α-L-Arabinofuranosidases (Abfs) are frequent components of enzyme systems that breakdown plant cell walls, specifically targeting polysaccharides such as arabinoxylans. Usually Abfs, which are auxiliary enzymes that act in synergy with β-D-xylanases, remove α-L-Araf residues from O-2 or O-3 mono-substituted D-Xylp subunits. Other quite rare Abfs (designated AXH-d) release single α-L-Araf moieties from O-2 and O-3 disubstituted D-Xylp subunits.
Novel chromogenic carbohydrate substrates that release a 4-nitrocatechol (4NTC) upon hydrolysis have been synthesized allowing the reliable and specific detection of their target enzymes in different screening modes. Indeed, these substrates can be used for both the high-throughput identification (in cells) and characterization (in liquid-phase assays) of Abfs, thus avoiding the need for distinct solid- and liquid-phase substrates. Simple microtiter plate-based assays were developed and dual-wavelength tracking provides a means to determine kinetic parameters.
Most originally, compounds that bear two vicinal α-L-Araf moieties directly bound to 4NTC or through a linker arm, have proved to be ideal substrates to detect enzymes that are able to accommodate bis-arabinofuranosylated moieties (AXH-d activity), and, as such, are the first of their kind. Furthermore, the use of these substrates to study more general Abf-mediated hydrolysis has provided new insight concerning the mode of action of these enzymes.
Acknowledgements: This work was supported by Midi-Pyrénées region grants DAER-Recherche 10008500 (to V.B.).
1 Borsenberger V, Ferreira F, Pollet A, Dornez E, Desrousseaux M-L, Massou S, Courtin CM, O’Donohue MJ, Fauré R ChemBioChem 2012 13(13):1885-1888.
2 Borsenberger V, Dornez E, Desrousseaux M-L, Courtin CM, O'Donohue MJ, Fauré R Tetrahedron Lett. Accepted.
3 Borsenberger V, Massou S, Dornez E, Desrousseaux M-L, Tenkanen M, Courtin CM, Dumon C, O'Donohue MJ, Fauré R In preparation.
