The Structure of the Complex between a Branched Pentasaccharide and Thermobacillus xylanilyticus GH-51 Arabinofuranosidase Reveals Xylan-Binding Determinants and Induced Fit †,‡ Gabriel Pae ¨s, §,| Lars K. Skov, ⊥ Michael J. O’Donohue, §,# Caroline Re ´mond, § Jette S. Kastrup, ∇ Michael Gajhede, ∇ and Osman Mirza* ,∇ INRAsUMR FARE 614, 8, rue Gabriel Voisin, BP 316, 51688 Reims cedex 2, France, NoVozymes A/S, KrogshøjVej 36, DK-2880 BagsVaerd, Denmark, and Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, UniVersity of Copenhagen, UniVersitetsparken 2, 2100 Copenhagen, Denmark ReceiVed March 12, 2008; ReVised Manuscript ReceiVed May 21, 2008 ABSTRACT: The crystal structure of the family GH-51 R-L-arabinofuranosidase from Thermobacillus xylanilyticus has been solved as a seleno-methionyl derivative. In addition, the structure of an inactive mutant Glu176Gln is presented in complex with a branched pentasaccharide, a fragment of its natural substrate xylan. The overall structure shows the two characteristic GH-51 domains: a catalytic domain that is folded into a (/R) 8 -barrel and a C-terminal domain that displays jelly roll architecture. The pentasaccharide is bound in a groove on the surface of the enzyme, with the mono arabinosyl branch entering a tight pocket harboring the catalytic dyad. Detailed analyses of both structures and comparisons with the two previously determined structures from Geobacillus stearothermophilus and Clostridium thermocellum reveal important details unique to the Thermobacillus xylanilyticus enzyme. In the absence of substrate, the enzyme adopts an open conformation. In the substrate-bound form, the long loop connecting -strand 2 to R-helix 2 closes the active site and interacts with the substrate through residues His98 and Trp99. The results of kinetic and fluorescence titration studies using mutants underline the importance of this loop, and support the notion of an interaction between Trp99 and the bound substrate. We suggest that the changes in loop conformation are an integral part of the T. xylanilyticus R-L-arabinofuranosidase reaction mechanism, and ensure efficient binding and release of substrate. Harnessing of the renewable carbon reserves contained within plant biomass is regarded to be one of the major technological challenges of the 21st century. To achieve this goal several strategies are being considered. Among these, a biotechnological approach that includes the application of polysaccharide-hydrolyzing enzymes is promising, because of the enormous potential for innovation in the various aspects of the industrial biotechnology sector. Hemicelluloses represent 20-35% of plant biomass and are the second most abundant source of renewable carbon after cellulose (1). Heteroxylans are the most common hemicelluloses and complex polysaccharides composed of a -D-xylose backbone that can be substituted with various neutral (arabinose, galactose) and acidic sugars (methyl- glucuronic acid), as well as with acetyl groups and phenolic acids (2). Due to the high complexity and structural vari- ability of heteroxylans, enzymatic hydrolysis is achieved using a vast array of enzymes that include endoxylanases (EC 3.2.1.1) and debranching enzymes such as R-L-arabino- furanosidases (EC 3.2.1.55). R-L-Arabinofuranosidases (Abf 1 ) are exo-acting enzymes that hydrolyze R-(1f2), R-(1f3) and/or R-(1f5) bonds and release arabinofuranosyl moieties from a wide variety of arabinose-containing substrates, including arabinan, ara- binogalactans, heteroxylans and synthetic compounds such as p-nitrophenyl-R-L-arabinofuranoside (pNP-R-L-Araf) (3). According to the glycoside-hydrolase classification system, Abfs are present in six families (3, 43, 51, 54, 62 and † This work was facilitated by the award of a Marie-Curie fellowship to G.P. and financial support from DANSYNC. ‡ The structures have been deposited in the RCSB Protein Data Bank (www.pdb.org) with pdb codes 2vrk and 2vrq for the Tx-Abf-Se and Tx-Abf-Glu176Gln:X4A structure, respectively. * To whom correspondence should be addressed: Osman Mirza, University of Copenhagen, Faculty of Pharmaceutical Sciences, Depart- ment of Medicinal Chemistry, Universitetsparken 2, 2100 Copenhagen, Denmark. E-mail: om@farma.ku.dk. Phone: +45 35336175. Fax: +45 35336100. § INRAsUMR FARE 614. | Present address: METabolic EXplorer S.A., Biopo ˆle, Clermont- Limagne, 63360 Saint-Beauzire, France. ⊥ Novozymes A/S. # Present address: INSA/INRAsUMR 792, 135, avenue de Rangueil, 31077 Toulouse cedex 04, France. ∇ University of Copenhagen. 1 Abbreviations: Abf, R-L-arabinofuranosidase; Ct-Abf, GH-51 R-L- arabinofuranosidase from Clostridium thermocellum; Gs-Abf, GH-51 arabinofuranosidase from Geobacillus stearothermophilus; pNP-Ara, p-nitrophenyl-R-L-arabinofuranose; pNP-R-L-Araf, p-nitrophenyl-R-L- arabinofuranoside; rms, root-mean-square; Tx-Abf, GH-51 R-L-ara- binofuranosidase from Thermobacillus xylanilyticus; Tx-Abf-Glu176Gln, inactive Glu176Gln mutant of Tx-Abf; Tx-Abf-Glu176Gln:X4A, complex between Tx-Abf-Glu176Gln and X4A; Tx-Abf-Se, a seleno- methionyl derivative of wild type Tx-Abf; Tx-Abf-Trp248Ala, Trp248Ala mutant of Tx-Abf; vdW, van der Waals; X4A, pentasaccharide O-- D-xylopyranosyl-(1f4)-O-R-L-arabinofuranosyl-(1f3)-O--D-xylopy- ranosyl-(1f4)-O--D-xylopyranosyl-(1f4)-O--D-xylopyranose. Biochemistry 2008, 47, 7441–7451 7441 10.1021/bi800424e CCC: $40.75  2008 American Chemical Society Published on Web 06/19/2008