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

TY - JOUR

T1 - Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases

AU - McGregor, Nicholas G.S.

AU - Artola, Marta

AU - Nin-Hill, Alba

AU - Linzel, Daniël

AU - Haon, Mireille

AU - Reijngoud, Jos

AU - Ram, Arthur

AU - Rosso, Marie Noëlle

AU - Van Der Marel, Gijsbert A.

AU - Codeé, Jeroen D.C.

AU - Van Wezel, Gilles P.

AU - Berrin, Jean Guy

AU - Rovira, Carme

AU - Overkleeft, Herman S.

AU - Davies, Gideon J.

N1 - © 2020 American Chemical Society

PY - 2020/3/11

Y1 - 2020/3/11

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85081945438&partnerID=8YFLogxK

U2 - 10.1021/jacs.9b11351

DO - 10.1021/jacs.9b11351

M3 - Article

C2 - 32053363

AN - SCOPUS:85081945438

SN - 0002-7863

VL - 142

SP - 4648

EP - 4662

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

IS - 10

ER -