title: Active Site Mapping of Xylan-Deconstructing Enzymes with Arabinoxylan Oligosaccharides Produced by Automated Glycan Assembly.
abstract: Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized resin. Arabinofuranose and glucuronic acid residues have been selectively attached to the backbone using fully orthogonal 2-(methyl)naphthyl (Nap) and 2-(azidomethyl)benzoyl (Azmb) protecting groups at the C2 and C3 hydroxyls of the xylose building blocks. The arabinoxylan oligosaccharides are excellent tools to map the active site of glycosyl hydrolases involved in xylan deconstruction. The substrate specificities of several xylanases and arabinofuranosidases were determined by analyzing the digestion products after incubation of the oligosaccharides with glycosyl hydrolases.
authors.affiliation: Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195, Berlin, Germany.
authors.forename: Deborah
authors.initials: D
authors.lastname: Senf
authors.affiliation: Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany.
authors.forename: Colin
authors.initials: C
authors.lastname: Ruprecht
authors.affiliation: Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195, Berlin, Germany.|Current address: Institute of Organic Chemistry, Johannes Gutenberg-Universität Mainz, Duesbergweg 10-14, 55128, Mainz, Germany.
authors.forename: Goswinus H M
authors.initials: GH
authors.lastname: de Kruijff
authors.affiliation: Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany.|Current address: Rosario Chemistry Institute (IQUIR, CONICET-UNR), Suipacha 570, S2002LRK, Rosario, Argentina.
authors.forename: Sebastian O
authors.initials: SO
authors.lastname: Simonetti
authors.affiliation: Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195, Berlin, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Schuhmacher
authors.affiliation: Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195, Berlin, Germany.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Seeberger
authors.affiliation: Department of Biomolecular Systems, Max-Planck-Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany.|Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195, Berlin, Germany.
authors.forename: Fabian
authors.identifier: http://orcid.org/0000-0003-2206-6636
authors.initials: F
authors.lastname: Pfrengle
mesh_terms: D001439:Bacteroides / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D020134:Catalytic Domain; D020633:Cellvibrio / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D060327:Solid-Phase Synthesis Techniques; D013379:Substrate Specificity; D014990:Xylans / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D014995:Xylosidases / Q000737:chemistry / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D014990:Xylans; C038910:glucuronoxylan; C085118:arabinoxylan; D006026:Glycoside Hydrolases; D014995:Xylosidases
keywords: arabinoxylan; carbohydrates; enzymes; plant cell wall; solid-phase synthesis