title: A ¹H NMR study of the specificity of α-l-arabinofuranosidases on natural and unnatural substrates.
abstract: BACKGROUND
  The detailed characterization of arabinoxylan-active enzymes, such as double-substituted xylan arabinofuranosidase activity, is still a challenging topic. Ad hoc chromogenic substrates are useful tools and can reveal subtle differences in enzymatic behavior. In this study, enzyme selectivity on natural substrates has been compared with enzyme selectivity towards aryl-glycosides. This has proven to be a suitable approach to understand how artificial substrates can be used to characterize arabinoxylan-active α-l-arabinofuranosidases (Abfs).
  
  
  METHODS
  Real-time NMR using a range of artificial chromogenic, synthetic pseudo-natural and natural substrates was employed to determine the hydrolytic abilities and specificity of different Abfs.
  
  
  RESULTS
  The way in which synthetic di-arabinofuranosylated substrates are hydrolyzed by Abfs mirrors the behavior of enzymes on natural arabinoxylo-oligosaccharide (AXOS). Family GH43 Abfs that are strictly specific for mono-substituted d-xylosyl moieties (AXH-m) do not hydrolyze synthetic di-arabinofuranosylated substrates, while those specific for di-substituted moieties (AXH-d) remove a single l-arabinofuranosyl (l-Araf) group. GH51 Abfs, which are supposedly AXH-m enzymes, can release l-Araf from disubstituted d-xylosyl moieties, when these are non-reducing terminal groups.
  
  
  CONCLUSIONS
  The present study reveals that although the activity of Abfs on artificial substrates can be quite different from that displayed on natural substrates, enzyme specificity is well conserved. This implies that carefully chosen artificial substrates bearing di-arabinofuranosyl d-xylosyl moieties are convenient tools to probe selectivity in new Abfs. Moreover, this study has further clarified the relative promiscuity of GH51 Abfs, which can apparently hydrolyze terminal disubstitutions in AXOS, albeit less efficiently than mono-substituted motifs.
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: Vinciane
authors.initials: V
authors.lastname: Borsenberger
authors.affiliation: Laboratory of Food Chemistry and Biochemistry, KU Leuven, Kasteelpark Arenberg 20 bus 2463, B-3001 Leuven, Belgium.
authors.forename: Emmie
authors.initials: E
authors.lastname: Dornez
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Desrousseaux
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Massou
authors.affiliation: Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry, University of Helsinki, P.O. Box 27, FI-00014 Helsinki, Finland.
authors.forename: Maija
authors.initials: M
authors.lastname: Tenkanen
authors.affiliation: Laboratory of Food Chemistry and Biochemistry, KU Leuven, Kasteelpark Arenberg 20 bus 2463, B-3001 Leuven, Belgium.
authors.forename: Christophe M
authors.initials: CM
authors.lastname: Courtin
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Dumon
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: O'Donohue
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France. Electronic address: regis.faure@insa-toulouse.fr.
authors.forename: Régis
authors.initials: R
authors.lastname: Fauré
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D006026:Glycoside Hydrolases
keywords: Arabinoxylan arabinofuranohydrolases; Di-arabinofuranosylated substrates; Enzymatic hydrolysis; NMR; Screening; Specificity