10), 4648-4662
Journal of the American Chemical Society, 2020, ⟨10.1021/jacs.9b11351⟩
ISSN: 0002-7863
1520-5126
Popis: 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.
Databáze: OpenAIRE
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