Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-L-Arabinofuranosidases - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Journal of the American Chemical Society Année : 2020

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

Résumé

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.

Dates et versions

hal-02482205 , version 1 (17-02-2020)

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Citer

Nicholas Mcgregor, Marta Artola, Alba Nin-Hill, Daniel Linzel, Mireille Haon, et al.. Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-L-Arabinofuranosidases. Journal of the American Chemical Society, 2020, ⟨10.1021/jacs.9b11351⟩. ⟨hal-02482205⟩
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