McGregor, Nicholas G.S., Artola, Marta, Nin-Hill, Alba et al. (12 more authors) (2020) Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases. Journal of the American Chemical Society. pp. 4648-4662. ISSN 1520-5126
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 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.
Metadata
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Copyright, Publisher and Additional Information: | © 2020 American Chemical Society | ||||
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Institution: | The University of York | ||||
Academic Units: | The University of York > Faculty of Sciences (York) > Chemistry (York) | ||||
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Depositing User: | Pure (York) | ||||
Date Deposited: | 15 Apr 2020 14:20 | ||||
Last Modified: | 06 Dec 2023 13:37 | ||||
Published Version: | https://doi.org/10.1021/jacs.9b11351 | ||||
Status: | Published | ||||
Refereed: | Yes | ||||
Identification Number: | https://doi.org/10.1021/jacs.9b11351 | ||||
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