Download MendeleyRational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining alpha-l-Arabinofuranosidases.
McGregor, N.G.S., Artola, M., Nin-Hill, A., Linzel, D., Haon, M., Reijngoud, J., Ram, A., Rosso, M.N., van der Marel, G.A., Codee, J.D.C., van Wezel, G.P., Berrin, J.G., Rovira, C., Overkleeft, H.S., Davies, G.J.(2020) J Am Chem Soc 142: 4648-4662
- PubMed: 32053363
- DOI: https://doi.org/10.1021/jacs.9b11351
- Primary Citation of Related Structures:
6SXR, 6SXS, 6SXT, 6SXU, 6SXV - PubMed 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 ¦Á-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify ¦Á-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic ¦Á-l-arabinofuranosides were sought. The conformational free energy landscapes of free ¦Á-l-arabinofuranose and several rationally designed covalent ¦Á-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 ¦Á-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of ¦Á-l-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of ¦Á-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 ¦Á-l-arabinofuranosidases.
Organizational Affiliation:
York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York YO10 5DD, U.K.
