| Autor: |
McGregor, Nicholas G. S., Artola, Marta, Nin-Hill, Alba, Linzel, Daniël, Haon, Mireille, Reijngoud, Jos, Ram, Arthur, Rosso, Marie-Noëlle, van der Marel, Gijsbert A., Codée, Jeroen D. C., van Wezel, Gilles P., Berrin, Jean-Guy, Rovira, Carme, Overkleeft, Herman S., Davies, Gideon J. |
| Zdroj: |
Journal of the American Chemical Society; March 2020, Vol. 142 Issue: 10 p4648-4662, 15p |
| Abstrakt: |
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. nigergrown 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: |
Supplemental Index |
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